Probing the role of the active-site Cysteine of Azurin by site-directed mutagenesis

The coordination chemistry and electron-transfer properties of a single-site mutant of the mononuclear copper electron-transfer protein azurin from Pseudomonas aeruginosa have been studied. The active-site cysteine at position 112 was replaced by an aspartate (Cys112Asp) to assess directly the importance of this ligand to the structure-function properties of azurin. Although the mutant protein retains a high-affinity copper-binding active site, the absorption and EPR spectra of Cu[superscript II]Cys112Asp azurin are quite distinct from those of the wild-type protein and indicate the presence of a normal (type 2) copper center. A Cu[superscript II/I] reduction potential of 180 mV vs. NHE (pH 7.0) was obtained through a redox titration experiment with cytochrome c[subscript 551]. The Co[superscript II] derivative of Cys112Asp azurin was prepared and found to be amenable to paramagnetic NMR spectroscopy. In conjunction with electronic absorption data, the NMR data were used to generate a computer model of the Co[superscript II] active-site structure in which the metal is coordinated by two histidines (His46 and His 117), a polypeptide backbone carbonyl oxygen (of Gly 45), and an out-of-plane, asymmetrically-bound bidentate aspartate (Asp 112) in an overall distorted square pyramidal geometry. The 2.4-Å resolution X-ray crystal structure of Cu[superscript II] Cys112Asp azurin is reported and confirms this ligand set and geometry. Ni[superscript II]-substituted Cys112Asp azurin was also made, but unlike Cu[superscript II] and Co[superscript II], the Ni[superscript II] ion is much less tightly bound by the protein. The electronic spectroscopy of Ni[superscript II] Cys112Asp azurin suggests the existence of some bonding interaction with the thioether sulfur atom of Met121. In contrast to Cys112-containing azurins, laser-induced intramolecular electron-transfer reactions from the reduced copper center of Asp 112-containing azurins to surface histidine-bound bis(bipyridy1)(imidazole) ruthenium(III) labels could not be observed. However, the kinetics of intermolecular protein-protein electron transfer between Cys112Asp and wild-type azurins were recorded by stopped-flow spectrophotometry. Analysis of the bimolecular kinetic data suggests that the Cys-to-Asp mutation has diminished significantly the electronic coupling between the copper and ruthenium centers in the intramolecular electron-transfer systems