Effects of charged amino acid mutations on the bimolecular kinetics of reduction of yeast iso-1-ferricytochrome c by bovine ferrocytochrome b5.
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 > 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, Asp60-Lys86, 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-Tml72, 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.
Department of Chemistry, Tennessee Technological University, Cookeville 38505.
S H
SH
Northrup
K A
KA
Thomasson
C M
CM
Miller
P D
PD
Barker
L D
LD
Eltis
J G
JG
Guillemette
S C
SC
Inglis
A G
AG
Mauk
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003574:Cytochrome c Group / Q000235:genetics / Q000378:metabolism*; D015786:Cytochromes b5 / Q000378:metabolism*; D045304:Cytochromes c; D006418:Heme / Q000378:metabolism; D006639:Histidine; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D029701:Saccharomyces cerevisiae Proteins; C003712:trimethyllysine; D006418:Heme; D006639:Histidine; D045304:Cytochromes c; D015786:Cytochromes b5; D008239:Lysine