Role of ligand substitution on long-range electron transfer in azurins

Azurin contains two potential redox sites, a copper centre and, at the oppo site end of the molecule, a cystine disulfide (RSSR). Intramolecular electr on transfer between a pulse radiolytically produced RSSR- radical anion and the blue Cu(II) ion was studied in a series of azurins in which single-sit e mutations were introduced into the copper ligand sphere. In the Met121His mutant, the rate constant for intramolecular electron transfer is half tha t of the corresponding wild-type azurin. In the His46Gly and His117Gly muta nts, a water molecule is co-ordinated to the copper ion when no external li gands are added. Both these mutants also exhibit slower intramolecular elec tron transfer than the corresponding wild-type azurin. However, for the His 117Gly mutant in the presence of excess imidazole, an azurin-imidazole comp lex is formed and the intramolecular electron-transfer rate increases consi derably, becoming threefold faster than that observed in the native protein . Activation parameters for all these electron-transfer processes were dete rmined and combined with data from earlier studies on intramolecular electr on transfer in wild-type and single-site-mutated azurins. A linear relation ship between activation enthalpy and activation entropy was observed. These results are discussed in terms of reorganization energies, driving force a nd possible electron-transfer pathways.