Redox reactivity of the type 1 (blue) copper protein amicyanin from Thiobacillus versutus with inorganic complexes

Abstract

Electron-transfer reactions of the type 1 copper protein amicyanin from Thiobacillus versutus with [Co(terpy)₂]²⁺(terpy = 2, 2′:6′, 2″-terpyridine) as a reductant for AmCu^II, and [Fe(CN)₆]^3– and [Co(phen)₃]³⁺(phen = 1, 10-phenanthroline) as oxidants for AmCu^I have been explored by stopped-flow kinetic studies at 25 °C, I= 0.100 M (NaCl). The reaction with [Co(terpy)₂]²⁺ is a straightforward single-stage process, whereas both the oxidaltions give biphasic kinetics. The first stages of the latter exhibit an active-site ‘switch-off’ mechanism previously assigned as a protonation (and dissociation) of the His-96 ligand of AmCu^I. From the studies with [Co(phen)₃]³⁺ an acid dissociation constant pK_a of 6.6 and rate constant at the higher pH of 7.1 × 10³ M ^–1 s^–1 are obtained. The reaction with [Fe(CN)₆]^3–gives similar behaviour with a pK_a, of 6.6, and from the fitting procedure adopted, a rate constant at the higher pH of 8.3 × 1 0⁶ M^–1s^–1, which is at the upper limit of the stopped-flow range. The second stage of the reaction is explained by the formation of a less-reactive form of AmCu^I in amounts of between 5 and 40% depending on the pH. The rate law in the case of [Co(phen)₃]³⁺ is independent of oxidant concentration, shows little dependence on pH and gives rate constants (a) in the range 0.07–0.12 s^–1. For the more reactive [Fe(CN)₆]^3– the rate equation is k_2obs=a+b[Fe(CN)₆^3–], with a varying between 0.1 3 and 0.1 5 s^–1 and b= 680 M^–1s^–1. The similar values of a for the two reactions are consistent with a common rate-controlling intramolecular isomerisation step. The bimolecular rate constant b is ≈ 10⁴ times smaller than the rate constant for the first stage of the [Fe(CN)₆]^3– oxidaltion at pH ≈ 8. The nature of the structural differences between the two forms of AmCu^I is considered.