Structural investigations of the Cu-A centre of nitrous oxide reductase from Pseudomonas stutzeri by site-directed mutagenesis and X-ray absorption spectroscopy

Nitrous oxide reductase is the terminal component of a respiratory chain th at utilizes N2O in lieu of oxygen. It is a homodimer carrying in each subun it the electron transfer site, Cu-A, and the substrate-reducing catalytic c entre, Cu-Z. Spectroscopic data have provided robust evidence for Cu-A as a binuclear, mixed-valence metal site. To provide further structural informa tion on the Cu-A centre of N2O reductase, site directed mutagenesis and Cu K-edge X-ray absorption spectroscopic investigation have been undertaken. C andidate amino acids as ligands for the Cu-A centre of the enzyme from Pseu domonas stutzeri ATCC14405 were substituted by evolutionary conserved resid ues or amino acids similar to the wild-type residues. The mutations identif ied the amino acids His583, Cys618, Cys622 and Met629 as ligands of Cu-1, a nd Cys618, Cys622 and His626 as the minimal set of ligands for Cu-2 of the Cu-A centre. Other amino acid substitutions indicated His494 as a likely li gand of Cu-Z, and an indirect role for Asp580, compatible with a docking fu nction for the electron donor. Cu binding and spectroscopic properties of r ecombinant N2O reductase proteins point at intersubunit or interdomain inte raction of Cu-A and Cu-Z. Cu K-edge X-ray absorption spectra have been reco rded to investigate the local environment of the Cu centres in N2O reductas e. Cu K-edge Extended X-ray Absorption Fine Structure (EXAFS) for binuclear Cu chemical systems show clear evidence for Cu backscattering at approxima te to 2.5 Angstrom. The Cu K-edge EXAFS of the Cu-A centre of N2O reductase is very similar to that of the Cu-A centre of cytochrome c oxidase and the optimum simulation of the experimental data involves backscattering from a histidine group with Cu-N of 1.92 Angstrom, two sulfur atoms at 2.24 Angst rom and a Cu atom at 2.43 Angstrom, and allows for the presence of a furthe r light atom (oxygen or nitrogen) at 2.05 Angstrom. The interpretation of t he Cu-A EXAFS is in line with ligands assigned by site-directed mutagenesis . By a difference spectrum approach, using the Cu K-edge EXAFS of the holoe nzyme and that of the Cu-A-only form, histidine was identified as a major c ontributor to the backscattering. A structural model for the Cu-A centre of N2O reductase has been generated on the basis of the atomic coordinates fo r the homologous domain of cytochrome c oxidase and incorporating our curre nt results and previous spectroscopic data.