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Regulation of ferrochelatase Simpson, Denyse Marie 1977

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THE REGULATION OF FERROCHELATASE by DENYSE MARIE SIMPSON B.Sc, University of Santa Clara, 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES • DEPARTMENT OF BIOCHEMISTRY THE UNIVERSITY OF BRITISH COLUMBIA We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1977 Denyse M. Simpson 1977. In p resent ing t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements fo r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e fo r reference and study. I f u r t h e r agree t h a t permiss ion fo r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . BIOCHEMISTRY Department of ' The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date A U G - 2 9 ' 1 9 7 7 ABSTRACT Regulatory factors a f f e c t i n g ferrochelatase a c t i v i t y were studied and an attempt was made to determine the role of ferrochelatase i n the regulation of heme biosynthesis. Ferrochelatase was found to have a value of 0.105 mM f o r the porphyrin substrates;, proto and mesoporphyrin IX and a K m -3 value of 8.30 x 10 mM for ferrous ion, i t s metal substrate. The V m a x values for proto and mesoporphyrin IX were 12.05 and 28.57 units/mg, respectively, and that of ferrous ion wa^2.89-units/mg. Ferrochelatase exhibited feedback product i n h i b i t i o n by hemin i n concentrations between 1 and 10 uM and stimulation of ferrochelatase a c t i v i t y by hemin at concentrations above 20 uM. Concentrations of ferrous ion exceeding 0.25 mM were found to i n h i b i t ferrochelatase a c t i v i t y , i n d i c a t i n g that the enzyme i s subject to substrate i n h i b i t i o n . The iodoacetamide sensitive binding s i t e of ferrochelatase was determined to be on the inside of the inner mitochondrial membrane in contact with the matrix. Ferrochelatase a c t i v i t y was found to be sen^ s i t i v e to i t s membrane environment,in p a r t i c u l a r i t was dependent on the hydrophobic portion of the phospholipids for a c t i v i t y rather than t h e i r hydrophilic head groups. This was demonstra-ted i n experiments i n which the l i p i d s were removed from submitochondrial p a r t i c l e s or detergent-solubilized preparations of rat l i v e r mitochondria by acetone extraction, and ferrocheTa^ tase a c t i v i t y reconstituted jgy^  the addition of l i p i d s . Reactivation was found to be a function of the unsaturation of the a c y l c h a i n o f e i t h e r the f a t t y a c i d or p h o s p h o l i p i d . C h o l e s t e r o l was found to i n c r e a s e a c t i v i t y below 2 8°C and to decrease a c t i v i t y above 45°C. D i s c o n t i n u i t i e s were seen i n Ar r h e n i u s p l o t s of f e r r o c h e l a t a s e a t 37°C f o r subm i t o c h o n d r i a l p a r t i c l e s and a t 28.5°C f o r detergentT-solubilized p r e p a r a t i o n s . F e r r o c h e l a t a s e was shown to have an abs o l u t e requirement f o r c a l c i u m ions but t h i s was not a requirement of the f e r r o c h e l a t a s e p r o t e i n , r a t h e r , i t was mediated through some e f f e c t o f c a l c i u m on the membrane. F e r r o c h e l a t a s e was observed t o have an absolute requirement f o r f e r r o u s i o n as metal s u b s t r a t e and to be able to u t i l i z e f e r r i c i o n o n l y i n the presence of e l e c t r o n donors such as NADH, NADPH, s u c c i n a t e , «*-glycerol phosphate o r c h o l i n e c h l o r i d e . The recovery^ of e l e c t r o n s from the donors f o r i r o n r e d u c t i o n was dependent upon the presence of t h e i r r e s p e c t i v e dehydrogenases and was independent o f r e s p i r a t i o n or r energy p r o c e s s e s . In a d d i t i o n to p r o v i d i n g r e d u c i n g equiva-l e n t s f o r i r o n r e d u c t i o n , the e l e c t r o n donors a l s o s t i m u l a t e d f e r r o c h e l a t a s e a c t i v i t y . M i t o c h o n d r i a from a n a e r o b i c a l l y grown Saccharomyces c e r e v i s i a e were found t o have hi g h ferro - r c h e l a t a s e a c t i v i t y but to have no i r o n r e d u c t i o n a c t i v i t y , whereas mito c h o n d r i a from a e r o b i c a l l y grown S. c e r e v i s i a e possessed both h i g h f e r r o c h e l a t a s e and i r o n r e d u c i n g a b i l i t i e s . The appearance o f i r o n r e d u c t i o n a b i l i t y d u r i n g r e s p i r a t o r y a d a p t a t i o n o f y e a s t was found t o c o r r e l a t e c l o s e l y w i t h the appearance of r e s p i r a t o r y enzymes and to be one of the f i r s t a c t i v i t i e s d e t e c t e d a f t e r the onset of a e r o f o i o s i s . Conven-t i o n a l techniques were i n s u f f i c i e n t to separate the f e r r o c h e l a t a s i v and i r o n reductase a c t i v i t i e s , although an assay based on the r e d u c t i o n of PMS by f e r r o u s i o n was used to q u a n t i t a t e i r o n r e d u c t i o n a c t i v i t y independently. V CONTENTS Page 1) ABSTRACT .. .. .. .. .. .. i i 2) LIST OF TABLES .. .. .. .. .. v i 3) - LIST OF FIGURES .. .. .. .. .. v i i i 4) ABBREVIATIONS .. .. .. .. .. x 5) ACKNOWLEDGEMENTS .. .. .. .. .. x i i 6) INTRODUCTION .. .. .. .. .. 1 7) MATERIALS .. .. .. .. .. .. 6 8) METHODS .. .. .. .. .. .. 7 9) EXPERIMENTAL RESULTS _ PART ONE KINETICS OF FERROCHELATASE INTRODUCTION .. .. .. . . 15 RESULTS .. . . . . . . 18 DISCUSSION .. .. .. .. 30 PART TWO THE LOCATION OF FERROCHELATASE INTRODUCTION .. .. . . . . 37 RESULTS .. . . . . . . 38 DISCUSSION .. .. .. .. 41 PART THREE MEMBRANE REQUIREMENTS OF FERROCHELATASE INTRODUCTION .. .. .. .. 43 RESULTS .. .. .. .. 45 DISCUSSION .. .. .. .. 57 PART FOUR REGULATORY FACTORS^ INTRODUCTION .. .. . . . . 60 RESULTS .. .. . . . . 62 DISCUSSION .. .. .. .. 89 10) CONCLUSION .. .. .. .. .. 95 BIBLIOGRAPHY .. .. .. .. .. 100 v i LIST OF TABLES TABLE PAGE I THE EFFECT OF FATTY ACIDS" ON FERROCHELATASE ACTIVITY .. .. .. .. .. .. 4 6 II EFFECT OF PURE PHOSPHOLIPIDS ON FERROCHELATASE ACTIVITY' .. .. .. .. .. .. 47 I I I EFFECT OF CHOLESTEROL ON THE ACTIVATION OF FERROCHELATASE BY PHOSPHOLIPIDS .. .. 49 IV EFFECT OF CHOLESTEROL ON THE ACTIVATION OF FERROCHELATASE BY DIPALMITOYL PHOSPHATIDYLCHOLINE 50 V EFFECT OF DIVALENT CATIONS ON THE ACTIVITY OF FERROCHELATASE IN DIALYZED, DETERGENT-SOLUBILIZED PREPARATIONS • .. . . . . . . . . 54 VI EFFECT OF CALCIUM ON THE ACTIVITY OF FERROCHELATASE IN LINOLEIC ACID VESICLES .. .. .. 55 VII EFFECT OF THE SOURCE OF IRON ON THE ACTIVITY OF FERROCHELATASE .. .. .. .. .. 56 V I I I EFFECT OF THE OXIDATION STATE OF IRON ON THE ACTIVITY OF FERROCHELATASE .. .. .. 63 IX EFFECT OF ELECTRON DONORS ON THE UTILIZATION OF FERRIC ION BY FERROCHELATASE IN SMP AND DETERGENT-SOLUBILIZED PREPARATIONS .. .. .. 67 X IRON REDUCING ACTIVITY IN MITOCHONDRIA PREPARED FROM AEROBICALLY-GROWN YEAST .. .. .. 6 8 XI FERROCHELATASE ACTIVITY AND IRON REDUCING ACTIVITY OF MITOCHONDRIA PREPARED FROM ANAE RO BlCALLY-GROWN "YJE.Z\.ST • • • • • • • • • • 73 v i i TABLE PAGE XII EFFECT OF RESPIRATORY INHIBITORS, UNCOUPLERS AND ATP ON IRON REDUCTION .. .. .. 77 XIII INHIBITION OF SUCCINATE IRON REDUCTION ACTIVITY BY MALONATE .. .. ..' .. .. 7.8 XIV LOSS OF NADH STIMULATION OF FERROCHELATASE ACTIVITY FOLLOWING,GEL FILTRATION ON SEPHADEX,G-150 79 XV EFFECT OF HEAT TREATMENT ON FERROCHELATASE ACTIVITY .. .. .w . . . . . . 81 XVI- ELUTION OF SUCCINATE DEHYDROGENASE, FERROCHELATASE AND F e 2 + -PMS REDUCTASE ACTIVITIES FROM A SEPHADEX G-150 COLUMN .. .. .. .. .. 87 v i i i LIST OF FIGURES F i g u r e Page 1 The Heme B i o s y n t h e t i c pathway .. .. .. 3 2 A Double R e c i p r o c a l P l o t of the A c t i v i t y of F e r r o c h e l a t a s e i n Submitochondrial P a r t i c l e s of Rat L i v e r with V a r y i n g c o n c e n t r a t i o n s of Ferrous Ion 2 0 3 A Double R e c i p r o c a l P l o t o f F e r r o c h e l a t a s e i n Submitochondrial P a r t i c l e s of Rat L i v e r w i t h v a r y i n g c o n c e n t r a t i o n s of pr o t o and mesoporphyrin IX .. 22 4 The e f f e c t of heme c o n c e n t r a t i o n on f e r r o c h e l a t a s e a c t i v i t y .. .. .. .. .. .. 2 4 5 The e f f e c t o f heme c o n c e n t r a t i o n on f e r r o c h e l a t a s e a c t i v i t y .. .. .. .. .. .. 2 7 6 A double r e c i p r o c a l p l o t of the e f f e c t of f e r r o u s i o n c o n c e n t r a t i o n on f e r r o c h e l a t a s e a c t i v i t y i n the presence and absence of 5 uM hemin .. .. 29 7 A scheme of the c o n t r o l exerted by heme on i t s b i o s y n t h e t i c pathway .. .. .. .. 35 8 The e f f e c t o f iodoacetamide on f e r r o c h e l a t a s e a c t i v i t y i n f u l l m i t o p l a s t s and i n n e r membrane v e s i c l e s .. .. .. . . . . .. 40 9 Ahi-irenius p l o t s of the e f f e c t of temperature on the a c t i v i t y of f e r r o c h e l a t a s e of sub m i t o c h o n d r i a l p a r t i c l e s and d e t e r g e n t - s o l u b i l i z e d p r e p a r a t i o n s 52 10 The e f f e c t o f e l e c t r o n donors on the u t i l i z a t i o n o f f e r r i c i o n by f e r r o c h e l a t a s e .. .. 65 The e f f e c t of temperature on f e r r o c h e l a t a s e a c t i v i t y and i r o n r e d u c t i o n .. .. Ten hour and two hour time courses of r e s p i r a t o r y a d a p t a t i o n C o - e l u t i o n o f f e r r o c h e l a t a s e and s t i m u l a t o r from a Sephadex G-150 column Sephadex G—150 e l u t i o n p r o f i l e of s u c c i n a t e 2+ dehydrogenase, f e r r o c h e l a t a s e and Fe ^ PMS reductase a c t i v i t i e s X ABBREVIATIONS AD ALA AS ATP BSA Co A Coenz Q Cys-H CO DCPIP Det. S o l DNP EDTA FAD FADH 2 FC F p d F P S FMN IM IAA khz ^ - a m i n o l e v u l i n i c a c i d d f - a m i n o l e v u l i n i c a c i d c f - a m i n o l e v u l i n i c a c i d synthetase adenosine t r i p h o s p h a t e bovine serum albumin coenzyme A, B-mercaptoethylaminopantothenj^s a c i d coenzyme Q, ubiquinone c y s t e i n e i n the f r e e s u l f h y d r y l form coporporphyrinogen I I I oxidase 2 , 6 - d i c h l o r o i n d o p h e n o l . d e t e r g e n t s o l u B i l i z e d p r e p a r a t i o n of r a t l i v e r m i t o c h o n d r i a 2 , 6 - d i n i t r o p h e n o l ethylenediamine t e t r a a c e t a t e disodium s a l t f l a v i n - a d e n i n e d i n u c l e o t i d e f l a v i n - a d e n i n e d i n u c l e o t i d e , dihydrogen form f e r r o c h e l a t a s e the f l a v o p r o t e i n between NADH dehydrogenase and Co Q i n complex I of the r e s p i r a t o r y c h a i n f l a v o p r o t e i n e l e c t r o n c a r r i e r between s u c c i n a t e dehydro-genase and Co Q i n complex I I of the r e s p i r a t o r y c h a i n f l a v i n mononucleotide the i n n e r m i t o c h o n d r i a l membrane iodoacetamide k i l o h e r t z x i the Michaelis-Menton constant expressed i n milimolar concentration units MW molecular weight expressed i n Daltons NADH dihydrogen nicotinamide adenine dinucleotide NADPH dihydrogen nicotinamide adenine dinucleotide phosphate OM the outer mitochondrial membrane P.H. the pyridine hemochrome assay PMS phenazine methosulphate PD protoporphyrinogen IX dehydrogenase RLM rat l i v e r mitochondria SDH succinate dehydrogenase SMP submitochondrial p a r t i c l e s of rat l i v e r mitochondria TTA thenoyltriflouroacetone UD uroporphyrinogen III decarboxylase US uroporphyrinogen III synthetase V m a x . the maximum v e l o c i t y as expressed i n units of enzyme a c t i v i t y per mg protein x i i ACKNOWLEDGEMENTS Any c o n t r i b u t i o n I may have made i s due to the example of e x c e l l e n c e s e t f o r me by my mentor, Rozanne Poulson. INTRODUCTION 2 The sequence of reactions by which heme i s synthesized was o r i g i n a l l y elucidated by Shemin and his colleagues i n a series of elegant experiments i n which i s o t o p i c a l l y l a b e l l e d proto-porphyrin of hemoglobin was achieved by the administration of lab e l l e d glycine to animals x ~ . The series of enzymic steps from the condensation of glycine and succinyl-CoA to form cS-aminolevulinic acid to the in s e r t i o n of iron into protoporphy-r i n IX to y i e l d heme are shown i n figure 1. Unequivocal evidence i s available for a l l steps although d e t a i l s of some of these reactions remain uncertain; namely, the precise manner of oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX, the mechanism of dehydrogenation of protoporphyrinogen IX to protoporphyrin IX and the manner of ins e r t i o n of iron into protoporphyrin IX to form heme. In mammals, the p r i n c i p a l s i t e s of heme biosynthesis are the l i v e r , where 70% of the heme formed serves as the prosthetic group for the mitochondrial and microsomal cytochromes, and the erythropoietic tissues. The rate of formation of porphyrins and heme i n both pro-caryotes and eucaryotes i s considered to be controlled by the f i r s t enzyme of the heme biosynthetic pathway^ -^, <J-amino-l e v u l i n i c acid synthetase (succinyl CoA: glycine c-succinyl transfease (decarboxylating) EC 2.3.1.37). This enzyme has recently been p u r i f i e d ^ . ALA-synthetase i s present i n the l i v e r mitochondria under normal conditions i n very low l e v e l s , apparently just s u f f i c i e n t to meet the heme requirements of the c e l l . A wide range of chemicals and steroids can upset t h i s 3 GLYCINE + SUCCINYL-SCoA 8-AMINOLEVULINIC ACID SYNTHASE -» S -AMINOLEVULINIC ACID S -AMINOLEVULINIC ACID DEHYDRATASE PORPHOBILINOGEN COPROPORPHYRINOGEN III ^ UROPORPHYRINOGEN I SYNTHASE + UROPORPHYRINOGEN III COSYNTHASE UROPORPHYRINOGEN UROPORPHYRINOGEN I SYNTHASE UROPORPHYRINOGEN I UROPORPHYRINOGEN III DECARBOXYLASE COPROPORPHYRINOGEN OXIDASE . PROTOPORPHYRINOGEN IX P R 0 T O P O R P H Y R I N O G E N ^ PROTPORPHYRIN IX DEHYDROGENASE FERROCHELATASE Fe-PROTOPORPHYRIN IX Mg-PROTOPORPHYRIN HEME, CYTOCHROMES PROSTHETIC GROUPS CHLORINS 4 d e l i c a t e balance leading to increases i n the hepatic l e v e l of the enzyme and to porphyrin and porphyrin precursor accumulation and e x c r e t i o n ^ - l 2 . These chemically induced porphyrias have attracted much attention because of the biochemical s i m u l a r i t i e s to the genetically determined porphyric diseases i n man. However, the diverse patterns of porphyrin accumulation i n inher-i t e d and chemically-induced porphyrias cannot be explained by the loss of regulation of ALA-synthetase alone. Thus, i t seems l i k e l y that there are other points of regulation i n this pathway. As described i n t h i s thesis, the finding that ferrochelatase, the l a s t enzyme in the pathway, also exhibits regulatory control may contribute to our understanding of the i n t r i c a t e regulatory mechanisms c o n t r o l l i n g the heme biosynthetic pathway. Ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1), also c a l l e d heme synthetase, which i s a membrane bound enzyme, catalyzes the incorporation of ferrous ion into protoporphyrin IX to form protoheme"''. A number of i n d i r e c t pieces of evidence led us to postulate that the enzyme, which has been l i t t l e studied, might serve an important regulatory function i n 15 heme synthesis. In protoporphyria , protoporphyrin IX accumu-lates and may indicate a l e s i o n i n the enzyme, ferrochelatase which u t i l i z e s i t as a substrate; ferrochelatase a c t i v i t y i s enhanced by chemicals which produce several forms of chemical porphyria, and the enzyme exhibits feedback product i n h i b i t i o n . The present study was undertaken to investigate the factors regulating ferrochelatase a c t i v i t y i n the mitochondria of r a t l i v e r and Saccharomyces cerevisiae. The location of the enzyme 5 and k i n e t i c constants were determined and substrate and product i n h i b i t i o n studies were performed. Environmental (membrane) factors necessary for optimum a c t i v i t y as well as other metabo-l i t e s a f f e c t i n g regulation were defined. The data indicate that ferrochelatase possesses unique regulatory mechanisms and suggest a ro l e for ferrochelatase i n other diverse enzyme func-tions within the mitochondrion. 6 MATERIALS Protoporphyrin IX dimethyl-ester, mesoporphyrin IX d i -methyl-ester, bovine hemin, D-mannitol, HEPES (N-2-hydroxy-ethyl-piperazine N-2-ethane sulfonic acid), bovine serum albumin, d i g i t o n i n , iodoacetamide, bovine heart c a r d i o l i p i n , c i s - l i n o l e i c acid, reduced nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate, Blue Dextran, cyclohexamide, chloramphenicol, adenosine triphosphate, phen-azine methosulfate and Antimycin A^ were obtained from Sigma Chemical Co. L-(+) cysteine-HCl (H20) was obtained from MCB Manufacturing Chemicals. Sucrose (a n a l y t i c a l grade) was pur-chased from Mallinkrodt. Distearoyl -L - o c - L e c i t h i n , d i o l e o y l -L-<x-lecithin and d i l i n o l e o y l - L - ° t - l e c i t h i n were obtained from Supelco Inc. Stearic acid, o l e i c acid//^-!f'-dipalmitoyl-L-«-l e c i t h i n and bovine phosphatidyl serine were products of Calbiochem. Linolenic acid was obtained from Hormel Foundation. Sephadex G-25 and G-150 were purchased from Pharmacia Fine Chemicals. Succinic acid disodium s a l t (succinate) was obtained from Eastman Organic Chemicals. Choline chloride, ergosterol, dinitrophenol and malonic acid disodium s a l t (malonate) were purchased from Matheson, Coleman and B e l l . Tween 80 and 2,6-dichloroindophenol (DCPIP) were obtained from Fisher S c i e n t i f i c Co. Glasperlin 0.45-0.50 mm were obtained from VWR S c i e n t i f i c . Yeast extract was purchased from Difco Laboratories. Nitrogen gas was obtained from Canadian Liquid A i r . ot-glycerol phosphate was a g i f t of Dr. D. E. Vance and thenoyltriflouroacetone was a g i f t of Dr. P. D. Bragg. 7 METHODS PREPARATION OF RAT LIVER MITOCHONDRIA Male wistar rats (22 0 g each), which had been starved 2 4 h, were k i l l e d and th e i r l i v e r s removed immediately and c h i l l e d i n cold buffer containing 5 mM Tris-HCl, pH 7.5, and sucrose, 0.25 M. Mitochondria were prepared from homogenates of r a t l i v e r by the 1 c method of Hogeboom . PREPARATION OF ENZYME EXTRACTS FROM RAT LIVER MITOCHONDRIA Submitochondrial p a r t i c l e s were prepared by resuspending the mitochondrial p e l l e t s i n two volumes of 50 mM Tris-HCl, pH 7.5, and subjecting the suspension to ultrasonic v i b r a t i o n for 5 min. at 20 kHz using a Bronwill Biosonik O s c i l l a t o r cooled by a rapi d l y flowing stream of cold water. The s o n i c a l l y disrupted suspension v/as centrifuged at 10,000 x g for 10 min. The supernatant was removed and recentrifuged at 100,000 x g for 1 h. The p e l l e t was resuspended i n two volumes of 50 mM Tris-HCl, pH 7.5 and designated the submitochondrial p a r t i c l e preparation. The detergent-solubilized extract was prepared by resuspend-ing the mitochondrial p e l l e t i n 50 mM Tris-HCl, pH 7.5 containing 0.8% KC1 and 1% cholate and subjecting the suspension to ultrasonic v i b r a t i o n for 5 min, at 2 0 kHz. After incubation i n an ice bath for 30 min. mitochondrial debris was removed by centrifugation at 20,000 x g for 20 min. The supernatant was recentrifuged at 176,000 x g for 1 h i n a Spinco 60 T i rotor. The r e s u l t i n g high speed supernatant constitutes the detergent-s o l u b i l i z e d preparation. 8 PREPARATION OF MITOPLASTS FROM RAT LIVER MITOCHONDRIA Mitoplasts were prepared by the method of Schnaitman and 17 18 Greenawalt as modified by Chan et a l A volume of mito-chondrial suspension was s t i r r e d (100 mg protein/ml) gently. An equal volume of 1.2% d i g i t o n i n stock solution was added and the mixture s t i r r e d for 15 min. This solution was d i l u t e d 1:4 by the addition of 3 volumes of i s o l a t i o n medium containing sucrose, 70 mM; D-mannitol, 220 mM; HEPES buffer, 2.0 mM; BSA, 0.5 mg/ml and adjusted to pH 7.4 just p r i o r to use. The di l u t e d suspension was homogenized b r i e f l y using a hand operated glass homogenizer and centrifuged at 10,000 x g for 10 min. The supernatant was c a r e f u l l y removed. The sediment was resuspended i n two volumes of i s o l a t i o n medium and resedimented at 10,000 x g. The p e l l e t was resuspended i n 50 mM Tris-HCl, pH 7.5 and desig-nated the mitoplast preparation. PREPARATION OF INNER MITOCHONDRIAL MEMBRANE PARTICLES Inner mitochondrial membrane v e s i c l e s were formed by subjecting the mitoplast preparation to sonic treatment f o r 5 min. at 20 kHz using a Bronwill Biosonic O s c i l l a t o r . The s o n i c a l l y disrupted suspension was centrifuged at 100,00 x g f o r 1 h. The p e l l e t was resuspended i n two volumes of 50 mM Tris-HCl, pH 7.5 and constitutes the inner mitochondrial membrane p a r t i c l e s . PREPARATION OF DIALYZED ENZYME PREPARATIONS FROM MITOCHONDRIA Dialyzed enzyme preparations were obtained by d i a l y z i n g aliquots of enzyme preparations, prepared as stated e a r l i e r , f or 20 h against 2 changes of 20 mM Tris-HCl, pH 7.5, containing 20 mM 2-mercaptoethanol and either 0.1 or 1.0 mM EDTA. 9 PREPARATION OF LIPID-DEPLETED ENZYME PREPARATIONS L i p i d depleted enzyme preparations were obtained by a modification of the acetone extraction procedure described by Lester and F l e i s c h e r ^ 9 . Two volumes of cold 90% aqueous acetone were added i n drops to andenzyme preparation at 0°C. After s t i r r i n g for 20 min., the protein was c o l l e c t e d by centrifugation at 10,000 x g for 10 min. The p e l l e t was spread on Whatman No. 50 f i l t e r paper and dried for 2 h at 4°C. The p e l l e t was redissolved i n tuo volumes of 50 mM Tris-HCl, pH 7.5 and desig-nated the acetone extract of lipid-depleted preparation. PREPARATIONS OF SUSPENSIONS OF FATTY ACIDS AND PHOSPHOLIPIDS Each suspension of f a t t y acid of phospholipid was prepared by adding 0.2 ml of 0.5 M Tris-HCl, pH 7.5, to the f a t t y acid or phospholipid and a g i t a t i n g the suspension on a Vortex mixer fo r 10 min. i n the presence of glass beads (0.45-0.50 mm diameter) . Protoporphyrin IX (0.8 ml, 0.1 mM) was then added and incorporated into the l i p i d phase and t h i s was followed by addition of 0.5 ml of resuspended acetone extract obtained from detergent-solubilized enzyme preparation. The reaction was started by the addition of 0.5 ml of 0.4 mM FeS0 4 > Assays contained 10 mM f a t t y acid or 1 mM phospholipid each. CULTURE MEDIA FOR SACCHAROMYCES CEREVISIAE The yeasts were grown either aerobically or anaerobically i n 20 the media described by Lindenmeyer and Smith , modified by the exclusion of l a c t a t e . Aerobically the c e l l s were grown on 4% (w/v) glucose at 29°C i n 500 ml of medium in a 4 l i t e r f l a s k on a c i r c u l a t i n g shaker at 100 rpm. For anaerobic growth the i n i t i a l glucose concentration was 4% (w/v). The inoculum was 1 0 taken from an aerobic starter culture and added to one l i t e r of medium i n an a i r - t i g h t two l i t e r flask f i t t e d with i n l e t and out-l e t tubes. After inoculation, the medium was thoroughly flushed with oxygen-free nitrogen for 60 min. and the c e l l s grown at 29°C with shaking at 1 0 0 rpm. PREPARATION OF CELL SUSPENSIONS AND FREEZE-THAWED MITOCHONDRIA FROM YEAST Cultures were cooled to 4°C i n i c e water and harvested by centrifugation. The freshly harvested c e l l s were washed twice with ice water and coll e c t e d by centrifugation. Mitochondria from c e l l s grown either aerobically or anaerobically were prepa-2 1 red by the method of Yu et a l , except that a sucrose:Tris-HCl buffer (o . 2 5 M sucrose, 0 . 0 5 M Tris-HCl, pH 7 . 5 ) was substituted. The crude mitochondrial f r a c t i o n was washed three times with sucrose:Tris-HCl buffer and stored at 20°C. The freeze-thawed mitochondrial p e l l e t was resuspended at a concentration of 20 mg of protein/ml of 0 . 0 5 M Tris-HCl, pH 7 . 5 and used as the mito-chodrial f r a c t i o n . RESPIRATORY ADAPTATION OF YEAST S. cerevisiae c e l l s were grown anaerobically i n glucose s a l t s medium to an absorbance of A 6 4 Q = 2 . 0 and harvested and washed i n cold nitrogen-saturated water under nitrogen. The c e l l s were resuspended i n adaptation medium. Adaptation medium I contained: yeast extract, 0 . 5 % ; KH 2P0 4, 0 . 9 % ; MgS04, 0 . 0 5 5 ; CaCl 2, 0.4% (NH 4) 2S0 4, 0 . 0 6 % and glucose, 5%. The c e l l suspension was incubated at 2 9°C with shaking at 1 0 0 rpm under a constant stream of nitrogen gas for 2 h. The c e l l s were 11 harvested under nitrogen with nitrogen-saturated water. Washed c e l l s were suspended i n water (one gram wet weight/ml water) and f i v e grams of c e l l s added to 2 l i t e r s of fresh adaptation medium I I . Medium two contained yeast extract, 0.2%; KI-^PO^ 0.9%, MgS04, 0.025%; CaCl 2, 0.035% (NH 4)S0 4, 0.06% and glucose, 1%. C e l l s were incubated at 29°C with shaking at 250 rpm under a i r . At timed i n t e r v a l s , c e l l s were harvested and mitochondria prepared as described above. Respiratory adaptation was stopped by the addition of cyclohexamide (0.1 irig/ml) , and chloram-phenicol (1 mg/ml) which were added to the adaptation medium 15 min. p r i o r to harvesting. ASSAY OF FERROCHELATASE ACTIVITY Ferrochelatase a c t i v i t y was assayed by the pyridine hemo^ 22 chrome procedure e s s e n t i a l l y as described by porra and Ross . The reaction mixture contained Tris-HCl, pH 7.5, 100 Mm; cysteine, 10 mM; 0.1 mM FeSO^; proto or mesoporphyrin IX, 0.1 mM and 0.5 ml of enzyme preparation. The t o t a l volume of a l l assays was 2 ml. One unit of ferrochelatase a c t i v i t y i s defined as the amount that catalyzes the formation of one nmole of heme from either proto or mesoporphyrin IX and ferrous ion i n 1 h under standard conditions. S p e c i f i c a c t i v i t y i s expressed as units per mg protein. ASSAY OF SUCCINATE DEHYDROGENASE Succinate dehydrogenase a c t i v i t y was assayed by following the succinate dependant reduction of PMS and DCPIP. The reaction mixture (2.57 ml) contained: assay mixture (0.1 M KH 2P0 4, pH 7.4; 0.1 M KCN; 4 mg/50ml DCPIP; 40 mg/50 ml PMS); suc-cinate, 1.5 mM; and ezyme preparation, 20 1. One unit of succinate dehydrogenase a c t i v i t y i s defined as the amount which catalyzes the formation of 1 nmole of reduced PMS i n 1 sec. under standard assay conditions. ASSAY OF FERROUS ION-PMS REDUCTASE Ferrous ion-PMS reductase a c t i v i t y was assayed by measuring the ferrous ion dependant reduction of PMS/DCPIP i n the presence of Antimycin A^. The reaction mixture (3 ml) contained: assay mixture (same as for succinate dehydrogenase), 2.8 ml; antimycin A3, 3 u l ; FeSO^, 0.013 mM; the enzyme preparation, 50 u l . One unit of ferrous ion-PMS reductase a c t i v i t y i s defined as the amount catalyzing the formation of 1 nmole of reduced PMS i n one second under standard assay conditions. ASSAY OF ELECTRON-DONOR DEPENDANT IRON REDUCTION Electron-donar dependant iron reduction a c t i v i t y was assayed by measuring the amount of heme formed with f e r r i c ion as metal substrate in the presence of either succinate, NADH, or NADPH. The reaction was i d e n t i c a l to that used i n the ferro -x chelatase assay with the exception that f e r r i c chloride was used i n place of ferrous sulfate, no cysteine was present i n the incubation mixture and the mixture contained either succinate, 3 mM; NADH, 3 mM or NADPH, 1 mM. ASSAY OF SUCCINATE STIMULATING ACTIVITY The a b i l i t y of succinate to stimulate ferrochelatase a c t i v i t y was assayed by measuring the amount of heme formed with succinate present in the assay mixture. The incubation mixture was i d e n t i c a l to that used to assay ferrochelatase with the exception that 3 mM succinate was present. PROTEIN DETERMINATION 23 P r o t e i n c o n t e n t was measured by t h e method o f Lowry e t a l u s i n g c r y s t a l l i n e b o v i n e serum albumi n ( F r a c t i o n V) as s t a n d a r d . THE PREPARATION OF PORPHYRINS The d i m e t h y l e s t e r s o f p r o t o p o r p h y r i n I X and m e s o p o r p h y r i n IX were h y d r o l y z e d w i t h 7.0 N HC1 f o r 5 h a t room t e m p e r a t u r e i n the d a r k . The a c i d was e v a p o r a t e d under reduced p r e s s u r e and the f r e e p o r p h y r i n was d i s s o l v e d i n 0.01 N KOH c o n t a i n i n g 20% e t h a n o l t o make a s o l u t i o n a t 1.5 mM w h i c h was s t o r e d a t -20°C. EXPERIMENTAL RESULTS 15 PART ONE KINETICS OF FERROCHELATASE INTRODUCTION The precise mechanism by which ferrochelatase f a c i l i t a t e s the combination of the two substrates ferrous ion and proto-porphyrin IX i s unknown. Product i n h i b i t i o n studies with hemin of Rhodopseudomonas spheroides established the regulatory role of ferrochelatase but as yet no substrate i n h i b i t i o n studies have been done to determine the reaction sequence. Because ferrochelatase has not been p u r i f i e d i t i s not known whether the low s p e c i f i c a c t i v i t y i n r a t l i v e r mitochondria (approximately 3 nmoles of heme formed per mg protein per hour at 37°C using the pyridine hemochrome assay method) i s due to a r e l a t i v e l y inactive enzyme or a highly active enzyme present i n small quantities. Michaelis-Menton constants from widely d i f -ferent sources are evidence of the great d i v e r s i t y of ferrochela-tase enzymes which d i f f e r vastly i n t h e i r a f f i n i t i e s for t h e i r substrates. The d i f f i c u l t y i n c o r r e l a t i n g k i n e t i c data, and indeed a l l data on ferrochelatase, i s the fact that three greatly d i f f e r e n t assay methods are presently employed by workers. The radioassays 5 9 employing [ Fe] as iron substrate followed by c r y s t a l l i z a t i o n of 25 ? 1 heme by the method of either Chu and Chu or Labbe and Nishida^" are considered less r e l i a b l e k i n e t i c a l l y , possibly producing an • - e f f e c t at the active s i t e , than the pyridine hemochrome method of Porra and Ross , which has been used throughout these 16 experiments. Even within the i n d i v i d u a l assay procedures there has been a wide variety of substrates used. For example, 21 Jones and Jones i n t h e i r k i n e t i c studies on ferrochelatase employed deuteroporphyrin and cobalous ion as substrates, whereas 29 Porra and Lascelles used ferrous ion and mesoporphyrin. Types. of enzyme preparation and s o l u b i l i z a t i o n techniques have also d i f f e r e d leaving one even more skeptical of comparing k i n e t i c constants; J o n e s ^ s o l u b i l i z e d spinach chloroplast p a r t i c u l a t e 31 ferrochelatase with 1% Tween 20, Yoneyama et a l s o l u b i l i z e d rat l i v e r mitochondria ferrochelatase with 1% cholate and isotonic KC1 and used the 6,00 0x<^ xh supernatant. In a l l of the following experiments ferrochelatase was considered s o l u b i l i z e d when treated with 0.8% KC1 w/v and 1% cholate and the 100, 000x5>xA supernatant used. However, several pieces of information now lead us to believe that the enzyme was not t r u l y s o l u b i l i z e d under these conditions. F i r s t , the recovery of ferrochelatase a c t i v i t y from the mitochondrial membrane i s only 50%, the other 50% remains i n the 100,000 xcyxh p e l l e t . When the mitochondria are incubated overnight with 1% cholate and 0.8% KC1 at -20°C no p e l l e t i s formed upon centrifugation, only a dense black o i l which layers out at the bottom of the centrifuge tube. Given enough time, however, the s o l u b i l i z a t i o n technique w i l l comple-t e l y disrupt the membrane. Second, the i r r e g u l a r i t y of the elution p r o f i l e from Sephadex G-150 (figures 12 and 14) and Sepharose 4B suggest v e s i c l e s of d i f f e r i n g size aire being sepa-rated, a l l of which contain some ferrochelatase. The peak for succinate dehydrogenase i s sharper and more well defined (figure 14) suggesting that i t was truely s o l u b i l i z e d under the conditions used. F i n a l l y , the studies of heat treatment on mitochondrial extracts yielded more ferrochelatase a c t i v i t y i n the p e l l e t a f t e r heating for one hour at 4 5°C, than i n the supernatant (table 12) suggesting ferrochelatase has been pulled down into the 10,000X0.*/? p e l l e t by i t s association with some heat dena-tured proteins. These observations a l l suggest that the " s o l u b i l i z e d " preparation i s a membrane dispersed preparation and consists of rA\CEL=te.T,--s' or v e s i c l e s of mitochondrial phospholipid and cholate with ferrochelatase and other membrane bound enzymes imbedded. I w i l l , however, continue to ref e r to the preparation as the detergent-solubilized preparation. For the reasons mentioned above, i t was considered necessary to determine k i n e t i c constants for rat l i v e r mitochondrial f e r -rochelatase of our detergent-solubilized preparations, under our assay conditions using FeSO^ as iron substrate and proto- or mesoporphyrin IX as porphyrin substrates. To obtain informa-t i o n on the regulation of ferrochelatase and i t s possible role i n the regulation of heme biosynthesis i n rat l i v e r mitochondria, the range of concentrations under which hemin exerted end product i n h i b i t i o n was investigated. To determine the possible reaction sequence, the e f f e c t of ferrous ion at con-centrations greater than saturation was studied. 18 RESULTS KINETIC CONSTANTS OF FERROCHELATASE The a c t i v i t y of ferrochelatase in submitochondria p a r t i c l e s was assayed i n the presence of 0.1 mM protoporphyrin IX and concentrations of ferrous sulfate between 0.10 mM and 0.01 mM. The V „ = v and K of ferrochelatase for ferrous ion were deter-IlldX III mined by a double r e c i p r o c a l p l o t (Figure 2). The K m was determined to be 8.30 x 10~ 3 mM and the V m a x 2.89 units/mg. The a c t i v i t y of ferrochelatase was assayed i n the presence of 0.1 mM ferrous sulfate and concentrations of eithe r proto or mesoporphyrin IX between 0.10 mM and 0.01 mM. The V m a x and K m of ferrochelatase for proto and mesoporphyrin substrates were determined from double r e c i p r o c a l plots (Figure 3) . The V" m a x for mesoporphyrin IX was found to be 2 8.57 units/mg and 12.05 units/mg protein for protoporphyrin IX. The K^ for both por-phyrins was determined to be 0.105 mM. The plots presented i n figures 3 and 2 also indicate that ferrochelatase reaction follows a sequential type mechanism. THE EFFECT OF HEMIN ON FERROCHELATASE Ferrochelatase a c t i v i t y i n the submitochondrial p a r t i c l e s was assayed i n the presence of from 1.0 uM to 100 uM hemin. When present i n concentrations up to 10 uM, hemin i n h i b i t e d ferrochelatase a c t i v i t y , whereas i t stimulated a c t i v i t y when present i n concentrations above 10 uM. In the presence of 10 uM hemin, ferrochelatase a c t i v i t y was i n h i b i t e d 50% (Figure 4). The product stimulation above 10 uM was due, i n part, to the 19 Figure 2 A double r e c i p r o c a l p l o t of the a c t i v i t y of ferrochelatase i n submitochondrial p a r t i c l e s of rat l i v e r with varying concen-t r a t i o n of ferrous ion. Assays were performed under standard conditions as described i n Methods with a range of ferrous sulfate concentrations from 0.1 mM to 0.01 mM and 0.10 mM protoporphyrin IX as porphyrin substrate. The ordinate values are the r e c i p r o c a l of s p e c i f i c a c t i v i t y (units/mg protein) and the abscissa values are the r e c i p r o c a l of ferrous ion concentration. The intercept on the ordinate axis i s 0.342/units/mg and the intercept on the abscissa axis i s -121/mM. 21 Figure 3 A double r e c i p r o c a l p l o t of the a c t i v i t y of ferrochelatase in submitochondrial p a r t i c l e s of rat l i v e r with varying con-centration of proto and mesoporphyrin IX. Assays were performed under standard conditions as described i n Methods with a range of proto and mesoporphyrin concentrations from 0.1 mM to 0.01 mM and with 0.1 mM ferrous s u l f a t e as metal substrate. The ordinate values are r e c i p r o c a l of s p e c i f i c a c t i v i t y (units/mg protein) and the abscissa values are r e c i p r o c a l of porphyrin concentration. The i n t e r -cept with the ordinate for mesoporphyrin IX i s 0.035/units/mg protein and for protoporphyrin IX i s 0.085/units/mg protein and the intercept with the abscissa for both proto and mesoporphy^ r i n i s -9.50/mM. o, ferrochelatase a c t i v i t y with protopor^ phyrin IX as substrate; • , ferrochelatase a c t i v i t y with mesoporphyrin IX as substrate. 1 V CD 6 ( p o r p h y r i n ) 23 Figure 4 The e f f e c t of hemin concentration on ferrochelatase activity-Assays were performed as described under Methods except for the addition of varying concentrations of hemin from 1 to 100 uM. Total reaction volume was 2 ml, the controls con-tained water i n place of hemin. The ordinate values are s p e c i f i c a c t i v i t y i n units of ferrochelatase a c t i v i t y per mg protein. The abscissa values are the concentrations of hemin in mM. S P E C I F I C A C T I V I T Y 4 6 8 10 u t i l i z a t i o n of hemin by ferrochelatase as substrate. Enzyme-free controls containing hemin gave n e g l i g i b l e absorbance read-ings under the assay conditions, but protoporphyrin-free controls containing hemin had high a c t i v i t i e s which were subtracted from the experimental r e s u l t s . Product i n h i b i t i o n by hemin was l i n e a r over the concentration range from 1.0 to 10.0 uM (Figure 5). THE EFFECT OF FERROUS ION ON FERROCHELATASE Ferrochelatase a c t i v i t y i n submitochondrial p a r t i c l e s was assayed over the range of ferrous sulfate concentrations from 0.1 mM to 1.0 mM i n the presence of protoporphyrin IX. This was repeated i n the presence of 5.0 uM hemin. Substrate i n h i b i t i o n by ferrous ion was observed over the iron concentra-t i o n range of 0.25 mM to 1.0 mM. At ferrous ion concentrations of 0.5 mM and 1.0 mM i n h i b i t i o n was 50% and 80%, respectively (Figure 6). In the presence of 5 uM hemin the same trend of substrate i n h i b i t i o n was observed but the maximum a c t i v i t y was lower due to the product i n h i b i t i o n by hemin at t h i s concen-t r a t i o n . Thus, i n the presence of 5 uM hemin, at ferrous ion concentrations of 0.5 mM and 1.0 mM enzyme a c t i v i t y was i n h i b i -ted 40% and 75% respectively. 26 Figure 5 The e f f e c t of hemin concentration on ferrochelatase a c t i v i t y Assays were performed as described under Methods except for the addition of hemin. The ordinate values are s p e c i f i c a c t i v i t y and the abscissa values are the concentrations of hemin. S P E C I F I C A C T I V I T Y V 2 4 6 8 1 0 o — » 28 Figure 6 A double r e c i p r o c a l p l o t of the e f f e c t of ferrous i o n concen-t r a t i o n on f e r r o c h e l a t a s e a c t i v i t y i n the presence and absence of 5 uM hemin. Assay c o n d i t i o n s were as described under Methods except th a t the ferrous i o n concentration was v a r i e d from 0.1 mM to 1.0 mM and one set of samples contained 5 uM hemin. The or d i n a t e values are the r e c i p r o c a l s of s p e c i f i c a c t i v i t y and the a b s c i s s a values are the r e c i p r o c a l s of ferrous i o n concen-t r a t i o n , o, f e r r o c h e l a t a s e a c t i v i t y ; •, f e r r o c h e l a t a s e a c t i v i t y i n the presence of 5 M hemin. 29 30 DISCUSSION The studies described here indicate that the ferrochela-tase reaction proceeds through a sequential mechanism, shows product i n h i b i t i o n at concentrations of hemin below 10 uM, substrate i n h i b i t i o n at ferrous ion concentrations above 0.25 mM and has a lower a f f i n i t y for i t s porphyrin substrate than i t s metal substrate. The K m of ferrochelatase i n rat l i v e r mitochondria for proto and mesoporphyrin IX of 105 jj'M i s cl o s e l y comparable to the K m of 100 ^ iM determined by Yoneyama et a l ^ 2 . The a f f i n i t y f or iron as determined i n th i s work, i s much greater having a K m of 8.30 uM compared with a value of 60 uM as deter-59 mined by Yoneyama, who used an. [ Fe] assky. The constants 59 determined for iron using [ Fe] did not take into account any e f f e c t of the isotope and are probably less r e l i a b l e than those determined i n our studies. Maximum ve l o c i t y constants of 2.89 units/mg for iron and 28.57 and 12.05 units/mg were obtained for meso and protoporphyrin IX, respectively. The greater a c t i v i t y of ferrochelatase with meso than protoporphyrin r e f l e c t s i t s greater maximum v e l o c i t y rather than i t s greater a f f i n i t y for mesoporphyrin, since the values of proto and mesopor-phyrin IX are identical.* The a f f i n i t y of bone-marrow ferrochelatase for protopor-32 phyrin IX as determined by Bottomly i s much greater than the l i v e r enzyme. She obtained values of 1.80 uM for protopor^ phyrin IX which might be an ind i c a t i o n of the a c t i v i t y of the _ 2 pathway. The K m value for iron was found to be 1.7 x 10 mM, higher than that for the l i v e r enzyme, that i s , the r e l a t i v e a f f i n i t i e s for the two substrates are reversed. However, the K m for iron was determined using the [ Fe] assay. A tabula-t i o n of values from d i f f e r e n t enzyme sources shows vast differences i n the k i n e t i c properties of the various ferrochela-tases. These values may well be a r e f l e c t i o n of properties of the enzyme other than s t r u c t u r a l or i o n i c differences of the active s i t e . The product i n h i b i t i o n of ferrochelatase i n Rhodopseudo-25 monas spheroides was discussed by Jones and co-workers i n 1970 2+ Using Co and deuteroporphyrin as substrates i n c e l l - f r e e extracts of R. spheroides they found i n h i b i t i o n by protoheme at concentrations up to 120 uM. The present studies indicate that the regulatory features of the eukaryotic mitochondrial system are very much more complex than those observed by Jones in the b a c t e r i a l system. We found i n h i b i t i o n by hemin to be 50% at 10 uM with increasing a c t i v i t y at hemin concentrations above 10 uM. At concentrations above 50 uM hemin stimulates ferrochelatase a c t i v i t y . The possible regulatory si g n i f i c a n c e of t h i s two-way e f f e c t must be considered i n l i g h t of the fact that heme regulates both the end and the beginning of the pathway. Concentrations of heme as small as 0.2 uM repress the synthesis of ALA- synthetase at some post - t r a n s c r i p t i o n a l 34-35 step i n chick embryo l i v e r . At much higher concentrations, about 35 uM hemin i n h i b i t s the a c t i v i t y of p u r i f i e d ALA-synthetase a c t i v i t y 3 6 . Thus, heme controls the pathway by 32 j Enzyme Source Assay Fe K Values (M) 3+ Proto Meso RLM P.H. 8. 3 105 105 ! 31 ! RLM [ 5 9Fe] 60 100 32 ; Bone Marrow [5 9Fe] 60 170 18 . . 33 S. i t e r s o n i i P.H. 20 47 30 Spinach Chloroplast [5 9Fe] 8. 0 0. 2 0.4 25 R. spheroides P.H. 6. 13 21. 3 P.H. i s the pyridine hemochrome assay A tabulation of the K m values of ferrochelatase for i t s metal and pophyrin substrates i s o l a t e d from various sources. 33 feedback i n h i b i t i o n i n four s p e c i f i c ways. Namely below 10 uM hemin i n h i b i t s ferrochelatase a c t i v i t y and represses ALA-synthetase synthesis, above 50 uM i t stimulates ferrochelatase a c t i v i t y and i n h i b i t s ALA-synthetase a c t i v i t y , thus allowing the f i n e s t control possible. When heme i s formed and released from the mitochondrion the c e l l u l a r l e v e l builds up and at 0.2 uM i t represses the formation of new ALA-synthetase enzyme. Although i t i s not known what intramitochondrial heme concentra-t i o n a 0.2 uM cytoplasmic corresponds to, i t appears that the f i r s t control point i s the de novo synthesis of ALA-synthetase. The second control i s the 5 0% i n h i b i t i o n of ferrochelatase a c t i v i t y when the heme concentration reaches 10 uM. A 50% reduction i n ferrochelatase a c t i v i t y would s t i l l allow substan-t i a l formation of heme. If the concentration of protoporphyrin i s high, the l e v e l of heme w i l l continue to r i s e u n t i l i t i s eventually present at a concentration s u f f i c i e n t to i n h i b i t ALA-synthetase a c t i v i t y i n the mitochondrion and to stimulate ferrochelatase a c t i v i t y and thereby reduce the l e v e l of proto^ porphyrin. The following scheme (Figure 7) shows how v/e envision the coarse and fine controls of the heme biosynthetic pathway and the order in which they occur. A l l of these data suggest that heme biosynthesis, regulated by endproduct feedback v i a ALA-synthetase and ferrochelatase, i s a f i n e l y controlled pathway. S t i l l another source of control of heme biosynthesis i s substrate i n h i b i t i o n which was observed i n the presence of excess ferrous ion. Ferrous ion exhibits substrate i n h i b i t i o n 34 Figure 7 A scheme of the control exerted by heme on i t s biosyn-t h e t i c pathway. Numbers refer to the order i n which i n h i b i t i o n or stimulation occurs depending on the concentration of heme required. } S - ALA COPROGEN S - A L A - ^ OJ on 36 at concentrations greater than 0.25 mM. It i s d i f f i c u l t to determine whether th i s i s important i n vivo, i t may only occur in v i t r o . A possible explanation i s that ferrochelatase plays some role i n the uptake of iron into the mitochondrion. I t has been shown that heme i n h i b i t s the energy-dependent uptake of iron into the mitochondrion. I t i s possible that the intramito-chondrial concentration of iron i s s u f f i c i e n t l y high to prevent heme synthesis thereby maintaining the l e v e l of heme below; that required to i n h i b i t the uptake of iron into the mitochondrion. 37 PART TWO THE LOCATION OF FERROCHELATASE INTRODUCTION Liver mitochondria have been known to contain ferroche-37 latase a c t i v i t y since 1959 . Studies by Jones and Jones showed that microsomes contained no ferrochelatase and that a c t i v i t y was r e s t r i c t e d to the mitochondrion. Employing the 38 swellxng-shrinking-sonication method of Scottocasa et a l they showed that ferrochelatase was bound to the inner mito-39 4 0 chondrial membrane ' It i s known that ferrochelatase contains an active sulfhydryl residue i n i t s active s i t e . The purpose of the following experiments was to determine whether the iodoacetamide-sensitive binding s i t e l i e s on the inside of the inner mitochondrial membrane, facing the matrix, or on the outside of the inner membrane facing the intermembrane space. 38 RESULTS Mitoplasts were assayed for ferrochelatase a c t i v i t y i n the presence of 0.1 mM to 7.0 mM iodoacetamide. The maximum i n h i b i t i o n of enzyme a c t i v i t y obtained i n the mitoplast prepara-tion was 35.24% at 7.0 mM iodacetamide. Mitoplasts were then sonicated to form inner membrane p a r t i c l e s and ferrochelatase a c t i v i t y was assayed i n these p a r t i c l e s i n the presence of from 0.1 mM to 7.0 mM iodoacetamide. The maximum i n h i b i t i o n of ferrochelatase a c t i v i t y obtained i n the inner membrane p a r t i c l e preparation was 82.48% at 7.0 mM iodoacetamide. Graphs of the percent i n h i b i t i o n versus iodoacetamide concentration show the greater i n h i b i t i o n observed i n the sonicated mitoplasts than i n the f u l l mitoplasts. Thus, the evidence indicates that the iodoacetamide-sensitive binding s i t e i s on the inside of the inner mitochondrial membrane facing into the matrix (Figure 8)» th i s data i s not compelling evidence but taken i n conjunction 84 with the findings of Jones and Jones presents an argument favoring the location of ferrochelatase on the inside surface of the mitochondrion. 39 Figure 8 The e f f e c t of iodoacetamide on the ferrochelatase a c t i v i t y i n f u l l mitoplasts and inner membrane p a r t i c l e s . Assays were performed under standard conditions as described in Methods except that the mixture contained iodo-acetamide at the concentrations indicated i n the figure. Graph 1 The ordinate values are percent i n h i b i t i o n of ferrochelatase a c t i v i t y Graph 2 The ordinate values are s p e c i f i c a c t i v i t y i n units/mg protein. •, mitoplast preparation; o, inner membrane v e s i c l e s . PERCENT INHIBITION 41 DISCUSSION The location of the iodoacetamide-sensitive binding s i t e of ferrochelatase on the inner membrane and the evidence that i t i s deeply bound in the membrane do not preclude the possi-b i l i t y that ferrochelatase spans the inner membrane. It i s not known to what extent the enzyme i s exposed to the extra-membrane environment and what bulk i s i n t r i n s i c to the membrane. The exact location of a l l the substrate and postulated a l l o s t e r i c e f fector binding s i t e s on the enzyme would help elucidate the problem of substrate permeability i n the case of iron and proximity i n the case of protoporphyrin IX and heme. Iron uptake by mitochondria has been shown to proceed by 41 42 mechanisms similar to those operating i n whole c e l l s ' F e r r i t i n binds to the mitochondrion and releases iron i n an energy-dependant step. This step i s in h i b i t e d by hemin. Previously, i t was ten t a t i v e l y suggested that ferrochelatase might be involved. At the present time we can only speculate as to the way i n which iron gets from the outer membrane to some s i t e on the inner membrane to be available for heme bio-synthesis. If the iodoacetamide-sensitive binding s i t e of ferrochelatase represents the iron binding s i t e , then iron ions have to pass through two membrane systems. I t could i n fact be the permeation of the inner membrane which represents the energy-dependant step. At any rate, iron from the outer mem-brane and protoporphyrin IX from the v i c i n i t y of the inner 42 membrane must come together on the ferrochelatase molecule and an es s e n t i a l sulfhydryl residue (s) instrumental i n th i s reaction i s accesible to the matrix of the mitochondrion. 4 3 PART THREE MEMBRANE REQUIREMENTS OF FERROCHELATASE INTRODUCTION 4 3 ^ 4 5 I t has been suggested that phospholipids are involved in the function of ferrochelatase since the enzyme a c t i v i t y can be restored i n l i p i d - f r e e extracts by the addition of phospho-l i p i d s and since enzymic a c t i v i t y i s stimulated by organic solvents. The enzyme extracted from erythrocyte stroma was shown to require l i p i d to be activated more strongly by a c i d i c phospholipids such as :y>.,.,..;x.T.-a\ ie ; - i r . . _ l . ' ., c a r d i o l i p i n , phosphatidic acid and phosphatidyl i n o s i t o l than by choline containing l i p i d s such as l e c i t h i n of sphingomyelin. Lysophospholipids were found to be the most active. This l a t e r data suggested that detergency or f l u i d i t y of the a c t i v a t i n g phospholipids was important. Since ferrochelatase i s a deeply embedded membrane bound enzyme i t seemed l i k e l y that the state of the membrane environment could e f f e c t a c t i v i t y and even be an important regulatory factor. This i s evidenced by the fact that cholate dispersion of the membrane leads to greater a c t i v i t y . It was hoped to determine the exact mechanism of action of phos-pholipids i n the ferrochelatase reaction and the extent to which the enzyme i s dependant upon the state of the membrane in which i t i s bound. The studies reported here suggest the membrane environment might a f f e c t enzyme structure since i) only phospho-44 l i p i d s which contained unsaturated acyl chains acted as cofactors of ferrochelatase i i ) the ac t i v a t i o n of ferrochelatase; by phospholipids was independent of the nature of the polar head group and i i i ) Arrhenius plots of ferrochelatase a c t i v i t y were segmented, the t r a n s i t i o n temperature being dependent on the state of the non-polar environment. 45 RESULTS EFFECT OF FATTY ACIDS ON FERROCHELATASE ACTIVITY IN LIPID-DEPLETED PREPARATIONS L i p i d d e p l e t i o n of m i t o c h o n d r i a l preparations r e s u l t e d i n a marked decrease i n the l e v e l of f e r r o c h e l a t a s e a c t i v i t y . Enzyme a c t i v i t y could be r e s t o r e d i n the acetone e x t r a c t s by the a d d i t i o n of f a t t y a c i d (Table I ) . The e f f e c t i v e n e s s of the various f a t t y acids t e s t e d was i n the f o l l o w i n g order, l i n o l e n i c a c i d (18:3)> l i n o l e i c a c i d (18 : 2) >^ o l e i c a c i d (18 :1) y s t e a r i c a c i d (18:0). None of the f a t t y acids had any e f f e c t on f e r r o -chelatase i n the d e t e r g e n t - s o l u b i l i z e d preparations p r i o r to acetone e x t r a c t i o n . EFFECT OF PHOSPHOLIPIDS ON FERROCHELATASE ACTIVITY IN LIPID-DEPLETED PREPARATIONS In a d d i t i o n to f a t t y a c i d s , phospholipids a l s o a c t i v a t e d f e r r o c h e l a t a s e i n l i p i d - f r e e preparations (Table I I ) . The extent of a c t i v a t i o n of f e r r o c h e l a t a s e i n acetone e x t r a c t s og mit o c h o n d r i a l preparations by pure s y n t h e t i c p h o s p h a t i d y l -c h o l i n e v e s i c l e s was c o r r e l a t e d w i t h the degree of uns a t u r a t i o n of the a c y l chains, thus d i l i n o l e o y l l e c i t h i n ^ > d i o l e o y l l e c i t h i n d i s t e a r o y l l e c i t h i n . Submersion of the r e a c t i o n mixtures i n an u l t r a s o n i c bath f o r 1 min. d i d not a f f e c t the degree or p a t t e r n of a c t i v a t i o n of f e r r o c h e l a t a s e i n d i c a t i n g t h a t the d i f f e r e n c e s observed were not due to d i f f e r e n c e s i n the s t a t e of d i s p e r s i o n of the phospholipids (cf. r e f . 31). Again, the a d d i t i o n of 46 TABLE I EFFECT OF FATTY ACIDS ON FERROCHELATASE ACTIVITY The fa t t y acid suspensions were prepared as described under Methods. Protoporphyrin IX was used as porphyrin substrate. The concentration of f a t t y acid i n each assay was 10 mM. Fatty acid (10 mM) Spe c i f i c a c t i v i t y (units/mg protein) None 1.00 Stearic acid ( 0 ) a 3.00 Oleic acid (1) 4.12 L i n o l e i c acid (2) 5.94 Linolenic acid (3) 6.12 Number of double bonds per molecule 47 TABLE II EFFECT OF PURE PHOSPHOLIPIDS ON FERROCHELATASE ACTIVITY The suspensions of phospholipids were prepared i n the same manner as described under Methods. Mesoporphyrin IX was used as porphyrin substrate. The concentration of phospholipid in each assay was 1 mM. Phospholipid S p e c i f i c a c t i v i t y (1 mM) (units/mg protein) None 6.70 Distearoyl l e c i t h i n 7.72 Dioleoyl l e c i t h i n 8.14 D i l i n o l e o y l l e c i t h i n 10.09 48 p h o s p h o l i p i d t o d e t e r g e n t - s o l u b i l i z e d enzyme p r e p a r a t i o n s p r i o r t o acetone e x t r a c t i o n had no a f f e c t on enzyme a c t i v i t y . A comparable s t u d y o f s e v e r a l c o m m e r c i a l l y a v a i l a b l e p h o s p h o l i p i d s , w h i c h d i f f e r e d g r e a t l y i n f a t t y a c i d c o m p o s i t i o n and p o l a r head groups, i n d i c a t e d t h a t t he n a t u r e o f t h e p o l a r head group was n o t a s i g n i f i c a n t d e t e r m i n a n t o f t h e f e r r o c h e l a - ? t a s e a c t i v i t y ( T a ble I I I ) . I n a l l c a s e s , t h e e x t e n t o f a c t i v a ^ t i o n o f f e r r o c h e l a t a s e i n the acetone e x t r a c t s by t h e s e p h o s p h o l i p i d s was d e c r e a s e d markedly i n the p r e s e n c e o f 10% c h o l e s t e r o l (Table I I I ) . As shown i n Ta b l e IV, the degree o f a c t i v a t i o n o f f e r r o c h e l a t a s e by pure d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e d e c r e a s e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f c h o l e s t e r o l a t 45° and i n c r e a s e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f c h o l e s -t e r o l a t 22.5°. EVIDENCE FOR LIPID-DETERMINED ACTIVATION OF FERROCHELATASE F e r r o c h e l a t a s e a c t i v i t y o f s u b m i t o c h o n d r i a l p a r t i c l e s and d e t e r g e n t - s o l u b i l i z e d p r e p a r a t i o n s was maximal a t 45°. o ° A r r h e n i u s p l o t s f o r t h e temp e r a t u r e range between 25 and 60 o f t he s u b m i t o c h o n d r i a l and d e t e r g e n t - s o l u b i l i z e d p r e p a r a t i o n s showed d e f i n i t e i n f l e c t i o n p o i n t s a t 37° and 26.5°, r e s p e c t i v e l y ( F i g u r e 9), s u g g e s t i n g t h a t t he t r a n s i t i o n i s dependant on the s t a t e o f the n o n - p o l a r environment o f the enzyme. The a c t i v a -t i o n energy (E) f o r f e r r o c h e l a t a s e o f s u b m i t o c h o n d r i a l p a r t i c l e s was 21,500 c a l o r i e s p e r mole below 37°, and 4,530 c a l / m o l e above 37°, and f o r f e r r o c h e l a t a s e o f d e t e r g e n t - s o l u b i l i z e d p r e p a r a t i o n s i t was 49,400 c a l o r i e s p e r mole below 28.5° and 4,570 c a l o r i e s p e r mole above 2 8.5°. 49 TABLE III EFFECT OF CHOLESTEROL ON THE ACTIVATION OF FERROCHELATASE BY PHOSPHOLIPIDS The suspensions of phospholipids were prepared i n the same manner as the f a t t y acid suspensions described i n Table I. Assays were performed under standard conditions as described i n Methods with protoporphyrin IX as the porphyrin substrate. An acetone extract of a detergent-solubilized preparation was used as the source of enzyme. The concentration of cholesterol in each assay was 10% (w/v). Compound S p e c i f i c a c t i v i t y (units/mg protein) None 1. 57 Phosphatidyl serine 2.47 Phosphatidyl serine + cholesterol 1. 63 Phosphatidyl choline 2.69 Phosphatidyl choline + cholesterol 2.07 Ca r d i o l i p i n 2.02 C a r d i o l i p i n + cholesterol 1.85 50 TABLE IV EFFECT OF CHOLESTEROL ON THE ACTIVATION OF FERROCHELATASE BY DIPALMITOYL PHOSPHATIDYL CHOLINE The suspension of dipalmitoyl phosphatidyl choline was prepared i n the same manner as the f a t t y acid suspensions described i n Table I. Standard assay conditions were used as described i n Methods except that the incubations were carr i e d out at 45° or 22.5°, as indicated i n the Table. Mesoporphyrin IX was used as the porphyrin substrate and an acetone extract of a detergent-solubi&dized preparation was used as the source of enzyme. Cholesterol S p e c i f i c a c t i v i t y (units/mg protein) (%) Temperature 2 2 . 5 0 45° 0 2. 77 5.50 10 2.88 4.43 25 3.23 3.57 50 3.27 2.10 51 Figure 9 Assays were performed under standard conditions as described i n Methods except that the temperature of the incubation was varied. Protoporphyrin IX was used as the porphyrin substrate. The ordinate values are the log of nmoles of p-i-otoheme formed per 1 hour per mg of protein, o, submitochondrial p a r t i c l e s preparation; •, detergent-s o l u b i l i z e d preparation. 3.2 3 . 4 3 . 6 T x 10 • 3 EFFECT OF LIPIDS ON THE CALCIUM REQUIREMENT OF FERROCHELATASE Ferrochelatase a c t i v i t y was stable to d i a l y s i s for 20 hours against a buffer containing 0.1 mM EDTA, whereas d i a l y s i s against 1 mM EDTA or gel f i l t r a t i o n through a Sephadex G-25 column equilib r a t e d with 50 mM Tris-HCl buffer, pH 7.5, resulted i n a t o t a l loss of enzyme a c t i v i t y (Table V). A c t i v i t y was not restored by the addition of nucleotides or coenzymes (FADH^, NADH, NADPH, ATP, succinate, fumarate), but the addition of 0.5 mM C a C l 2 to the assay mixture resulted i n a complete reac-t i v a t i o n of the enzyme. MgCl^ (2 mM) effected a p a r t i a l , but variable, reactivation of the enzyme. The calcium requirement of ferrochelatase was also inves-tigated i n reconstituted l i p i d - f r e e preparations. A detergents s o l u b i l i z e d enzyme extract and a submitochondrial preparation were each dialyzed for 20 hours against 1 mM EDTA. An acetone extract was then prepared from each of the dialyzed preparations and the ferrochelatase a c t i v i t y reconstituted with l i n o l e i c acid. Both preparations were assayed in the presence and absence 2+ of calcium ions. The results (Table VI) indicated that Ca was not required for a c t i v i t y when the enzyme was i n a model l i p i d environment. This conclusion was supported by the finding that the ferrous s a l t of oxalic acid, a chelator with a high a f f i n i t y for 2+ Ca , was a good substrate for ferrochelatase of acetone extracts reactivated with l i n o l e i c acid (Table VII), but a very poor substrate for ferrochelatase of detergent-solubilized preparations. 54 TABLE V EFFECT OF DIVALENT CATIONS ON THE ACTIVITY OF FERROCHELATASE IN DIALYZED, DETERGENT-SOLUBILIZED PREPARATIONS Samples of detergent-solubilized enzyme preparation were dialyzed for 20 h against buffer containing 0.1 mM or 1 mM EDTA. The samples were then assayed for ferrochelatase a c t i v i t y as described i n Methods except for the addition of 0.5 mM CaCl^ or 2 mM MgCl 2 as indicated i n the table. The porphyrin substrate was mesoporphyrin IX. Treatment S p e c i f i c a c t i v i t y (units/mg protein) None 10. 32 Dialyzed vs. 0.1 mM EDTA 10.21 Dialyzed vs. 1 mM EDTA 0.0 Dialyzed vs. 1 mM EDTA and assayed 10.13 i n presence of 0.5 mM CaCl 2 Dialyzed vs. 1 mM EDTA and assayed i n presence of 2 mM MgCl 0 3.11 55 TABLE VI EFFECT OF CALCIUM ON THE ACTIVITY OF FERROCHELATASE IN LINOLEIC ACID VESICLES Detergent-solubilized and submitochondrial enzyme preparations were each dialyzed for 20 h against buffer con-taining 1 mM EDTA. An acetone extract was then prepared from each preparation and the ferrochelatase a c t i v i t y reconstituted with a suspension of l i n o l e i c acid as described i n Table I. Standard assay conditions were used as described i n Methods except for the addition of 0.5 mM CaCl as indicated i n the table. Protoporphyrin IX was used as the porphyrin substrate. Treatment S p e c i f i c A c t i v i t y (units/mg protein) Acetone extract of submitochondrial 4.0 preparation + l i n o l e i c acid Acetone extract of submitochondrial 5.1 2 + preparation + l i n o l e i c acid + Ca Acetone extract of detergent- 6.1 s o l u b i l i z e d preparation + l i n o l e i c acid Acetone extract of detergent- 5.2 s o l u b i l i z e d preparation + l i n o l e i c acid + Ca 56 TABLE VII EFFECT OF THE SOURCE OF IRON ON THE ACTIVITY OF FERROCHELATASE Standard assay conditions were used as described i n Methods except that the source of iron was varied as indicated i n the table. The porphyrin substrate was protoporphyrin IX and the enzyme source was either a detergent-solubilized preparation or a l i n o l e i c acid reactivated acetone extract of th i s preparation as indicated i n the table. Enzyme source Iron source S p e c i f i c a c t i v i t y (units/mg protein) Detergent-solubilized preparation 3.93 FeSO 4.93 4 FeC 20 4 1.90 L i n o l e i c acid r e a c t i -vated acetone extract 3. 93 FeSO 4 4. 65 FeC 20 4 4.45 57 DISCUSSION The studies described here indicate that the extent of act i v a t i o n of ferrochelatase by phospholipids i s d i r e c t l y related to the number of double bonds i n the acyl chain of the phospholipid. Since the unsaturation breaks up the hydro-phobic interactions leading to a more f l u i d hydrocarbon core our r e s u l t s suggest that ferrochelatase a c t i v i t y i s dependant on a f l u i d hydrophobic phase. Cholesterol i s known to increase membrane f l u i d i t y below the t r a n s i t i o n temperature and 46 to decrease the f l u i d i t y above the t r a n s i t i o n temperature Thus, the observation that ferrochelatase a c t i v i t y at 45°, which i s well above the t r a n s i t i o n temperature of 37°, decreased with increasing concentrations of cholesterol whereas at 22.5° enzyme a c t i v i t y increased with increasing concentrations of cholesterol i s consistent with t h i s i n t e r p r e t a t i o n . I t has been shown that hydration i s correlated with the f l u i d i t y of the hydrocarbon core^ 7. The e f f e c t of f l u i d i t y ^ on ferrochelatase a c t i v i t y might, therefore, r e f l e c t the a b i l i t y of water to penetrate the l i p i d phase or i t might indicate a requirement for hydration of polar groups. A l t e r n a t i v e l y , the f l u i d i t y of the hydrophobic phase might f a c i l i t a t e favourable enzymic conformation or i t might act by allowing more mobility within the membrane. Studies of the calcium requirement of ferrochelatase indicate that i t does not depend on the formation of a complex between enzyme, metal and substrate for a c t i v i t y since gel 58 f i l t r a t i o n of an enzyme preparation followed by l i p i d removal and rea c t i v a t i o n gave an enzyme-lipid system which was indepen-dent of calcium ions. Instead, i t would appear that the e f f e c t 2+ of Ca on ferrochelatase a c t i v i t y i s mediated v i a and e f f e c t of the metal on the membrane. Calcium ions could a f f e c t the membrane i n any of three ways; by acting as a chelator and bridging phospholipids by t h e i r head groups either to each other or to the enzyme, by screening the surface charge of the phospholipid b i l a y e r or by a l t e r i n g the phase c h a r a c t e r i s t i c s of the membrane. The explanation involving calcium ions as a bridge of chelator i s unsatisfactory because reconstitution of the lipid-depleted enzyme preparations occurred i n the presence of monovalent ions which lack any chelating a b i l i t y . The explanation of calcium a f f e c t i n g the membrane by screening the surface charge seems unlikel y for several reasons. F i r s t , 48 Lowe and P h i l l i p s described an " e l e c t r o s t a t i c e f f e c t " on which the surface charge of a m i s c e l l or possibly a membrane, could a t t r a c t the po s i t i v e ferrous ions to f a c i l i t a t e the reaction; second, i t does not account for the calcium requirement of ferrochelatase since i n the model fat t y acid and phospholipid environments polar group charges are balanced by monovalent ions; and t h i r d , i t i s not consistent with the finding of Sawada 49 et a l that negative charges of the phospholipid head groups activate ferrochelatase i n chicken erythrocyte. However, i t i s possible that the f i n a l argument applies only to the enzyme system i n avian erythrocyte, since our findings indicate that, in r a t l i v e r , the act i v a t i o n of ferrochelatase by^ phospholipids i s independent of the p a r t i c u l a r charge on the polar head group 59 of the molecule. The t h i r d explanation, i n which calcium a f f e c t s the phase c h a r a c t e r i s t i c s of the membrane applies to ves i c l e s of pure phosphatidyl g l y c e r o l and phosphatidyl serine in which i t has been demonstrated that 1 mM Ca abolishes the phase t r a n s i t i o n between 0-70°C^. I t i s not known whether 2+ Ca plays a s i g n i f i c a n t role i n determining the phase charac-t e r i s t i c s of the native mitochondrial membrane which i s composed of 76% protein and a wide variety of phospholipids. Indeed, u n t i l now i t was not known that the phase c h a r a c t e r i s t i c s of the membrane affected ferrochelatase a c t i v i t y . However, t h i s seems probable since the i n f l e c t i o n point at 37°C i n the \Arrhenius p l o t of ferrochelatase a c t i v i t y i n submitochondrial p a r t i c l e s was lowered to 2 8.5° after disruption of the hydro-phobic phase with cholate. This indicates that the t r a n s i t i o n r e s u l t s from alterations i n the l i p i d environment of the enzyme. 2+ The p o s s i b i l i t y e xists that Ca e f f e c t s an adequate environment in the native mitochondrial membrane for ferrochelatase by modifying the phase c h a r a c t e r i s t i c s of the membrane. 60 PART FOUR REGULATORY FACTORS INTRODUCTION It has been known for some time that ferrochelatase u t i l i -51 52 zes iron'ions only i n the reduced form ' . However, i t has been shown by Jones and coworkers that f e r r i c ions can serve as metal substrate for ferrochelatase of both avian erythrocytes and r a t l i v e r mitochondria i f the incubation mixture i s sup-53 54 25 plemented with NADH or succinate ' ' , NADH being the more e f f e c t i v e electron donor. In these studies, the reduction of iron from the f e r r i c to the ferrous state was measured by the consequent uptake of 0 2 with an oxygen electrode and was found to procede much more rapidl y i n sonicated mitochondria than i n f u l l mitochondria. This suggests a permeability b a r r i e r to f e r r i c ions and again points i n d i r e c t l y to a possible role for ferrochelatase in iron transport. The iron reducing a c t i v i t y was found to be l o c a l i z e d exclusively i n the membrane f r a c t i o n . The electron donor dependent reduction of ir o n was found to be ins e n s i t i v e to Antimycin A and rotenone suggesting that reducing equivalents are not dependent on electron transport complexes I or III for v i a b i l i t y . However, cytochrome b was oxidized by f e r r i c chloride and submitochondrial p a r t i c l e s when rotenone and Antimycin were used to i s o l a t e t h i s region of the r e s p i r a -61 tory chain. Thus, there i s an obvious contradiction here. S p e c i f i c a l l y , how can cytochrome b be instrumental i n iron reduction when complex I (NADH-Coenz Q reductase) a c t i v i t y i s unrelated to iron reduction? To obviate t h i s discrepancy, Jones suggested that there i s some flavoprotein capable of accepting electrons;:; from Fp^ or Fp g of complex I or II through either NADH dehydrogenase or succinate dehydrogenase, and that th i s flavoprotein i s connected to Coenz Q through Fp^ or Fp g and that cytochrome b i s oxidized by Coenz Q during iron reduc-t i o n . In an attempt to define more pr e c i s e l y the mechanism by which f e r r i c ion i s reduced to ferrous ion a more detailed study of the relationship between iron reduction and the respiratory chain was undertaken. The studies described here shed much doubt on the scheme proposed by Jones. The data indicate that iron reduction i s independent of complexes I and II of the respiratory chain and that NADH dehydrogenase and succinate dehydrogenase, which are necessary to recover reducing equivalents, donate th e i r electrons to an acceptor, perhaps a flavoprotein, quite d i s t i n c t from the complexes of the r e s p i r a -tory chain. I t was also found that a protein, ferrous ion-PMS reductase, i s capable of the ferrous ion dependant reduction of PMS. In addition, evidence i s presented which suggests that there i s a physical connection between the system responsible for iron reduction and ferrochelatase i t s e l f . 62 RESULTS THE REQUIREMENT OF FERROCHELATASE FOR FERROUS ION A s o l u b i l i z e d preparation of ferrochelatase from rat l i v e r mitochondria was assayed with FeSO^ and F e C l 3 as iron sources. Assays containing ferrous ion were carried out i n the presence and absence of 10 mM cysteine to determine i f cysteine is; s u f f i c i e n t to keep ferrous ion reduced throughout the assay. In the absence of cysteine, ferrous.ion i s rapidly oxidized under the assay conditions and was a poor substrate for f e r r o -chelatase which exhibited only 22% of the a c t i v i t y observed i n the presence of cysteine. Cysteine i s therefore necessary to maintain iron i n the reduced form during the incubation period. F e r r i c ion was found to be a very poor substrate for ferrochela-tase, which exhibited only 21% of the a c t i v i t y obtained with ferrous ion a metal substrate i n the presence of cysteine (Table VIII) . EFFECT OF ELECTRON DONORS ON UTILIZATION OF FERRIC ION BY FERROCHELATASE Submitochondrial p a r t i c l e s prepared from r a t l i v e r were assayed using f e r r i c chloride as metal source i n the presence of various concentrations of succinate, NADH OR NADPH. These electron donors were found to confer iron reducing a b i l i t y upon either ferrochelatase or some other enzymatic en t i t y (Figure 10). The iron reducing a c t i v i t y was shown to be enzyme dependent. No ferrous ion was formed aft e r incubation of f e r r i c ion with succinate, NADH or NADPH at 40°C for 2 h as determined by 63 TABLE VIII EFFECT OF THE OXIDATION STATE OF IRON ON THE ACTIVITY OF FERROCHELATASE Assays were performed under standard conditions as described i n Methods with modifications as indicated i n the table. Iron source S p e c i f i c a c t i v i t y (units/mg) FeS0 4 (+ cys-H) FeS0 4 (- cys-H) FeCl, (- cys-H) 5.03 1.11 1.07 64 F i g u r e 10 E f f e c t of e l e c t r o n donors on u t i l i z a t i o n of f e r r i c i o n by-f e r r o c h e l a t a s e Assays were performed under standard c o n d i t i o n s as des-^ c r i b e d i n Methods except no c y s t e i n e was present and f e r r i c c h l o r i d e was used as the i r o n source of ferrous^ s u l f a t e . The o r d i n a t e values are s p e c i f i c a c t i v i t y - and the a b s c i s s a values are the concentrations of e l e c t r o n donor. •, f e r r o c h e l a t a s e a c t i v i t y i n the presence of NADFH; o, f e r r o c h e l a t a s e a c t i v i t y i n the presence of NADH; f e r r o c h e l a t a s e a c t i v i t y i n the presence of succinate. SPECIFIC ACTIVITY 66 o-phenanthroline, an indicator s p e c i f i c for ferrous ion. The iron reducing a b i l i t y was temperature dependent and was rapidly denatured above 60°C. A s o l u b i l i z e d preparation of rat l i v e r mitochondria was assayed for ferrochelatase with f e r r i c chloride as iron source i n the presence of 3 mM succinate, 5 mM <X-glycerolphosphate or 3 mM choline chloride. Each of these electron donors stimula-ted the u t i l i z a t i o n of f e r r i c ion by ferrochelatase (Table IX). Indeed, of the electron donors tested, a l l those which provide reducing equivalents for r e s p i r a t i o n through flavoproteins are capable of confering iron reducing a b i l i t y . The effectiveness of the electron donors i n s o l u b i l i z e d preparations was greater than that observed i n submitochondrial p a r t i c l e s (Table X). This may r e f l e c t the a v a i l a b i l i t y of a binding s i t e which i s exposed i n the s o l u b i l i z e d preparation but hidden i n submito-chondrial p a r t i c l e s . Ferrochelatase a c t i v i t y assayed with f e r r i c ion as substrate i n the presence of the electron donors succinate, NADH or NADPH was greater than ferrochelatase a c t i -v i t y with ferrous ion as substrate. Thus, i n addition to th e i r role i n iron reduction, electron donors stimulate f e r r o -chelatase a c t i v i t y . When succinate stimulating a c t i v i t y was assayed with ferrous ion as iron source and succinate as electron donor i t was observed to stimulate ferrochelatase 1.67 times. However, the p o s s i b i l i t y was not excluded that a separate stimulator which bound the electron donor also stimulated ferrochelatase. 67 TABLE IX EFFECT OF ELECTRON DONORS ON THE UTILIZATION OF FERRIC ION BY FERROCHELATASE IN SMP AND DETERGENT-SOLUBILIZED PREPARATIONS Assay conditions were as described under Methods i n the section e n t i t l e d ; Assay of electron donor-dependent iron reduction. Enzyme preparation Electron donor Sp. Act Stimulation Det. Sol (FeS0 4/cysH) - 10.30 Det. Sol (FeCl 3) - 1.20 Ot-G- P, (5 mM) 5.54 4.62x Cholne -Cl,33mM, 5. 12.75 10.63 Succinate, 3 mM 15.82 13.18 NADH, 3 mM 15.26 12.72 NADPH, 1 mM 15.31 12.76 SMP (FeS04+cysH) - 9.27 SMP (FeCl 2) - 1.20 0 SMP (Fecl 3) Succinate, 4 mM 4.34 3. 62x NADH, 4 mM 6.24 5.20x NADPH, 1 mM 11.12 9.27x 68 TABLE X IRON REDUCING ACTIVITY IN MITOCHONDRIA PREPARED FROM AEROBIC-ALLY GROWN YEAST Mitochondria were prepared from aerobically-grown yeast as described under Methods. Ferrochelatase a c t i v i t y was assayed under standard conditions. Mesoporphyrin IX was used as porphyrin substrate. Incubation was at 29°C. Iron source and electron donor S p e c i f i c a c t i v i t y FeS0 4 36.20 F e C l 3 + 3 mM Succinate 60. 45 F e C l 3 + 1 mM NADPH 40.83 F e C l 3 1. 97 69 THE EFFECT OF TEMPERATURE ON FERROCHELATASE AND IRON REDUCTION A s o l u b i l i z e d preparation of rat l i v e r mitochondria was assayed for ferrochelatase a c t i v i t y with f e r r i c chloride as iron source i n the presence and absence of 3 mM succinate at various temperatures. In the presence of succinate, enzyme a c t i v i t y increased l i n e a r l y up to 43°, where i t l e v e l l e d o f f . In the absence of succinate, ferrochelatase a c t i v i t y remained very low u n t i l a temperature of 43° was reached. Above 45°, ferrochela-tase a c t i v i t y with f e r r i c ion as metal substrate increased greatly, suggesting that there had been a b a r r i e r to iron reduc-tion which had been overcome by temperature (Figure 11). Ferrochelatase a c t i v i t y when assayed with ferrous ion as metal substrate was found to be stimulated by temperature i n the same manner as that assayed with f e r r i c ion as the metal substrate i n the presence of 3 mM succinate. FERROCHELATASE AND IRON REDUCING ABILITY OF S. CEREVISIAE The yeast system suggested i t s e l f as a means of c o n t r o l l i n g r e s p i r a t i o n while studying iron reduction. Yeast c e l l s were grown aerobically on 3% g l y c e r o l as described i n Methods, har-vested and the mitochondria prepared. The f u l l mitochondria were assayed for ferrochelatase with f e r r i c ion as metal subst-rate in the presence of either succinate (3 mM) or NADH (1 mM). I t was found that aerobic yeast mitochondria possessed high l e v e l s of ferrochelatase a c t i v i t y as well as electron donor stimulated iron reduction a c t i v i t y . Mitochondria i s o l a t e d from yeast grown anaerobically on 4% glucose were assayed for ferrochelatase with ferrous ion as 70 Figure 11 The e f f e c t of temperature on ferrochelatase activity- and iron reduction. Ferrochelatase i n detergent-solubilized preparation was assayed under standard conditions as described i n Methods. •, ferrochelatase a c t i v i t y i n the presence of 3 mM succinate; o, ferrochelatase a c t i v i t y . TEMPERATURE (°C) 72 metal substrate and found to have high l e v e l s of ferrochelatase a c t i v i t y . However, when these preparations were assayed with f e r r i c ion as metal substrate i n the presence of electron donors no ir o n reducing a b i l i t y was detected (Table XI). Ferrochela-tase was not stimulated by succinate i n mitochondria prepared from anaerobically grown c e l l s . THE DEVELOPMENT OF IRON REDUCING ACTIVITY DURING RESPIRATORY ADAPTATION OF YEAST The development of iron reducing a c t i v i t y during respiratory adaptation was followed over a 10 h period. Anaerobically-grown yeast c e l l s were harvested and introduced into a r e s p i r a -tory adaptation medium as described i n Methods. The adaptation process was stopped at 0, 2.5, 5.0, 7.5, and 10.0 hours by the addition of cyclohexamide and chloramphenicol. The c e l l s were harvested and mitochondria prepared. The mitochondria were then assayed for iron reducing a b i l i t y with f e r r i c ion as metal substrate i n the presence of 3 mM succinate. Succinate depen-dent iron reducing a c t i v i t y developed to 80% of maximum within the f i r s t 2.5 h afte r the onset of respiratory adaptation (Figure 14). A 2 h time course performed i n an i d e n t i c a l manner but with samples taken at 0, 0.25, 0.5, 1.0, 1.5 and 2.0 hours aft e r the st a r t of the adaptation process showed that iron reducing a b i l i t y begins to develop af t e r 15 minutes of respiratory adaptation. There appears to be a short lag period (Figure 12). IRON REDUCTION ACTIVITY IS NOT ENERGY OR RESPIRATION DEPENDENT Submitochondrial a r t i c l e s prepared from r a t l i v e r mito-chondria were assayed for ferrochelatase with f e r r i c ion as metal substrate and succinate as electron donor i n the presence TABLE XI FERROCHELATASE ACTIVITY AND IRON REDUCING ACTIVITY OF MITOCHONDRIA PREPARED FROM ANAEROBICALLY GROWN YEAST Mitochondria were prepared from anaerobically-grown yeast as described i n Methods and assayed for ferrochelatase with f e r r i c and ferrous ions as metal substrate Iron source and electron donors Sp e c i f i c a c t i v i t y FeSo 4 4 5 . 9 0 FeCl^ + 3 mM succinate F e C l 3 + 3 mM NADH FeCl-, + 1 mM NADPH 0 0 0 74 Figure 12 Twelve hour and two hour time courses of respiratory adaptation Respiratory adaptation of yeast c e l l s was carried out as described i n Methods. Ferrochelatase was assayed under standard conditions with FeCl^ as metal substrate and 3 mM succinate as electron donor. On both graphs the ordinate values are s p e c i f i c a c t i v i t y and the abscissa values are time of adaptation i n hours. A IIAII^W '^UI'VUC 76 of various respiratory i n h i b i t o r s and also the uncoupler,DNP. None were observed to have an e f f e c t on iron reduction or u t i l i z a t i o n by ferrochelatase. ATP did not stimulate i r o n reduction suggesting that the electron donors do not work through an energy dependent pathway. In addition, the electron donors can e f f e c t iron reduction i n the absence of viable r e s p i r a t i o n (Table XII). DEPENDANCE ON DEHYDROGENASES FOR IRON REDUCTION Submitochondrial p a r t i c l e s from rat l i v e r mitochondria were assayed for ferrochelatase a c t i v i t y with f e r r i c ion as metal substrate i n the presence of succinate (3 mM) and 1.0 mM, 6.0 mM and 12.0 mM malonate, which i s a known competitive i n h i b i t o r of succinate dehydrogenase. In h i b i t i o n by malonate was concen-t r a t i o n dependent and t h i s provides evidence that succinate dehydrogenase i s necessary for the recovery of reducing equiva-lents from succinate (Table XIII). G e l l f i l t r a t i o n of a detergent-solubilized preparation of RLM on a Sephadex G-150 column equilibrated with 50 mM Tris-HCl, pH 7.5, separated succinate" dehydrogenase (MW 300,000) and NADPH dehydrogenase (MW 50-60,000). I t was found that fractions assayed with the respective electron donors had retained suc c i -nate-dependent stimulation of iron reducing a c t i v i t y but had l o s t NADPH-dependent iron reducing a c t i v i t y (Table XIV). I t i s concluded, therefore, that NADPH dehydrogenase i s e s s e n t i a l to recover reducing equivalents from NADPH and donate them for iron reduction. THE EFFECT OF HEAT TREATMENT ON FERROCHELATASE A s o l u b i l i z e d preparation of rat l i v e r mitochondria was 77 TABLE XII. EFFECT OF RESPIRATORY INHIBITORS, UNCOUPLERS AND ATP ON IRON REDUCTION F e r r o c h e l a t a s e i n submitoc h o n d r i a l p a r t i c l e s of r a t l i v e r was assayed as d e s c r i b e d i n Methods w i t h F e C l ^ as metal s u b s t r a t e and the i n c l u s i o n of the i n h i b i t o r s and s t i m u l a t o r s d e s c r i b e d i n the t a b l e . I n h i b i t o r , s t i m u l a t o r , uncoupler S p e c i f i c a c t i v i t y None 1. 21 3 mM s u c c i n a t e 7. 29 3 mM su c c i n a t e + 1 mM Amtal 6. 93 3 mM s u c c i n a t e + 0.1 mM KCN 7. 09 3 mM s u c c i n a t e + 0.1 mM TTA 7. 19 3 mM NADH 5. 87 3 mM NADH + 0.1 mM TTA 5. 60 3 mM s u c c i n a t e + 60 mM DNP 7. 26 2+ 0.1 mM ATP + 0.01 mM Mg 4. 28 0.01 mM Mg 3. 10 78 TABLE XIII INHIBITION OF SUCCINATE DEPENDENT IRON REDUCTION BY MALONATE Submitochondrial p a r t i c l e s of rat l i v e r mitochondria were assayed under standard conditions with FeCl^ as iron source, 3 mM succinate as electron donor and various concen-trations of malonate. Malonate concentration S p e c i f i c a c t i v i t y 0 mM 6.85 1 mM 6.60 6 mM 5.43 12 mM 4.37 79 TABLE XIV LOSS OF NADPH STIMULATION OF FERROCHELATASE ACTIVITY FOLLOWING GEL FILTRATION ON SEPHADEX G-150 Fractions from the Sephadex G-150 column were assayed under standard conditions with succinate or NADPH as electron donors. Fraction Volume of eluent S p e c i f i c a c t i v i t y with 3 mM succinate S p e c i f i c a c t i v i t y with 1 mM N A D P H 0-12 60 ml 0 0 13 65 ml 14. 97 0 14 7 0 ml 15.55 0 15 75 ml 15.23 0 16 80 ml 14.29 0 17 85 ml 12.25 0 18-30 90- 140 ml 0 0 80 heated for 1 h at 54° u n t i l thick, gelatinous material was formed. The heat denatured p r e c i p i t a t e was c o l l e c t e d by cen-t r i f u g a t i o n at 10,000 x g for 10 min. The protein p r e c i p i t a t e was suspended i n a volume of 5 0 mM Tris-HCl, pH 7.5 to y i e l d the o r i g i n a l volume. The p e l l e t , the supernatant and a combi^ nation of both were assayed for ferrochelatase with ferious ion as metal substrate i n the presence of succinate. The reconstituted mixture of both supernatant and p e l l e t had a higher s p e c i f i c a c t i v i t y of ferrochelatase than either superna^ tant of p e l l e t alone (Table XV). The simple explanation of these data i s that some factor was more abundant i n one of the two fractions and stimulated ferrochelatase a c t i v i t y when reconstituted. The p e l l e t contained more ferrochelatase a c t i v i t y than the supernatant which probably indicates that ferrochelatase i s not t r u l y i n soluble form but i n eticejfe.ST'ie^. which are prec i p i t a t e d on heat treatment due to denaturation of the heat l a b i l e enzymes. Since ferrochelatase i s stable at 54° i t i s s t i l l an active enzyme though the rvcelles has been pre-c i p i t a t e d . Another possible explanation for the increased a c t i v i t y observed upon reconstitution i s that succinate dehydro-*-genease, which i s t r u l y s o l u b i l i z e d under these conditions i s present i n higher concentrations i n the supernatant and i s therefore available i n greater concentration when supernatant and p e l l e t are combined. GEL FILTRATION ON SEPHADEX G-150 OF FERROCHELATASE AND SUCCINATE STIMULATING ACTIVITIES Gel f i l t r a t i o n of detergent-solubilized r a t l i v e r mitocon-d r i a through a Sephadex G-150 column, equilibrated with 50 mM 81 TABLE XV EFFECT OF HEAT TREATMENT ON FERROCHELATASE ACTIVITY Ferrochelatase was heat treated as described i n the text and the p e l l e t , supernatant and combined fractions were assayed as described in Methods with the electron donors speci-f i e d i n the table. The s p e c i f i c a c t i v i t i e s are given. Electron Donor 0.5 ml P e l l e t 0.5 ml supernatant 0.25 ml P e l l e t + 0.25 ml Supt none 7.73 5. 80 6.19 3 mM succinate 8.27 6.20 12.58 3 mM NADH 8.43 8. 31 13.07 1 mM NADPH 8.43 ^a 12.35 a not determined 82 T r i s - H C l , pH 7.5, r e s u l t e d i n two major peaks of f e r r o c h e l a t a s e a c t i v i t y , which p a r a l l e l e d e x a c t l y the s u c c i n a t e s t i m u l a t i n g a c t i v i t y . T h i s suggests e i t h e r the s t i m u l a t o r i s a p a r t of f e r r o c h e l a t a s e enzyme or i t c o - e l u t e s e x a c t l y w i t h i t . The i r -r e g u l a r i t y of the e l u t i o n p r o f i l e (Figure 13) i s thought to be due to s e p a r a t i o n o f miceHeSnas of v a r i o u s s i z e s , a l l of which c o n t a i n f e r r o c h e l a t a s e . FERROUS ION DEPENDANT-PMS REDUCTION IN RAT LIVER MITOCHONDRIA A d e t e r g e n t - s o l u b i l i z e d p r e p a r a t i o n of r a t l i v e r m i t o c h o n d r i a was assayed f o r PMS r e d u c t i o n w i t h ferrous- i o n as the e l e c t r o n donor. At a f e r r o u s i o n c o n c e n t r a t i o n of 0.067 mM the r e d u c t i o n of PMS was r a p i d . In the presence of 0.013 mM f e r r o u s i o n 2+ and antimycin A^, Fe -PMS r e d u c t i o n was 57 nmoles/second/ assay. Gel f i l t r a t i o n on Sephadex G-150 of a d e t e r g e n t - s o l u b i l i z e d p r e p a r a t i o n r e s u l t e d i n succinatec. dehydrogenase, f e r r o c h e l a t a s e and ferrous-PMS reductase a c t i v i t i e s c o - e l u t i n g i n the same f r a c t i o n s (Figure 14 and Table XVI). 83 Figure 1 3 Co-elution of ferrochelatase and stimulator from a sephadex G - 1 5 0 column Sephadex G T - 1 5 0 was equilibrated with 5 0 mM Tris-HCl, pH 7 . 5 . The t o t a l bed volume was 2 4 3 ml and the void volume as determined by blue dextran was 6 0 ml. Five ml fractions were col l e c t e d and assayed for ferrochelatase and succinate stimulated ferrochelatase by the procedures outlined i n Methods. Both a c t i v i t i e s eluted immediately after the void volume. •, succinate stimulating a c t i v i t y ; o, ferrochelatase a c t i v i t y ; T , protein content. 84 A1IAI1DV DldD3dS 85 Figure 14 Sephadex G-150 e l u t i o n p r o f i l e of succinate dehydrogenase, 2 + ferrochelatase and Fe - PMS reductase a c t i v i t i e s A l l assays were performed as described under Methods. The ordinate values are a c t i v i t i e s of ferrochelatase in units, 2+ on the l e f t ; SDH a c t i v i t y i n units, on the r i g h t and Fe - PMS reductase on the f a r r i g h t . The abscissa values are f r a c t i o n numbers coll e c t e d from the column (5 ml f r a c t i o n s ) . •, ferrochelatase a c t i v i t y i n nmoles/fc ; O, succinate dehy-2+ drogenase a c t i v i t y i n nmoles/sec; A , Fe -VPMS reductase a c t i v i t y i n nmoles/sec. 86 • FERROCHELATASE ACTIVITY (nmoles/hr) o NO O CO o 4^  O O O T " — i — r i—r o T oo o 1 i—i—i—i—i—i—r — . * * • / / s s o _L_ NO O _J_ CO o _ l _ o Jt I— O 1 I o o _I_ Nl O 00 o I I SUCCINATE D E H Y D R O G E N A S E ACTIVITY (nmoles/sec) O 0 0 O I » I I I I I I | I I I I I I I 1 F e + 2 -PMS REDUCTASE ACTIVITY (nmoles/see) 87 TABLE XVI ELUTION OF SUCCINATE DEHYDROGENASE, FERROCHELATASE AND Fe -PMS REDUCTASE ACTIVITIES FROM A SEPHADEXG-1-50 COLUMN The column was equilibrated as described i n the text A l l assays are described i n Methods. 88 Fraction Units of Ferrochelatase A c t i v i t y Units of SDH A c t i v i t y Units of Fe 2 +-PMS Reductase A c t i v i t y 1 3.42 00 00 2 43.00 14.29 4.00 3 38. 38 15.71 2.48 4 50.59 47.14 4. 86 5 36.99 4. 86 6 42.63 11.43 5.81 7 38. 47 6.57 8 36.63 8.57 9 19.42 3.43 10 17.57 5.71 11 20.44 2.86 2.48 12 00 2.86 13 00 3.90 14 00 1.43 15 00 00 1.05 16 00 00 17 00 00 1.71 18 00 00 19 00 00 2.95 20 00 00 21 00 00 0.86 22 00 00 23 00 00 0. 67 24 00 00 00 25 00 00 00 26 00 00 00 27 00 00 00 89 DISCUSSION It i s a question of long standing i n t e r e s t how reduced iron becomes available for heme biosynthesis i n the mitocon-drion. The physiological iron c a r r i e r i n the blood, serum protein t r a n s f e r r i n , and the i n t r a c e l l u l a r iron storage and c a r r i e r protein, f e r r i t i n , both contain iron in the oxidized 55—58 plus three state . Since ferrous ion does not bind to t r a n s f e r r i n the b i o l o g i c a l removal of F e 3 + from t r a n s f e r r i n might involve reduction of transferrin-bound i r o n . However, 3+ extracts of l i v e r and r e t i c u l o c y t e s which a c t i v e l y reduce Fe 2+ 59 to Fe were inactive with t r a n s f e r r i n as substrate . Reduc-tion mechanisms have dominated thinking and experimentation on the release of iron from f e r r i t i n ^ . Michaelis-Menton et a l ^ reported that iron was l i b e r a t e d from f e r r i t i n i n v i t r o by the ft 2 reducing action of d i t h i o n i t e . Later, Tanaka obtained eviden-ce that release of iron from f e r r i t i n was accomplished by a flavoprotein and mechanisms were proposed on the basis of f e r -r i t i n accepting electrons from reduced xanthine oxidase to 63-65 66 release ferrous ion . Then i n 1971 Osaki and S i r i v e c h demonstrated an enzyme system which can reduce f e r r i t i n - F e 3 + i n homogenates of l i v e r from several vertebrates. The system 3+ catalyzes the reduction of ferritin-bound Fe to free ferrous ion by interaction with FAD, FMN or r i b o f l a v i n i n the presence of NADH. The i n v i t r o reduction of f e r r i t i n - f e r r i c ion by reduced f l a v i n nucleotides hasebeen characterized to some extent k i n e t i c a l l y and the r i b o f l a v i n moiety i s thought to .Erind to the 90 6 7 protein portion of the f e r r i t i n molecule . These studies were done with either p u r i f i e d f e r r i t i n or re t i c u l o c y t e preparations and represent the phenomenon of iron absorption at the plasma membrane l e v e l . I t i s not immediately apparant to what extent iron absorption across the mitochondrial membrane follows a simi l a r mechanism. The finding that iron reduction i n mito-chondrial preparations i s dependent upon reducing equivalents, in some cases f l a v i n s , was not surprising and the accruing evidence that the protein responsible for iron reduction i s a flavoprotein suggest a similar mechanism may be i n e f f e c t at both the plasma and mitochondrial membrane l e v e l s . No p h y s i c a l -chemical proof exists for the flavo-protein nature of the iron reducing a c t i v i t y but biochemical evidence led Jones to suggest the scheme already described i n the introduction to t h i s section. B r i e f l y , t h i s theory holds that some flavoprotein accepts elec-trons from either NADH dehydrogenase or succinate dehydrogenase and t h e i r flavoprotein intermediate.: and dir e c t s them into the respiratory chain at Coenz Q, cytochrome b being.oxidized i n the process. Recent experimentation sheds much doubt on t h i s scheme. F i r s t l y , iron reducing a c t i v i t y of submitochondrial p a r t i c l e s procedes e n t i r e l y independently of complexes I and II or the respiratory chain. A s p e c i f i c i n h i b i t o r of complex II between the flavoprotein FAD and Coenz Q or cytochrome b, thenoyl-triflouroacetone, did not i n h i b i t succinate dependent iron reduc-t i o n . This provides d i r e c t proof that electrons do not flow from Fp s to Coenz Q during the process of iron reduction. Rotenone did not i n h i b i t NADH dependent iron reduction. The respective dehydrogenases are necessary to recover.reducing equivalents but appear to donate t h e i r electrons to some accep-tor, perhaps a flavoprotein, quite d i s t i n c t form complexes of the respiratory chain. Further evidence of t h i s was given when Amytal, and i n h i b i t o r of complex I between FMN and coenz Q, and cyanide, an i n h i b i t o r of complex IV between cytochrome c and Q>2, also f a i l e d to i n h i b i t iron reduction. There was no energy dependence or requirement as the uncoupler DNP, did not i n h i b i t and ATP did not stimulate iron reduction. There appears, however, to be an electron acceptor, which seems to have some r e l a t i o n to the flavoproteins donating reducing equivalents to Coenz Q. Choline, Qt-glcerolphosphate, and NADPH are also capable of providing electrons for iron reduction and a l l have the common property of donating electrons for r e s p i r a t i o n through flavoproteins. Subsequently, i t was found that some protein, designated here as ferrous ion-PMS reductase, common to a l l these electron donors, i s capable of ferrous ion dependent re-duction of PMS. Succinate, NADH and NADPH also reduce PMS. Thus i n the presence of excess ferrous ion, CN"and antimycin, the enzyme w i l l catalyze the reverse reaction and take electrons from reduced iron and reduce the a r t i f i c i a l electron PMS. This does not prove that cytochrome b i s not involved i n iron reduction, but that the protein, ferrous ion-PMS reductase, unique from those donating to Coenz Q, i s involved. A time course of the development of ferrochelatase a c t i v i t y and iron reducing a c t i v i t y i n yeast mitochondria during res-piratory adaptation provides evidence on two important points. F i r s t , that anaerobically grown yeast, which do not contain functioning mitochondria, but only precursors or promitochondria, do not contain the entity responsible for iron reduction; they develope t h i s during the biogenesis and assembly of r e s p i r i n g mitochondria. The second s a l i e n t point i s that ferrochelatase i s r e s t r i c t e d i n a c t i v i t y by the l e v e l of iron reducing a c t i v i t y present i n the mitochondrion. F u l l y developed, r e s p i r i n g mitochondria require a l l the respiratory enzymes and cytochromes, thus, a heavy demand i s put on the enzymes responsible for heme synthesis and, of course, 74 reduced iron i s needed. Nejedly and Greshow have traced the sequential increase i n respiratory enzymes of S_. c e r v i s l a e during respiratory adaptation. Our studies show that iron reducing a c t i v i t y i s one of the f i r s t enzymes to reach a high l e v e l of a c t i v i t y , i t s synthesis s t a r t i n g 15 minutes afte r the onset of aerobiosis. Correlating Nejedly and coworker's data with my own, the f i r s t a c t i v i t y detected upon respiratory adapta-ti o n i s cytochrome oxidase, which reaches a two f o l d increase i n a c t i v i t y i n the f i r s t 15 minutes of aerobiosis. Iron reducing a c t i v i t y reaches a .3 f o l d increase i n a c t i v i t y a f t e r 3 0 minutes followed by NADH-cytochrome e reductase and succinate dehydro-genase both of which reach 2 f o l d increases i n a c t i v i t y a f t e r 30 and 45 minutes, respectively. Iron reductase, NADH-cytochrome c reductase and cytochrome oxidase a l l increase i n a stepwise manner with the f i r s t plateau s t a r t i n g at 60 minutes and the second r i s e i n a c t i v i t y at 90 minutes. Succinate dehydrogenase 93 a c t i v i t y increases e r r a t i c a l l y . Thus, there appears to be a s t r i k i n g c o r r e l a t i o n between respiratory and iron reducing a c t i v i t i e s . The finding that electron donors capable of providing reducing equivalents for iron reduction are also stimulators of ferrochelatase a c t i v i t y r a i s e the question of the intimate relationship of these two a c t i v i t i e s . The complex k i n e t i c s displayed by ferrochelatase suggested the p o s s i b i l i t y of an a l l o s t e r i c e f f e c t o r and i t seems l i k e l y that the presence or absence of electron donors could be a f f e c t i n g ferrochelatase i n such a manner. This would most l i k e l y be mediated through another general electron acceptors-donor, perhaps the very enzyme responsible for iron reduction. Whatever the actual connection between the two enzyme^acti-v i t i e s they are not e a s i l y separable by the conventional methods employed thus f a r . Iron reducing and ferrochelatase a c t i v i t i e s c o - s o l u b i l i z e , co-precipitate and co-elute from Sephadex G-150 and Sepharose 4B columns. These re s u l t s support the hypothesis that these a c t i v i t i e s are either functions of the same enzyme or are i n some physical contact within the mitochondrial membrane. As stated e a r l i e r , ferrochelatase may be present i n a n vce = or membrane material which contains other enzyme a c t i v i t i e s inr-eluding iron reducing a c t i v i t y , thus the connection could simply be proximity of the two enzyme a c t i v i t i e s i n the membrane. However, at t h i s time, I would hesitate to exclude the p o s s i b i l i t y of the two a c t i v i t i e s being functions of the same enzyme or enzyme system even though there i s no r e a l evidence for t h i s . 94 Nevertheless, the question s t i l l remains how does f e r r i -t i n , containing Fe"^+, bind the outer mitochondrial membrane but 2+ release Fe into the mitochondrion for use i n heme synthesis by ferrochelatase? The permeability b a r r i e r to f e r r i c ion i s overcome in the l i v i n g mitochondrion. Some ent i t y capable of extracting f e r r i c ions from f e r r i t i n , which i s bound to the outer membrane, yiel d s up ferrous ions to ferrochelatase i n the inner membrane for use i n heme synthesis. The data presented here indicate that the entity i s a p r o t e i n , l i t i s membrane bound, i t accepts electrons from a l l electron donors capable of yi e l d i n g electrons to the respiratory chain through flavoproteins, i t developes rapidly during the assembly of functional mito-chondria, and, as yet has not been separated from ferrochelatase a c t i v i t y . The relationship between these two important functions remains an i n t r i g u i n g problem. 95 CONCLUSION The porphyrias and anemias are two major disease states i n which heme biosynthesis i s disturbed. Such diseases pro-vide evidence of the delicacy of the control mechanisms regulating heme biosynthesis. The porphyrias are a group of diseases which are either hereditary or acquired i n o r i g i n and are characterized by abnormalities of heme biosynthesis and regulation. The anemias also are either hereditary or acquired and are characterized by abnormal iron metabolism which i n many cases i s confounded by aberant heme metabolism. Enzymic lesions with subsequent loss of regulation of heme biosynthesis have been postulated as possible causes for some types of porphyria and anemia. E x t r i c a t i o n of the o r i g i n of these d i s -eases w i l l require a detailed understanding of the i n t r i c a c i e s of the control mechanisms regulating both heme and iron metaboclism. Heme aff e c t s the disassociation of iron from the ir o n -68 69 t r a n s f e r r i n complex and i t s uptake into r e t i c u l o c y t e s ' and 70 71 erythroid c e l l s ' , thus the c e l l u l a r uptake of iro n i s decreased by conditions associated with excessive c e l l u l a r heme. Iron i s instrumental i n regulating heme biosynthesis at three points i n the heme pathway; f i r s t , iron exerts substrate i n h i b i t i o n over ferrochelatase at high iron concentrations, second, iron i s a cofactor f o r the enzyme coproporphyrinogen oxidase and t h i r d , ferrous ion i n h i b i t s uroporphyrinogen de-72 73 carboylase and cosynthetase i n the cytosol ' . There i s 96 also evidence that high intramitochondrial iron concentrations lead to st r u c t u r a l damage of c r i s t a e and peroxidation of 74 membrane phospholipids which would ultimately a f f e c t the i n ^ t e g r i t y of the heme biosynthetic system. Knowledge of the convergence of the heme and iron pathways and the control they exert over.each other has led to the successful elucidation of the factors underlying the diseases porphyria cutanea tarda and si d e r o b l a s t i c anemia. Porphyria cutanea tarda i s characterized biochemically by excessive hepatic heme synthesis and excretion of hepta and hexaporphyrins of the b i o l o g i c a l l y inactive isomer I series. The pattern of porphyrin excretion suggests an impaired a b i l i t y to decarboxy*? late uroporphyrinogens. Since iron i n h i b i t s decarboxylase and cosynthetase i t i s thought possible that t h i s form of porphyria might be associated with disordered iron metabolism, t h i s sup-position has now been demonstrated. Sideroblastic anemia, which i s characterized by the presence of sideroblasts which are erythroid precursors containing excessive intramitochondrial iron deposits, also appears to be a consequence of defective heme synthesis. Decreased heme synthesis r e s u l t s i n increased uptake of iron into the developing normoblast, which accumulates i n the mitochondria which re s u l t s gnegr.eatly enlarged mitochon-75 d r i a devoid of normal c r i s t a e . Another example of the e f f e c t of disruptions i n the regulation of heme biosynthesis and iron metabolism i s seen i n patients poisoned with lead. Lead t o x i c i t y may r e s u l t i n both.sideroblastic anemia and porphyria. Lead i n h i b i t s the enzymes ALA-dehydratase and ferrochelatase leading to porphyrin accumulation and iron over-loading of the mitochondrion''. In t h i s thesis several new poinfe%.of regulation of heme synthesis have been i d e n t i f i e d and the p o s s i b i l i t y - has been suggested that another metaSblic function, the reduction of iron, may exert control over heme formation. Thus, i n addition to end product i n h i b i t i o n and repression of ALA-synthetase by heme, we have now demonstrated a dual e f f e c t of heme upon fer-r rochelatase activity-; namely, i n h i b i t i o n at low heme concentra^ tions and stimulations at high concentrat±on%. To our knowledge of the effects of iron upon heme synthesis we can now add that ferrochelatase i s inh i b i t e d at high, concentrations of iron, leading to reduced Kerne concentrations which, may reduce entrance of iron into the c e l l . The demonstration of a d i s t i n c t iron reducing a c t i v i t y which can l i m i t heme formation suggests a c l o s e l y linked control between the entrance of iron into the mitochondrion, the a v a i l a b i l i t y of reducing equivalents and heme biosynthesis. The finding that electron donors stimulate ferrochelatase a c t i v i t y lends credence to t h i s possibility^. Without implying any temporal significance the following ordered sequence of events i s proposed. Transferrin, containing f e r r i c ion binds to the surface of the c e l l . F e r r i c ion i s yielded up into the c e l l where i t might combine with a p o f e r r i t i n to form f e r r i t i n . Iron might also e x i s t i n the c e l l bound to 78 79 some other c a r r i e r such as sucrose ' or another low molecular 80 81 82 weight protein , or even a non-protein chelator ' . Iron and i t s c a r r i e r bind to the outside of the mitochondrion and iron i s absorbed by some energy dependent process. F e r r i c ion then 98 comes i n contact with an ent i t y capable of iron reduction. This entity, perhaps a flavoprotein, has derived i t s reducing equivalents from electron donors which are capable of providing electrons for res p i r a t i o n and they- also provide electrons for iron reduction through t h e i r respective dehydrogenases. The iron reduction a c t i v i t y i s also in contact with cytochrome b which may be the l i n k by which i t makes contact with the dehy-drogenases. The iron reduction a c t i v i t y i s i n physical contact with the ferrochelatase a c t i v i t y which chelates*ferrous ion to protoporphyrin IX, derived from protoporphyrinogen dehydrogenase^ f also situated on the inner mitochondrial membrane. Heme i s released into the matrix of the mitochondrion. Heme and excess ferrous^ ion delivered across the mitochondrial membranes are able to accumulate and exert manifold instances of control over t h i s reaction sequence. Within the mitochondrion, heme can in-^ h i b i t the formation of S-ALA, i t s f i r s t committed precursor and can i n h i b i t the formation of more heme by 50%. Under the conditions when heme continues to be formed and the concentration exceeds 5 0 uM, heme can stimulate ferrochelatase to u t i l i z e the accumulated protoporphyrin IX, which, as far as i s known, has no regulatory properties. Iron, accumulated within the mitochon^ drion can i n h i b i t ferrochelatase at concentrations greater than 0.25 mM. Outside the mitochondrion, heme can i n h i b i t the de novo synthesis of S-ALA synthetase when present i n concentra-tions as low as 0.1 uM and can i n h i b i t the uptake of iron into the c e l l . Irpn can i n h i b i t uroporphyrinogen decarboxylase and cosynthetase i n the cy-tosol and excess iron i s also sequestered by f e r r i t i n and extruded from the c e l l by\ micropinocytotic 99 vehicles. If a l l these mechanisms are functional, iron loading and porphyrin accumulation w i l l not occur. However, abnormal states w i l l occur i f any s i t e of regulation i s malfunctioning. More general metabolic control exerted by the a v a i l a b i l i t y of reducing equivalents provided by anabolic reactions affects heme biosynthesis through the iron reductase a c t i v i t y which, i n turn, l i m i t s heme production. .This completes a cycle of i n t r a c e l l u l a r metabolic control. Enzymic regulation other than metabolic ef f e c t o r s has also been demonstrated. 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