Biochemistry zyxwvu 1993, 32, zyxwvu 6613-6623 6613 Effects of Charged Amino Acid Mutations on the Bimolecular Kinetics of Reduction of Yeast Iso- 1 -ferricytochrome zyxw c by Bovine Ferrocytochrome b5t Scott H. Northrup,’ Kathryn A. Thomasson, and Cynthia M. Miller Department of Chemistry, Tennessee Technological University, Cookeville, Tennessee 38505 Paul D. Barker,$ Lindsay D. Eltis,g J. Guy Guillemette, Stephen C. Inglis, and A. Grant Mauk’ Department of Biochemistry, University zyxwvut of British Columbia, Vancouver, British Columbia, Canada, V6T 123 Received December 9, 1992; Revised Manuscript Received March 18, 1993 zyxwvutsrqponmlkjihgfedcbaZYXWV ABSTRACT: The reduction of wild-type yeast iso- 1-ferricytochrome c (ycytc) and several mutants by trypsin- solubilized bovine liver ferrocytochrome b5 (cytb5) has been studied under conditions in which the electron- transfer reaction is bimolecular. The effect of electrostatic charge modifications and steric changes on the kinetics has been determined by experimental and theoretical observations of the electron-transfer rates of ycytc mutants K79A, K’72A, K79A/K’72A, and R38A (K’ is used to signify trimethyllysine (Tml)). A structurally robust Brownian dynamics (BD) method simulating diffusional docking and electron transfer was employed to predict the mutation effect on the rate constants. A realistic model of the electron-transfer event embodied in an intrinsic unimolecular rate constant is used which varies exponentially with donor- acceptor distance. The BD method quantitatively predicts rate constants over 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 and those predicted by BD descend in the following order: native ycytc zyxwvuts > K79A > K‘72A > K79A/K‘72A. Variant R38A was studied at a different ionic strength than this series of mutations, and the theory agreed with experiment in predicting a smaller rate constant for the mutant. In all cases the predicted effect of mutation was in the correct direction, but not as large as that observed. The BD simulations predict that the two proteins dock through essentially a single domain, with a distance of closest approach of the two heme groups in rigid body docking typically around 12 A. Two predominant classes of complexes were calculated, the most frequent involving the quartet of cytb5/ycytc interactions, Glu48-Arg13, Glu56-Lys87, Asp6GLys86, and heme-Tml72, having an average electrostatic energy of -13.0 kcal/mol. The second most important complexes were of the type previously postulated (Salemme, 1976; Mauk et al., 1986; Rodgers et al., 1988) with interactions Glu44-Lys27, Glu48-Arg13, Asp60-Tm172, and heme-Lys79 and having an energy of -6.4 kcal/mol. The ionic strength dependence of the bimolecular reaction rate was well reproduced using a discontinuous dielectric model, but poorly so for a uniform dielectric model. The quantification of physical and chemical factors that determine electron-transfer rates between cytochromes is a topic of considerable interest (Marcus & Sutin, 1985; King et al., 1985; Gray, 1986; Northrup et al., 1988, 1990; Mayo et al., 1986; Hoffman & Ratner, 1987; Hazzard et al., 1987; Summers & Erman, 1988; Wendoloski et al., 1987; Dixon et al., 1989,1990). Thereductionof yeast iso-1-ferricytochrome c (ycytc) by trypsin-solubilized ferrocytochrome b5 (cytb5) is an attractive prototypical system for the study of such factors, in that these proteins are small, soluble, structurally well- characterized (Bushnell et al., 1990; Mathews et al., 1972, 1986), and readily modified by site-directed mutagenesis (Pielak et al., 1985; von Bodman et al., 1986; Funk et al., 1990). We recently studied this reaction (using the native f This work has been made possible by Grants GM34248 (S.H.N.) and GM33804 (A.G.M.)from theNational Institutesof Health, by Grant MT1706 from the Medical Research Council of Canada (to Michael Smith), by Grant 241 59-B6 (S.H.N.)from thePetroleumResearch Fund as administered by the American Chemical Society, and by the Camille and Henry Dreyfus Scholar/Fellow Program for Undergraduate Insti- tutions (at TTU). S.H.N. is a Camille and Henry Dreyfus Scholar; K.A.T. is a Camille and Henry Dreyfus Fellow. * Authors to whom correspondence should be addressed. t Present address: Cambridge Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, U.K. I Resent address: Gesellschaft Biotechnologisch Forschung, Mascherod- er Weg 12, D-3330 Braunschweig, Germany. horse heart ferricytochrome c (hcytc)) as a function of pH, temperature, and ionic strength to gain greater insight into the structural and functional properties that determine the rate of electron transfer under second-orderreaction conditions (Eltis, et al., 1991). We demonstrated that one can make semiquantitative predictions of ionic strength and pH depen- dence on this system using the Brownian dynamics (BD) simulation method and a dipolar sphere model of the proteins (Northrup et al., 1986, 1990). Using that simple theory, we provided an interpretative framework for understanding the role of electrostatics, ionic strength, temperature and pH effects, and geometric reactive criteria in the bimolecular rates observed in the experiments. However, a simple three-charge model is expected to fail when used to predict the detailed effects of point mutations involving charged amino acid residues. In this present study, we have evaluated the effect of electrostatic charge modifications and steric changes on the bimolecular kinetics of the reduction of ycytc by cytb5 by experimental and theoretical observations of the electron- transfer rates of ycytc mutants K79A, K’72A, K79A/K’72A, and R38A (K’ or Tml denotes trimethyllysine). Tm172 and Lys79 are two key charged residues near the heme crevice which are involved in ionic contacts with cytochrome partners, while Arg38 lies outside of the heme-exposed face. Here we 0006-2960/93/0432-6613$04.00/0 0 1993 American Chemical Society