Kinetic studies on 1:1 electron-transfer reactions involving blue copper proteins. Part 9. Effects of chromium(III) modification on reactions of plastocyanin with [Co(phen)3]3+(phen = 1,10-phenanthroline), [Fe(CN)6]3–, [Co(dipic)2]–(dipic = dipicolinate)(oxidants) and [Ru(NH3)5(py)]2+(py = pyridine)(reductant)

Abstract

A 1:1 chromium(III)-modified form of parsley plastocyanin was obtained by reduction of oxidised protein, here referred to as PCu(II), with Cr²⁺(aq). At pH 7.5 a 21% decrease in rate constant is observed for the [Co(phen)₃]³⁺ oxidation of PCu(I)·Cr^III as compared to native protein. Biphasic kinetics (rate constants k₁ and k₂) are obtained for the same reaction at pH 5.8 suggesting that at this pH the Cr^III is attached in two ways. The rate constant k₁ is the same as that observed for native plastocyanin, with k₂ some 72% less. On attachment of a second mol of Cr^III by the same procedure to give PCu(I)·2Cr^III, uniphasic kinetics with a rate constant k₂ are observed. Within experimental error no effect of single modification is observed on rate constants for the [Fe(CN)₆]^3– oxidation at pH 7.5 and 5.8. At pH 5.8 the [Ru(NH₃)₅(py)]²⁺(py = pyridine) reduction of PCu(II)·Cr^III gives a net 22% decrease, which increases to 38% for PCu(II)·2Cr^III. No effect is observed for the [Co(dipic)₂]^–(dipic = dipicolinate) oxidation of PCu(I)·Cr^III also at pH 5.8. These observations support the belief that positively charged complexes [Co(phen)₃]³⁺(phen = 1,10-phenanthroline) and [Ru(NH₃)₅(py)]²⁺ react at a broad region on plastocyanin close to Tyr 83 and the negatively charged residues 42–45, whereas [Fe(CN)₆]^3– and [Co(dipic)₂]-react at a site close to His 87. The reduction potential for the PCu(II)–PCu(I) couple (370 mV) is decreased by ca. 20 mV on attachment of Cr^III.