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 AmCul have been explored by stopped-flow kinetic studies at 25 degrees C, I = 0.100 M (NaCl). The reaction with [Co(terpy)(2)](2+) is a straightforward single-stage process, whereas both the oxidations 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 AmCul. 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 x 10(3) 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 x 10(6) M(-1) s(-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 AmCul in amounts of between 5 and 40% depending on the pH. The rate law in the case of [Co(ph)(3)](3+) is independent of oxidant concentration, shows little independence on pH and gives rate constants (a) in the range 0.07-012 s(-1). For the more reactive [Fe(CN)(6)](3-) the rate equation is k(2abs) = a + b[Fe(CN)(6)(3-)], with a varying between 0.13 and 0.15 s(-1) and b = 680 M(-1) s(-1). The similar values of a for the two reactions are consistent with a common rate-controling intramolecular isomerisation step. The bimolecular rate constant b is approximate to 10(4) times smaller than the rate constant for the first stage of the [Fe(CN)(6)](3-) oxidation at pH approximate to 8. The nature of the structural differences between the two forms of AmCul is considered.