Type 1 blue copper protein amicyanin from Thiobacillus versutus: line-broadening effects of chromium(III) complexes and related studies

Abstract

The effect of redox-inactive cationic and anionic paramagnetic chromium(III) complexes on the ¹H NMR spectrum of the reduced type 1 blue copper protein amicyanin, AmCu^I, from Thiobacillus versutus has been studied as a means of defining sites for association at the protein surface. With [Cr(CN)₆]^3– two sites are detected, one at the adjacent hydrophobic patch close to the exposed imidazole of the co-ordinated His-96, and the other at Phe-92 which has Lys-59, Lys-60, Arg-69 and Arg-100 in close proximity and is adjacent to the active site-co-ordinated Cys-93. In contrast, the cationic complexes [Cr(NH₃)₆]³⁺ and [Cr(en)₃]³⁺(en = ethane-1,2-diamine) cause no significant line broadening and no preferred sites for association are detected. Kinetic stopped-flow studies on the competitive inhibition by [Cr(CN)₆]^3– of the [Fe(CN)₆]^3– oxidation of AmCu^I indicate that [Fe(CN)₆]^3– reacts at two sites, one of which is inhibited by [Cr(CN)₆]^3– and the other is unaffected by [Cr(CN)₆]^3–. It is suggested that the first of these corresponds to reaction at the Phe-92 site, contributing 25% to the reaction, and the second to reaction at His-96. Therefore, in its reaction with [Fe(CN)₆]^3– amicyanin has adjacent and remote binding sites.