Role of the Surface-Exposed and Copper-Coordinating Histidine in Blue Copper Proteins:  The Electron-Transfer and Redox-Coupled Ligand Binding Properties of His117Gly Azurin

• Published in: Journal of the American Chemical Society Vol. 122; no. 49; pp. 12186 - 12194
• Main Authors: Jeuken, Lars J. C, van Vliet, Pieter, Verbeet, Martin Ph, Camba, Raul, McEvoy, James P, Armstrong, Fraser A, Canters, Gerard W
• Format: Journal Article
• Language: English
• Published: American Chemical Society 13-12-2000
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja0006144

In many reduced blue copper proteins the C-terminal surface-exposed active-site histidine protonates at low pH and dissociates from the Cu atom. In this state, the proteins exhibit high reduction potentials and low oxidation rates. In contrast, the homologous histidine (117) of azurin does not protonate. This difference has been examined by studying the electrochemical behavior of an azurin mutant in which histidine 117 is replaced by glycine to create a cavity enabling external ligands to enter the protein and coordinate to the Cu. We show that the external ligands influence the electrochemical properties of the copper site, as studied with potentiometric titrations of protein solutions and with fast-scan and low-temperature cyclic voltammetry of protein films adsorbed on graphite electrodes. The reduction potential (E 0‘) of His117Gly azurin without external ligands is very high, at 670 ± 10 mV, but it decreases upon addition of Cl- or imidazole. The reduced form has little affinity for these ligands; however, under fast-scan or cryoscopic conditions (−70 °C, 70% methanol) the reduced form of the imidazole complex can be “trapped”, and a reversible redox couple is established. The electrochemical kinetics of the trapped state are very fast and similar to those of wild-type (wt) azurin. The reduction potential is ∼60 mV lower than for wt azurin under identical conditions. The dissociation constant K diss of the Cu(I)−imidazole complex lies between 14 and 69 M at 20 °C, while that of the Cu(I)−Cl- complex is estimated to be as high as 106 M. These very low affinities show that for wt azurin the covalent link between the imidazole side chain of His117 and the protein framework is crucial for maintaining this side chain as a ligand of the Cu(I) ion.