Protein–protein cross-reactions involving plastocyanin, cytochrome f and azurin: self-exchange rate constants and related studies with inorganic complexes

Abstract

Protein–protein cross-reactions involving acidic and basic plastocyanins, cytochrome f and azurin, and including reactions between two blue copper proteins, have been studied for the first time. The high reactivity of cytochrome, f, and properties of the basic Anabaena variabilis plastocyanin, are noted. Reduction potentials for different plastocyanins have been obtained at pH 7–9 and are in the range 340–380 mV. The application of Marcus theory has enabled self-exchange rate constants (25 °C) for acidic parsley (3.3 × 10³ M^–1 s^–1) and basic A. variabilis(5.9 × 10⁵ M^–1 s^–1) plastocyanins to be calculated at pH 7.5, I= 0.10 M (NaCl). The influence of overall charge on the PCu(I)–PCu(II) self-exchange, estimated as –8, –7 in the case of parsley and +1, +2 for A. variabilis, is noted. A mechanism for electron transfer involving contact of adjacent hydrophobic regions on the two plastocyanin surfaces giving a Cu ⋯ Cu separation of 12–14 Å is proposed. Rate constants for the cytochrome f(II) reduction of four PCu(II) plastocyanins (acidic parsley, spinach, Scenedesmus obliquus and basic A. variabilis) are a factor of ≈10² less in the case of the basic form. The rate constant for Pseudomonas aeruginosa azurin with cytochrome f has also been determined. The A. variabilis and azurin rate constants give a cytochrome f(II)–f(III) self-exchange of 5.0 × 10⁵ M^–1 s^–1 at pH 7.5, whereas that obtained from the cross-reaction with the more highly charged parsley PCu(II)(2.3 × 10⁸ M^–1 s^–1) requires correction for work terms, and is predictably out of line. Only one out of four cross-reactions of A. variabilis plastocyanin and azurin with [Co(phen)₃]³⁺(phen = 1,10-phenanthroline) and [Fe(CN)₆]^3– gives a satisfactory fit to the Marcus theory, indicating an influence of more localised protein charge on these reactions. The absence of a fully developed acidic patch region on the surface of A. variabilis plastocyanin is discussed.