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 AmCuII, and [Fe(CN)6]3– and [Co(phen)3]3+(phen = 1, 10-phenanthroline) as oxidants for AmCuI 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 AmCuI. From the studies with [Co(phen)3]3+ an acid dissociation constant pKa 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 pKa, 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 AmCuI 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 k2obs=a+b[Fe(CN)63–], 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 AmCuI is considered.