Metal co-ordination, protein-folding, and electron-transfer studies of wild-type and mutant azurin

Abstract

Azurin is a copper-containing protein that functions as an electron carrierin certain bacteria. Like other cupredoxins its three-dimensional structureis essentially that of a b-barrel. The interaction between the oxidizedcopper ion and the metal binding site of the protein results in a strongoptical absorption around 630 nm and a special combination of EPRparameters. The copper ion in azurin is co-ordinated by three strongligands (NNS) and two more weakly interacting groups (S and O).The properties of the metal binding site in azurin have been probed byreplacing the native copper ion for non-native cobalt and nickel ions, andthe resulting structures have by X-ray crystallography been determined. Themain difference compared to copper is a displacement of cobalt and nickeltoward the oxygen ligand. This makes these metal ions four co-ordinated.Spectroscopic and structural properties of the copper ion in azurin havealso been probed by binding small molecules to the metal. These moleculesinteract with the copper ion in 121X azurin mutants, but not in wild-typeazurin. The interaction of the mutants M121X (X = Gly, Ala, Val, Leu, andAsp) with oxygen- and nitrogen-containing ligands has been analyzed by EPRand optical spectroscopy, as well as by resonance Raman spectroscopy. TheX-ray structure of Ala121 and Ala121 + azide have been determined in orderto analyze the geometrical changes upon ligand binding. One end of theexogenous ligand azide interacts with the copper ion close to where thesulfur of Met121 is positioned in the wild-type structure. This structuredemonstrates that the exogenous ligand interaction can change theco-ordination of the copper ion from three to four strong ligands.The effect of removing the disulfide bond in azurin has been investigated.The major change upon replacing the cysteine residues in the disulfide bondfor alanine residues is a decreased folding stability. It is concluded thatthe disulfide contributes at least 20 kJmol-1 to the folding energy ofazurin.A possible function of aromatic amino acids in the electron transferreaction has been investigated by introducing an additional tryptophan inazurin. The tryptophan was engineered in a proposed electron transferpathway in van der Waals contact with the native tryptophan. Evidencesuggests that several aromatic amino acids stacked against each other mayinduce a more effective electron transfer between the donor and acceptorsite.