Kinetic studies on 1 : 1 electron-transfer reactions involving blue copper proteins. Part 6. Competitive inhibition of the [Co(phen)3]3+(phen = 1,10-phenanthroline) oxidation of parsley plastocyanin PCu(I) by redox-inactive complexes

Abstract

A search has been carried out for redox-inactive inorganic complexes which exhibit competitive inhibition (i.e. blocking) of the [Co(phen)₃]³⁺(phen = 1,10-phenanthroline) oxidation of negatively charged parsley plastocyanin, PCu(I), at 25 °C and I= 0.10 M (NaCl). Three complexes have been identified which give greater association (K_B/M^–1) with PCu(I) than that previously determined for [Cr(phen)₃]³⁺(176 M^–1). Association constants (M^–1) obtained at pH 7.5 are: [Co(NH₃)₃]³⁺, 470; [Pt(NH₃)₆]⁴⁺, 22 300; and [(NH₃)₅Co(NH₂)Co(NH₃)₅]⁵⁺, 16 000. Acid dissociation of [Pt(NH₃)₆]⁴⁺, pK_a 7.1, requires that a lower pH be used for maximum effectiveness of this complex. Partial blocking only is observed in all three cases, and it is concluded that the site for electron transfer involves a broad region of protein surface incorporating Tyr 83 and the negative patch of residues including 42–45. The size of the effect for the 4+ and 5+ complexes makes these complexes most appropriate for further studies in which use of this site is tested for. It has been demonstrated with [Pt(NH₃)₆]⁴⁺ that association with PCu(I) becomes less effective at pH < 6.6. The protein acid dissociation pK_a of 5.8 which is obtained is coincident with pK_a values reported previously for the inactivation of PCu(I). A possible explanation involving a co-operative effect between the active site and negative patch 42–45 is proposed. No similar competitive inhibition of the [Fe(CN)₆]^3– oxidation of PCu(I) by redox-inactive complexes [Zr(C₂O₄)₄]^4– and [Mo(CN)₈]^4– is observed at pH 7.5.