Characterization of a Tungsten-Substituted Nitrogenase Isolated from <i>Rhodobacter capsulatus</i><sup>,</sup>
Bibliographic record
Abstract
In the phototrophic non-sulfur bacterium Rhodobacter capsulatus, the biosynthesis of the conventional Mo-nitrogenase is strictly Mo-regulated. Significant amounts of both dinitrogenase and dinitrogenase reductase were only formed when the growth medium was supplemented with molybdate (1 microM). During cell growth under Mo-deficient conditions, tungstate, at high concentrations (1 mM), was capable of partially (approximately 25%) substituting for molybdate in the induction of nitrogenase synthesis. On the basis of such conditions, a tungsten-substituted nitrogenase was isolated from R. capsulatus with the aid of anfA (Fe-only nitrogenase defective) mutant cells and partially purified by Q-sepharose chromatography. Metal analyses revealed the protein to contain an average of 1 W-, 16 Fe-, and less than 0.01 Mo atoms per alpha(2)beta(2)-tetramer. The tungsten-substituted (WFe) protein was inactive in reducing N(2) and marginally active in acetylene reduction, but it was found to show considerable activity with respect to the generation of H(2) from protons. The EPR spectrum of the WFe protein, recorded at 4 K, exhibited three distinct signals: (i) an S = 3/2 signal, which dominates the low-field region of the spectrum (g = 4.19, 3.93) and is indicative of a tungsten-substituted cofactor (termed FeWco), (ii) a marginal S = 3/2 signal (g = 4.29, 3.67) that can be attributed to residual amounts of FeMoco present in the protein, and (iii) a broad S = 1/2 signal (g = 2.09, 1.95, 1.86) arising from at least two paramagnetic species. Redox titrational analysis of the WFe protein revealed the midpoint potential of the FeWco (E(m) < -200 mV) to be shifted to distinctly lower potentials as compared to that of the FeMoco (E(m) approximately -50 mV) present in the native enzyme. The P clusters of both the WFe and the MoFe protein appear indistinguishable with respect to their midpoint potentials. EPR spectra recorded with the WFe protein under turnover conditions exhibited a 20% decrease in the intensity of the FeWco signal, indicating that the cofactor can be enzymatically reduced only to a small extent. The data presented in the current study demonstrate the pivotal role of molybdenum in optimal N(2) fixation and provides direct evidence that the inability of a tungsten-substituted nitrogenase to reduce N(2) is due to the difficulty to effectively reduce the FeW cofactor beyond its semi-reduced state.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| 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) | 0.002 | 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 teacher head, 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".