Reactions of five spinach plastocyanin PCu(I) mutants with [Fe(CN)6]3–and [Co(phen)3]3+(phen = 1,10-phenanthroline) and related studies

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Abstract
Rate constants (25 °C) have been determined by stopped-flow spectrophotometry for the oxidation of five spinach plastocyanin (PCu) mutants inthe copper(I) form, with inorganic redox partners [Fe(CN)6]3– and [Co(phen)3]3+(phen = 1,10-phenanthroline), I= 0.100 M (NaCl). The mutants investigated, Leu12-Asn, Leu12Glu, Asp42Asn, Tyr83His and Tyr83Phe, incorporate changes at the Leu-12 position of the adjacent (to the Cu) hydrophobic site, and at the remote acidic patch residues Tyr-83 and Asp42. At pH > 7.5 the acid-dissociated glutamate (pKa 6.9) form of Leu12Glu impedes the [Fe(CN)6]3– oxidant which reacts ≈90% more slowly than with the native protein. A 2.2-fold increase in reactivity is however observed with [Co(phen)3]3+, which is consistent with a switch in reaction from the remote to adjacent site. This was confirmed by competitive inhibtion studies with a redox-inactive 6 + tetranuclear cobalt(III) complex. Also for this mutant the active-site His-87 pKa determined by 1H NMR studies is shifted to a higher apparent value of 6.1 as compared to 4.9 for native protein, which reflects the combined effect of active site/Glu-12 protonation equilibria. Variations in rate constants with pH are also explored for the Tyr83His mutant, where acid dissociation (pKa 8.4) affects reaction with both oxidants in a similar manner. At pH 7.5 no significant change in reactivity is observed with the Asp42Asn and Tyr83Phe mutants, while there is a five-fold enhancement in the reaction of the Leu12Asn mutant with [Fe(CN)6]3–. Rate constants were also determined for [Fe(CN)6]4– and [Co(phen)3]2+ reductions of the copper(II) mutant forms at pH 7.5. Reduction potentials PCu(II)/(I) for the five mutants are in the range 360–402 mV, as compared to 375 mV for native protein.