REDESIGN OF THE INTERIOR HYDROPHILIC REGION OF MITOCHONDRIAL CYTOCHROME-C BY SITE-DIRECTED MUTAGENESIS

Heme propionate-7 in cytochrome c is an ionizable group located in a r egion of the protein that is inaccessible to bulk solvent. Electrostat ic stabilization of this functional group appears to be achieved throu gh interaction of heme propionate-7 with several amino acid residues t hat occur within hydrogen-bonding distance of it. To investigate the f unctional and spectroscopic roles of the amino acid residues that cont ribute to the immediate environment of heme propionate-7, the followin g variant forms of yeast (Saccharomyces cerevisiae) cytochrome c have been prepared and characterized by electrochemical and spectrochemical analyses: Arg38Ala, Tyr48Phe, Ala38Phe, Tyr48Phe/Trp59Phe, and Arg38A la/Tyr48Phe/Trp59Phe. For each protein, the dependence of midpoint red uction potential and NMR spectrum on pH was determined, and the UV (25 0-450 nm) circular dichroic (CD) spectrum was measured. All of the var iant proteins exhibited decreased reduction potentials with the greate st difference (-65 to -70 mV) exhibited by the multiply mutated protei ns. The electrostatic properties of the variant proteins as reflected by the oxidation-state dependence of the His-39 pK(a) value were simil ar 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 res idue is replaced by Phe. The present results establish that the electr ochemical effects of eliminating hydrogen-bonding interactions with he me propionate-7 are not additive and that the functional modulation of cytochrome c through regulation of the heme propionate-7 dielectric e nvironment involves a complex combination of solvation effects and ele ctrostatic or hydrogen-bonding interactions.