Enforced interaction of one molecule of plastocyanin with two molecules of cytochrome c and an electron-transfer reaction involving the hydrophobic patch on the plastocyanin surface

Laser flash photolysis is used to study the photoinduced electron-transfer reaction cyt(III)// pc(II) + (3)Zncyt --> cyt(III)//pc(I) + Zncyt(+) at pH 7.0 and 25 degrees C. In the covalent (symbol //) complex cyt(III)//pc(II) the acidic patch in cupriplastocyanin is directly cross-linked to the basic patch in ferricytochrome c. The triplet state of zinc cytochrome c reduces the pc(II) moiety, not the cyt(III) moiety, of the covalent complex. The reaction is strictly bimolecular in the entire range of ionic strength studied, from 1.25 mM to 1.00 M. The two reactants interact only transiently, in a collisional complex, and do not form a persistent complex cyt(III)//pc(II)/Zncyt. Because noncovalent (symbol /) association of three separate protein molecules is far less probable than association of the covalent complex and another protein molecule, we conclude that, without the aid of covalent cross-links, one molecule of plastocyanin will not form a ternary complex with two molecules of cytochrome c, cyt/pc/cyt. Dependence of the rate constant on ionic strength is analyzed in terms of van Leeuwen theory of electrostatic interactions, which recognizes the importance of dipole moments of the proteins. This analysis shows that (3)Zncyt reacts with the hydrophobic patch in the pc(II) moiety of the covalent complex cyt(III)//pc(II). At high ionic strength, at which electrostatic interactions are practically abolished, the blue copper site is reduced with approximately equal rates via the hydrophobic patch in the pc(II) moiety of the complex and via the acidic patch in free pc(II). This is evidence that the two distinct patches on the plastocyanin surface are comparable in their intrinsic ''conductivity'' for electrons coming to the copper site. Positively charged and electroneutral redox partners tend to react at the acidic patch (although not necessarily at the initial docking site in this broad patch) for electrostatic, not electronic, reasons. Earlier theoretical studies disagreed about the relative electronic conductivities of the two patches. This experimental study corroborates very recent theoretical studies that found the two patches to be comparable in the efficiency of electron transfer.