Insights into blue copper proteins using spectroscopic methods

Abstract

By controlling the structure of the binding site, a protein can profoundly influence the properties of a metal center including its chemical potential, its kinetic reactivity and its binding constant.For example, it is evident from small molecule studies that electron transfer is much more facile in copper systems when minimal structural reorganization is required. To fill in some of the gaps in our knowledge about structural aspects of blue copper proteins, as summarized below, we have carried out a variety of physical studies on the proteins themselves as well as appropriate derivatives and relevant small molecules. The nickel(II) derivative of azurin, Ni(II)Az, has been prepared and shown to contain two unpaired electrons which virtually rules out the possibility of a planar nickel site. This is despite the pronounced tendency of nickel(II) to prefer planar binding of an N 2S 2 donor set involving thiolate sulfurs [1]. The paramagnetism and the fact that d-d bands are found in the near infrared argue for a pseudotetrahedral binding geometry and suggest that the metal binding site of azurin is rather rigidly defined by the peptide moiety. Further structural information has been obtained from NMR studies. Due to the rapid electron spin relaxation associated with tetrahedral nickel(II), we have been able to observed isotropically shifted resonances in the 1H NMR spectrum which can be attributed to protons of the ligand moieties [2]. These resonances shift discontinuously with pH in parallel with the C2 hydrogen of histidine-35, providing direct evidence that this protonation is coupled to a conformational change. 113Cd NMR studies indicate there is little difference between the binding sites of stellacyanin and azurin [3] even though stellacyanin lacks methionine which is one of the copper ligands of azurin. The NMR results are consistent with the suggestion that a closely related donor type, probably cystine sulfur, replaces methionine sulfur in the case of stellacyanin [4]. Finally metal replacements studies have also proved useful in studied of tree laccase. By substituting mercury(II) for copper(II) in the type 1 site we have for the first time been able to isolate the EPR spectrum of type 2 copper, allowing observation of ligand hyperfine splitting in the perpendicular region [5].