Two [Fe(IV)O Trp<sup>•</sup>] Intermediates in<i>M.</i><i>tuberculosis</i>Catalase-Peroxidase Discriminated by Multifrequency (9−285 GHz) EPR Spectroscopy: Reactivity toward Isoniazid
Bibliographic record
Abstract
We have characterized the intermediates formed in the peroxidase cycle of the multifunctional heme-containing enzyme KatG of M. tuberculosis. Selected Trp variants from the heme proximal (W321F) and distal (W107F and W91F) sides were analyzed together with the wild-type enzyme with regard to the reaction with peroxyacetic acid and hydrogen peroxide (in the catalase-inactive W107F). The 9 GHz EPR spectrum of the enzyme upon reaction with peroxyacetic acid showed the contribution of three protein-based radical species, two Trp* and a Tyr*, which could be discerned using a combined approach of multifrequency Electron Paramagnetic Resonance (EPR) spectroscopy with selective deuterium labeling of tryptophan and tyrosine residues and site-directed mutagenesis. Trp321, a residue in H-bonding interactions with the iron through Asp381 and the heme axial ligand His270, was identified as one of the radical sites. The 9 GHz EPR signal of the Trp321 radical species was consistent with an exchange-coupled species similar to the oxoferryl-Trp radical intermediate in cytochrome c peroxidase. On the basis of the possibility of distinguishing among the different radical intermediates of the peroxidase cycle in M. tuberculosis KatG (MtKatG), we used EPR spectroscopy to monitor the reactivity of the enzyme and its W321F variant with isoniazid, the front-line drug used in the treatment of tuberculosis. The EPR experiments on the W321F variant preincubated with isoniazid allowed us to detect the short-lived [Fe(IV)=O Por*+] intermediate. Our results showed that neither the [Fe(IV)=O Por*+] nor the [Fe(IV)=O Trp321*+] intermediates were the reactive species with isoniazid. Accordingly, the subsequent intermediate (most probably the other Trp*) is proposed to be the oxidizing species. Our findings demonstrate that the protein-based radicals formed as alternative intermediates to the [Fe(IV)=O Por*+] can play the role of cofactors for substrate oxidation in the peroxidase cyle of KatGs.
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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".