PHOTOINDUCED ELECTRON-TRANSFER FROM THE TRIPLET-STATE OF ZINC CYTOCHROME-C TO FERRICYTOCHROME B5 IS GATED BY CONFIGURATIONAL FLUCTUATIONS OF THE DIPROTEIN COMPLEX

The intracomplex electron-transfer reaction (3)Zncyt/b(5)(III) --> Zncyt(+)/b(5)(II) within electrostatic and covalent complexes of zinc(II) cytochrome c and ferricytochrome b(5) is studied by laser flash photolysis. Kinetic effects of protein cross-linking and of solution viscosity are interpreted in terms of dynamic mobility of the associated proteins with respect to each other. The rate constant for the monoexponential reaction in the electrostatic complex is 3.5 x 10(5) s(-1) in aqueous solution; this value is independent of protein concentration and ionic strength, but it decreases markedly as viscosity is raised by addition of glycerol or sucrose. The multiexponential reaction in the covalent complex was analyzed also in terms of the stretched exponential, exp[-(kt)(n)]. The best fit requires k = 6.8 X 10(4) s(-1) and n = 0.56 in aqueous solution; this rate constant is independent of protein concentration and ionic strength, but it decreases slightly as viscosity is raised. Fitting of the viscosity dependence to a simple two-state kinetic model yields a rate constant of 3.0 x 10(5) s(-1) for rearrangement of the electrostatic complex (3)Zncyt/b(5)(III) from the initial docking configuration to a different, more reactive, configuration. The corresponding rate constant for rearrangement of the electrostatic complex (3)Zncyt/pc(II) containing plastocyanin, determined previously, is 2.5 X 10(5) s(-1). Evidently, the intracomplex reaction in both electrostatic complexes is gated by a rearrangement process. Fitting of the viscosity dependences to two types of modified Krammers's equation shows that cross-links raise internal friction between the associated protein molecules from 3 cP in both electrostatic complexes Zncyt/b(5)(III) and Zncyt/pc(II) to 15 cP in the covalent complex Zncyt/b(5)(III); the rate constant for intracomplex electron transfer in the covalent complex (3)Zncyt/pc(II) is independent of viscosity, so that in this case internal friction is undefined. This study shows that electrostatic complexes which zinc(II) cytochrome c forms with plastocyanin and with cytochrome b(5) have similar dynamic properties even though plastocyanin has two distinct reactive patches on its surface, whereas cytochrome b(5) has only one such patch. The rearrangement process responsible for gating of the intracomplex electron-transfer reaction probably amounts to configurational fluctuations during which the exposed heme edge in zinc(II) cytochrome c remains within or near the broad acidic patch in plastocyanin or cytochrome b(5). Additional conformational fluctuation should be considered, especially in the Zncyt/b(5)(III) system, because covalent cross-linking of these two proteins does not completely abolish viscosity dependence of the intracomplex electron-transfer reaction. Configurational mobility of the associated proteins with respect to each other and conformational mobility of individual proteins should be distinguished in future experimental and theoretical studies of electron-transfer reactions between metalloproteins.