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The akaline conformational transitions of ferricytochrome c Rosell, Federico Ignacio


The alkaline conformational transitions of several yeast iso-l -ferricytochrome c variants were characterized with a variety of spectroscopic and physical techniques. The first group of variants consists of cytochromes in which selected lysyl residues were replaced by alanine. On the basis of 1H-NMR and EPR spectroscopy, it was shown that Lys73 is one of the residues that replaces Met80 as an axial ligand to the heme iron and results in the alkaline conformer IVa. Lys79 had been shown previously to coordinate the metal centre in a second alkaline conformer IVb (Ferrer, J.C., Guillemette, J.G., Bogumil, R., Inglis, S.C., Smith, M. & Mauk, A.G . (1993) J. Am. Chem. Soc., 115, 7507-7508). The absence of both lysyl residues from the cytochrome sequence (i.e., Lys73Ala/Lys79Ala double variant) results in the stabilization of the native conformation of the protein which persists at higher pH (pKa=10.5(3)). However, addition of amino group-containing compounds to solutions of this variant results in a protein adduct with spectroscopic features similar to those of the alkaline isomers. Furthermore, the conformational transition of the wild-type protein (pKa=8.70(2)) represents a weighted average of the transition of the native Met80-bound conformer to either the Lys73- (pKa=8.44(l)) or Lys79-bound (pKa=8.82(2)) conformers. pH-jump kinetics indicate that replacement of Met80 by Lys73 occurs with a greater rate constant (kf=160(5) s-1) than the replacement by Lys79 (kf=1.51(5) s-1) despite the more significant structural changes that must take place on the distal side of the protein to accommodate coordination of the heme iron by Lys73. This rearrangement of the molecule, however, does not change significantly the exposure of the heme group to solvent relative to the Lys79-bound isomer. Both alkaline conformers, IVa and IVb, have a similar midpoint reduction potential (-190 mV vs. SHE, pH ~8.7) suggesting that the dielectric constant of the heme cavity in the two conformers is comparable. The temperature and ionic strength dependence of the conformational equilibria of the Lys73Ala and Lys79Ala variants was studied. The formation of the Lys73-bound alkaline conformer exhibits a stronger temperature dependence than the isomerization involving Lys79. 'H-NMR shows that salts can significantly affect the distribution of alkaline conformers so that Lys73 ligation to the iron is favoured at low salt concentrations, whereas at higher concentrations, Lys79 coordination is preferred. The second group of variants includes cytochromes with amino acid replacements at highly conserved sites in the sequence (Thr78Ala, Tyr67Phe, Phe82Gly, Phe82Lys, Phe82Ser, Phe82Asp, Phe82Met, Phe82His, Phe82Trp, and Phe82Ile). In general, the Lys73-bound conformer is the favoured alkaline species in the Phe82Xxx variants, thus accounting in part for the generally decreased pKap of the transition of these variants. Unconventional cases are the Phe82His variant in which a non-native, bis-histidine ligation is observed that interferes with the normal alkaline transition, and the Phe82Trp variant in which a high-spin species is trapped at mildly alkaline pH. There is no indication at this time that Lys82 coordinates the heme iron. The variant with Phe at position 67 is unique among all the other cytochromes investigated in that the significantly stabilized native conformer appears to undergo the transition to primarily the Lys79- bound conformer. Finally, the alkaline transition of a cytochrome expressed with a newly developed bacterial expression system was characterized. During bacterial expression, Lys72 is not trimethylated as it is in yeast. The lack of this post-translational modification results in a third alkaline conformer in which this lysyl residue replaces Met80 as a heme ligand. Although this isomerization occurs more readily than that involving Lys73 or Lys79, the absence of the methionyl ligand in Tml72Lys/Met80Ala variant does not facilitate the formation of any of the three alkaline conformers. In this variant, the lysyl residues must compete with water and hydroxide for the axial heme iron coordination site, and the Lys72-bound conformer appears to be preferred alkaline species. [Scientific formulae used in this abstract could not be reproduced.]

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