Kinetic studies on 1:1 electron-transfer reactions involving blue copper proteins. Part 10. The assignment of binding sites in the reactions of plastocyanin (and azurin) with non-physiological protein redox partners

Abstract

One-equivalent reductions of plastocyanin, PCu(II)(estimated charge 8–), with cytochrome c(II)(8+), and with high-potential iron–sulphur protein in the reduced form, Hipip(r)(3–), have been studied at I= 0.10 M (NaCl). Three effects have been tested for. First the different responses of the reactions to pH in the range 4.5–8.8 are noted. Secondly competitive inhibition of the reaction cytochrome c(II)+ PCu(II), but not Hipip(r)+ PCu(II), with redox-inactive [Pt(NH₃)₆]⁴⁺ has been observed. In the third approach PCu(II) modified by attachment of Cr^III at residues 42–45 reacts more slowly with cytochrome c(II), but at the same rate with Hipip(r). All three approaches are consistent with cytochrome c reacting at the previously defined Tyr 83/42–45 site on plastocyanin. The responses with Hipip(r) suggest that this reductant reacts at a different binding site on PCu(II), which may be the same as that used by [Fe(CN)₆]^4–, namely the His 87 site. Since it has previously been demonstrated that plastocyanin reacts at the exposed haem edge of cytochrome c, it can be concluded that the Cu to Fe separation at the time of electron transfer is ca. 20 Å. Effects of pH on the reduction of azurin, ACu(II)(1–), with cytochrome c(II) and Hipip(r) are also considered.