EFFECTS OF CHARGED AMINO-ACID MUTATIONS ON THE BIMOLECULAR KINETICS OF REDUCTION OF YEAST ISO-1-FERRICYTOCHROME-C BY BOVINE FERROCYTOCHROME-B(5)

The reduction of wild-type yeast iso-1-ferricytochrome c (ycytc) and s everal mutants by trypsin-solubilized bovine liver ferrocytochrome b5 (cytb5) has been studied under conditions in which the electron-transf er reaction is bimolecular. The effect of electrostatic charge modific ations and steric changes on the kinetics has been determined by exper imental and theoretical observations of the electron-transfer rates of ycytc mutants K79A, K'72A, K79A/K'72A, and R38A (K' is used to signif y trimethyllysine (Tml)). A structurally robust Brownian dynamics (BD) method simulating diffusional docking and electron transfer was emplo yed to predict the mutation effect on the rate constants. A realistic model of the electron-transfer event embodied in an intrinsic unimolec ular rate constant is used which varies exponentially with donor-accep tor distance. The BD method quantitatively predicts rate constants ove r a considerable range of ionic strengths. Semiquantitative agreement is obtained in predicting the perturbing influence of the mutations on the rate constants. Both the experimentally observed rate constants a nd those predicted by BD descend in the following order: native ycytc > K79A > K'72A > K79A/K'72A. Variant R38A was studied at a different i onic strength than this series of mutations, and the theory agreed wit h experiment in predicting a smaller rate constant for the mutant. In all cases the predicted effect of mutation was in the correct directio n, but not as large as that observed. The BD simulations predict that the two proteins dock through essentially a single domain, with a dist ance of closest approach of the two heme groups in rigid body docking typically around 12 angstrom. Two predominant classes of complexes wer e calculated, the most frequent involving the quartet of cytb5/ycytc i nteractions, Glu48-Arg13', Glu56-Lys87, Asp60-Lys86, and heme-Tm172, h aving an average electrostatic energy of -13.0 kcal/mol. The second mo st important complexes were of the type previously postulated (Salemme , 1976; Mauk et al., 1986; Rodgers et al., 1988) with interactions Glu 44-Lys27, Glu48-Arg13, Asp60-Tm172, and heme-Lys79 and having an energ y of -6.4 kcal/mol. The ionic strength dependence of the bimolecular r eaction rate was well reproduced using a discontinuous dielectric mode l, but poorly so for a uniform dielectric model.