Arg-38 is an internal residue of mitochondrial cytochrome c that is close to heme propionate-7. Previous work comparing the behavior of cytochromes c from several species [Moore, G. R., Harris, D. E., Leitch, F. A., & Pettigrew, G. W. (1984) Biochim. Biophys. Acta 764, 331-342] has suggested that Arg-38 lowers the pKa of this propionate group and thereby accounts for the relative pH independence of the cytochrome c reduction potential from pH 5 to pH 8. The influence of Arg-38 on the oxidation-reduction equilibrium of yeast iso-1-cytochrome c has now been investigated by electrochemical, NMR, and theoretical analysis of six specifically mutated forms of this protein in which Arg has been replaced by Lys, His, Gln, Asn, Leu, or Ala. As the electron-withdrawing character of the residue at position 38 decreases, the reduction potential of the protein also decreases, with the largest decrease (ca. 50 mV) observed for the Ala variant. However, the variation in the reduction potentials of the mutants as a function of pH was similar to that observed for the wild-type protein. The effects of some of these mutations on the pKa values of His-33 and His-39 have been determined by NMR spectroscopy and found to be minimal. Calculations of the electrostatic free energy for the Leu-38 variant predict a decrease in the reduction potential of this mutant that is remarkably close to that observed experimentally. This work establishes that while Arg-38 contributes to the relatively high reduction potential of cytochrome c, this residue does not appear to be the sole functionality responsible for lowering the heme propionate-7 pKa.

Heme propionate-7 in cytochrome c is an ionizable group located in a region of the protein that is inaccessible to bulk solvent. Electrostatic stabilization of this functional group appears to be achieved through interaction of heme propionate-7 with several amino acid residues that occur within hydrogen-bonding distance of it. To investigate the functional and spectroscopic roles of the amino acid residues that contribute to the immediate environment of heme propionate-7, the following variant forms of yeast (Saccharomyces cerevisiae) cytochrome c have been prepared and characterized by electrochemical and spectrochemical analyses: Arg38Ala, Tyr48Phe, Ala38Phe, Tyr48Phe/Trp59Phe, and Arg38Ala/Tyr48Phe/Trp59Phe. For each protein, the dependence of midpoint reduction potential and NMR spectrum on pH was determined, and the UV (250-450 nm) circular dichroic (CD) spectrum was measured. All of the variant proteins exhibited decreased reduction potentials with the greatest difference (-65 to -70 mV) exhibited by the multiply mutated proteins. The electrostatic properties of the variant proteins as reflected by the oxidation-state dependence of the His-39 pKa value were similar to those of the wild-type protein. Previous indirect assignments of minima in the CD spectrum of cytochrome c at 282 and 289 nm to Trp-59 are confirmed by spectra of the variant cytochromes in which this residue is replaced by Phe. The present results establish that the electrochemical effects of eliminating hydrogen-bonding interactions with heme propionate-7 are not additive and that the functional modulation of cytochrome c through regulation of the heme propionate-7 dielectric environment involves a complex combination of solvation effects and electrostatic or hydrogen-bonding interactions.

The structures of variants of yeast iso‐1‐cytochrome c, in which the previously unchanged Tyr48 and Tyr48 + Trp59 have been replaced by Phe, have been characterised by NMR. The NMR data indicated that the structures of the variant cytochromes c are very similar to the wild‐type protein. In particular, the heme environment and interactions of the heme macrocycle were shown to be preserved. The observation of chemical shift differences have allowed for the assessment of conformational changes. The substitution of Trp59 by Phe may have caused a small conformational change, a manifestation of which is the observed chemical shift differences at His39, Val57 and Tyr74. The structural basis for the reduction in redox potential accompanying the amino acid substitutions is discussed and the proposal made that the changes in potential are a direct consequence of the side chain properties and do not result primarily from conformational changes.

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Mitochondria1 cytochrome c is a monohaem electron-transfer protein of 103-1 14 amino acids. Arginine-38 is an unvaried, internally located residue that interacts with the buried haem prioponate-7 (HP-7) [ 11. Previous work with related bacterial cytochromes has led to proposals that arginine-38 reduces the pK, of HP-7 to less than 4.5 [2] and has an important role in the oxidation-state-linked protein conformational change [ 31. To test these proposals mutants of yeast iso-1-cytochrome c have been constructed in which arginine-38 has been replaced by lysine, histidine, asparagine, glutamine, leucine and alanine (R. L. Cutler, M. Smith & A. G. Mauk, unpublished work). In the present communication we report 'H n.m.r. spectroscopic data showing that replacement of arginine-38 by alanine-38 does not significantly affect the interaction of HP-7 with the neighbouring tryptophan-59.Samples of ferricytochrome c were prepared for n.m.r. as previously described [4]. Both proteins contained the substitutions cysteine-102 to threonine-102; they differed in residue 38, which was arginine or alanine. N.m.r. spectra were recorded with a JEOL GX-400 n.m.r. spectrometer. Spectra showing nuclear Overhauser enhancements (NOE) [5] and two-dimensional correlated spectroscopy (2D-COSY ) spectra [6] were obtained using the standard JEOL PLEXUS packages. Fig. 1 shows the NOE difference spectrum of the alanine-38 ferricytochrome obtained upon saturation of a HP-7 PCH resonance. This is very similar to the NOE difference spectrum obtained for the arginine-38 protein and for horse ferricytochrome c [4]. The pattern of NOES show that the conformation of HP-7 is not significantly altered by the replacement of arginine-38 by alanine-38. The peak at 7.6 p.p.m. comes from the tryptophan 59 residue which is hydrogen-bonded to HP-7 in wild-type yeast cytochrome c [l].Abbreviations used: HP-7, haem propionate-7, NOE, nuclear Overhauser enhancement; 2D-COSY, two-dimensional correlated spectroscopy.