Structural investigations of the Cu<sub>A</sub> centre of nitrous oxide reductase from Pseudomonas stutzeri by site-directed mutagenesis and X-ray absorption spectroscopy
Bibliographic record
Abstract
Nitrous oxide reductase is the terminal component of a respiratory chain that utilizes N2O in lieu of oxygen. It is a homodimer carrying in each subunit the electron transfer site, CuA, and the substrate‐reducing catalytic centre, CuZ. Spectroscopic data have provided robust evidence for CuA as a binuclear, mixed‐valence metal site. To provide further structural information on the CuA centre of N2O reductase, site directed mutagenesis and Cu K‐edge X‐ray absorption spectroscopic investigation have been undertaken. Candidate amino acids as ligands for the CuA 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 Cu1, and Cys618, Cys622 and His626 as the minimal set of ligands for Cu2 of the CuA centre. Other amino acid substitutions indicated His494 as a likely ligand of CuZ, and an indirect role for Asp580, compatible with a docking function for the electron donor. Cu binding and spectroscopic properties of recombinant N2O reductase proteins point at intersubunit or interdomain interaction of CuA and CuZ. Cu K‐edge X‐ray absorption spectra have been recorded to investigate the local environment of the Cu centres in N2O 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 CuA centre of N2O reductase is very similar to that of the CuA 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 CuA 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 CuA‐only form, histidine was identified as a major contributor to the backscattering. A structural model for the CuA centre of N2O 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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".