Purification and Molecular Characterization of an [FeFe]-Hydrogenase from <i>Thermotoga hypogea</i>
Bibliographic record
Abstract
Thermotoga hypogea is a hyperthermophilic bacterium growing on carbohydrates and peptides, producing acetate, CO2 and H2 as major end products. Hydrogenase activity was detected in the cell-free extract of T. hypogea and a hydrogenase was purified to homogeneity. The purified enzyme was homotetrameric with a subunit of 65 kDa, and contained 16 atoms of Fe and 11.6 atoms of acid labile sulfur per mole subunit. The gene encoding the hydrogenase was sequenced, and four iron-sulfur clusters including the H-cluster were found to be present in the gene sequence, indicating it is an [FeFe]-hydrogenase. The enzyme was oxygen sensitive with a t1/2 of 3 min when exposed to air. It was thermostable with t1/2 values of approximately 40 and 15 min when incubated at 70°C and 85°C, respectively. The optimal temperature for the hydrogenase activity was about 85 °C. Both benzyl viologen (BV) and methyl viologen (MV) could serve as electron acceptors, and the apparent Vmax values were determined to be 1,142 and 607 μmol H2 oxidized min-1 mg-1, respectively. For H2 uptake activity, the apparent Km values for MV and BV were 0.17 and 0.24 mM, respectively. For H2 evolution activity, the apparent Km value for MV and apparent Vmax value were determined to be 1.1 mM and 192 μmol min-1 mg-1, respectively. Ferredoxin purified from T. hypogea served as electron carrier. The enzyme exhibited pH optima of 10.0 and 8.0 for the H2 uptake and evolution activities, respectively. The physiological function of the enzyme is proposed to catalyze the H2 evolution. Keywords: [FeFe]-hydrogenase, ferredoxin, iron-sulfur clusters, ferredoxin, hydrogen production, hyperthermophiles, Thermotoga hypogea.
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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) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
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".