Kinetic studies on 1 : 1 electron-transfer reactions involving blue copper proteins. Part 12. Reactions of spinach plastocyanin with inorganic redox partners

Abstract

Reactions of spinach plastocyanin have been studied at 25 °C, I= 0.10 M (NaCl), and a comparison is made with the previously studied parsley plastocyanin, which has 31 different residues in a single polypeptide chain of 99 amino-acid residues. Active-site protonations leading to a switch-off in reactivity of PCu (I) are notably different, as indicated by the acid dissociation constants pK_a for spinach (4.9 average) and parsley (5.5). Protonation of PCu(II) at the east-face binding site, which incorporates Tyr 83, gives similar pK_a′ values of 5.6 (spinach) and 5.8 (parsley). A number of other features are similar, including rate constants at pH 7.5 with [Fe(CN)₆]^3–, [Co(phen)₃]³⁺(phen = 1, 10-phenanthroline), and [Co(dipic)₂]^–(dipic = dipicolinate) as oxidants for PCu(I), and [Ru(NH₃)₅(py)]²⁺(py = pyridine) and [Co(terpy)₂]²⁺(terpy = 2, 2′:6′2″-terpyridyl) as reductants for PCu(II), which are all within ±33% of those for parsley plastocyanin, suggesting conservation of binding sites, reduction potential, and mechanism of electron transfer. With [Fe(CN)₆]^3– as oxidant, reaction is assigned to the His 87 (north) site, whereas with [Co(phen)₃]³⁺ reaction occurs at two (or more) binding sites with (from the effect of H⁺)ca. 70% of reaction at the Tyr 83 site. The oxidant [Ru(NH₃)₅(CH₃CN)]³⁺ has been used for the first time. Blocking with redox inactive [(NH₃)₅Co(NH₂)Co(NH₃)₅]⁵⁺(K_B= 6 300 M¹, pH 7.5) suggests that 91 % of this reaction is at the Tyr 83 site. From similar blocking experiments with [Co(phen)₃]³⁺ as oxidant the corresponding value is 80%(61% for parsley). Only mild association and blocking is observed with Mg²⁺(K≈ 38 M^–1).