Comparing the rates and the activation parameters for the forward reaction between the triplet state of zinc cytochrome c and cupriplastocyanin and the back reaction between the zinc cytochrome c cation radical and cuproplastocyanin.

This is a comparative study of the photoinduced (so-called forward) electron-transfer reaction 3Zncyt/pc(II) --> Zncyt+/pc(I), between the triplet state of zinc cytochrome c (3Zncyt) and cupriplastocyanin [pc(II)], and the thermal (so-called back) electron-transfer reaction Zncyt+/pc(I) --> Zncyt/pc(II), between the cation (radical) of zinc cytochrome c (Zncyt+) and cuproplastocyanin [pc(I)], which follows it. Both reactions occur between associated (docked) reactants, and the respective unimolecular rate constants are kF and kB. Our previous studies showed that the forward reaction is gated by a rearrangement of the diprotein complex. Now we examine the back reaction and complare the two. We study the effects of temperature (in the range 273.3-302.9 K) and viscosity (in the range 1.00-17.4 cP) on the rate constants and determine enthalpies (DeltaH), entropies (DeltaS), and free energies (DeltaG) of activation. We compare wild-type spinach plastocyanin, the single mutants Tyr83Leu and Glu59Lys, and the double mutant Glu59Lys/Glu60Gln. The rate constant kB for wild-type spinach plastocyanin and its mutants markedly depends on viscosity, an indication that the back reaction is also gated. The activation parameters DeltaH and DeltaS show that the forward and back reactions have similar mechanisms, involving a rearrangement of the diprotein complex from the initial binding configuration to the reactive configuration. The rearrangements of the complexes 3Zncyt/pc(II) and Zncyt+/pc(I) that gate their respective reactions are similar but not identical. Since the back reaction of all plastocyanin variants is faster than the forward reaction, the difference in free energy between the docking and the reactive configuration is smaller for the back reaction than for the forward reaction. This difference is explained by the change in the electrostatic potential on the plastocyanin surface as Cu(II) is reduced to Cu(I). It is the smaller DeltaH that makes DeltaG smaller for the back reaction than for the forward reaction.