Replacements of lysine 32 in yeast cytochrome c. Effects on the binding and reactivity with physiological partners

Lysine 32 has been previously implicated by chemical modification and modeling studies as a key component of the domain which controls recognition and binding of cytochrome c to its physiological partners, e.g. cytochrome b2, cytochrome c peroxidase, and cytochrome oxidase. In order to quantitate the importance of this residue, we have investigated the role of Lys-32 in the reactivity of cytochrome c in redox reactions in vitro and in vivo with protein partners by using a series of altered forms of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae in which Lys-32 is replaced by Leu-32, Gln-32, Trp-32, and Tyr-32. Leu-32 and Gln-32 represent substitutions which change charge without seriously affecting the steric bulk of the side chain or the stability of the protein. For the Leu-32- and Gln-32-altered proteins, steady state kinetic studies with cytochrome c peroxidase, cytochrome b2, and cytochrome oxidase showed that neither of the steady state kinetic parameters, Km nor Vmax, were substantially modified by mutation. Studies of single turnover kinetics with a small molecule (ascorbate) or within bound complexes with either cytochrome b5 or cytochrome c peroxidase demonstrated that redox kinetics are only slightly affected by these substitutions. NMR experiments demonstrated that the Gln-32-altered protein can still bind strongly to a physiological partner, cytochrome c peroxidase. Growth in lactate medium demonstrated that the activity in vivo compared with the normal value was reduced to only 85% with the Gln-32- and Leu-32-altered proteins and to 65% with the Trp-32- and Tyr-32-altered proteins. These findings suggest that the evolutionary invariance of Lys-32 reflects only small quantitative changes in the binding and reactivity of cytochrome c.