Structural investigations of the Cu_A centre of nitrous oxide reductase from <i>Pseudomonas stutzeri</i> by site‐directed mutagenesis and X‐ray absorption spectroscopy

Nitrous oxide reductase is the terminal component of a respiratory chain that utilizes N 2 O in lieu of oxygen. It is a homodimer carrying in each subunit the electron transfer site, Cu A , and the substrate‐reducing catalytic centre, Cu Z . Spectroscopic data have provided robust evidence for Cu A as a binuclear, mixed‐valence metal site. To provide further structural information on the Cu A centre of N 2 O reductase, site directed mutagenesis and Cu K‐edge X‐ray absorption spectroscopic investigation have been undertaken. Candidate amino acids as ligands for the Cu A centre of the enzyme from Pseudomonas stutzeri ATCC14405 were substituted by evolutionary conserved residues or amino acids similar to the wild‐type residues. The mutations identified the amino acids His583, Cys618, Cys622 and Met629 as ligands of Cu 1 , and 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 ligand of Cu Z , and an indirect role for Asp580, compatible with a docking function for the electron donor. Cu binding and spectroscopic properties of recombinant N 2 O reductase proteins point at intersubunit or interdomain interaction of Cu A and Cu Z . Cu K‐edge X‐ray absorption spectra have been recorded to investigate the local environment of the Cu centres in N 2 O reductase. Cu K‐edge Extended X‐ray Absorption Fine Structure (EXAFS) for binuclear Cu chemical systems show clear evidence for Cu backscattering at ≈ 2.5 Å. The Cu K‐edge EXAFS of the Cu A centre of N 2 O 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 Å, two sulfur atoms at 2.24 Å and a Cu atom at 2.43 Å, and allows for the presence of a further light atom (oxygen or nitrogen) at 2.05 Å. The interpretation of the 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 holoenzyme and that of the Cu A ‐only form, histidine was identified as a major contributor to the backscattering. A structural model for the Cu A centre of N 2 O reductase has been generated on the basis of the atomic coordinates for the homologous domain of cytochrome c oxidase and incorporating our current results and previous spectroscopic data.