REACTIONS OF 5 SPINACH PLASTOCYANIN PCU(I) MUTANTS WITH [FE(CN)6]3- AND [CO(PHEN)3]3-PHENANTHROLINE) AND RELATED STUDIES( (PHEN = 1,10)

Rate constants (25-degrees-C) have been determined by stopped-flow spe ctrophotometry for the oxidation of five spinach plastocyanin (PCu) mu tants in the 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). T he mutants investigated, Leu12-Asn, Leu12Glu, Asp42Asn, Tyr83His and T yr83Phe, incorporate changes at the Leu-12 position of the adjacent (t o the Cu) hydrophobic site, and at the remote acidic patch residues Ty r-83 and Asp-42. At pH >7.5 the acid-dissociated glutamate (pK(a) 6.9) form of Leu12Glu impedes the [Fe(CN)6]3- oxidant which reacts almost- equal-to 90% more slowly than with the native protein.. A 2.2-fold inc rease in reactivity is however observed with [Co(phen)3]3+, which is c onsistent with a switch in reaction from the remote to adjacent site. This was confirmed by competitive inhibition studies with a redox-inac tive 6 + tetranuclear cobalt(III) complex. Also for this mutant the ac tive-site His-87 pK(a) determined by H-1 NMR studies is shifted to a h igher apparent value of 6.1 as compared to 4.9 for native protein, whi ch reflects the combined effect of active site/Glu-12 protonation equi libria. Variations in rate constants with pH are also explored for the Tyr83His mutant, where acid dissociation (pK(a) 8.4) affects reaction with both oxidants in a similar manner. At pH 7.5 no significant chan ge in reactivity is observed with the Asp42Asn and Tyr83Phe mutants, w hile there is a five-fold enhancement in the reaction of the Leu12Asn mutant with [Fe(CN)6]3-. Rate constants were also determined for the [ 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.