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^ -^, 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