EFFECTS OF SINGLE AND DOUBLE MUTATIONS IN PLASTOCYANIN ON THE RATE-CONSTANT AND ACTIVATION PARAMETERS FOR THE REARRANGEMENT GATING THE ELECTRON-TRANSFER REACTION BETWEEN THE TRIPLET-STATE OF ZINC CYTOCHROME-C AND CUPRIPLASTOCYANIN

The unimolecular rate constant for the photoinduced electron-transfer reaction (3)Zncyt/pc(II) --> Zncyt(+)/pc(I) within the electrostatic c omplex of zinc cytochrome c and spinach cupriplastocyanin is k(F). We report the effects on k(F) Of the following factors, all at pH 7.0: 12 single mutations on the plastocyanin surface (Leu12Asn, Leu12Glu, Leu 12Lys, Asp42Asn, Asp42Lys, Glu43Asn, Glu59Gln, Glu59Lys, Glu60Gln, Glu 60Lys, Gln88Glu, and Gln88Lys), the double mutation Glu59Lys/Glu60Gln, temperature (in the range 273.3-302.9 K), and solution viscosity (in the range 1.00-116.0 cP) at 283.2 and 293.2 K. We also report the effe cts of the plastocyanin mutations on the association constant (K-a) an d the corresponding free energy of association (Delta G(a)) with zinc cytochrome c at 298.2 K. Dependence of kF on temperature yielded the a ctivation parameters Delta H-double dagger, Delta S-double dagger, and Delta G(double dagger). Dependence of k(F) on solution viscosity yiel ded the protein friction and confirmed the Delta G(double dagger) valu es determined from the temperature dependence. The aforementioned intr acomplex reaction is not a simple electron-transfer reaction because d onor-acceptor electronic coupling (H-AB) and reorganizational energy ( lambda), obtained by fitting of the temperature dependence of k(F) to the Marcus equation, deviate from the expectations based on precedents and because kF greatly depends on viscosity. This last dependence and the fact that certain mutations affect K, but not kF are two lines of evidence against the mechanism in which the electron-transfer step is coupled with the faster, but thermodynamically unfavorable, rearrange ment step. The electron-transfer reaction is gated by the slower, and thus rate determining, structural rearrangement of the diprotein compl ex; the rate constant kF corresponds to this rearrangement. Isokinetic correlation of Delta H-double dagger and Delta S-double dagger parame ters and Coulombic energies of the various configurations of the Zncyt /pc(II) complex consistently show that the rearrangement is a facile c onfigurational fluctuation of the associated proteins, qualitatively t he same process regardless of the mutations in plastocyanin. Correlati on of kF with the orientation of the cupriplastocyanin dipole moment i ndicates that the reactive configuration of the diprotein complex invo lves the area near the residue 59, between the upper acidic cluster an d the hydrophobic patch. Kinetic effects and noneffects of plastocyani n mutations show that the rearrangement from the initial (docking) con figuration, which involves both acidic clusters, to the reactive confi guration does not involve the lower acidic cluster and the hydrophobic patch but involves the upper acidic cluster and the area near the res idue 88.