The Ferric-Superoxo Intermediate of the TxtE Nitration Pathway Resists Reduction, Facilitating Its Reaction with Nitric Oxide
| Autor: | Jonathan D. Caranto, Maria F Martinez, Christopher P. Martin, Yousong Ding, Manyun Chen |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Iron
Hydroxylation Nitric Oxide Photochemistry Ferric Compounds Biochemistry Article Nitric oxide Ferrous Electron Transport Electron transfer chemistry.chemical_compound Cytochrome P-450 Enzyme System Nitration medicine Nitrates biology Autoxidation Chemistry Tryptophan Cytochrome P450 Nitro Compounds Kinetics Biocatalysis biology.protein Ferric Spectrophotometry Ultraviolet Oxidation-Reduction medicine.drug |
| Zdroj: | Biochemistry |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/acs.biochem.1c00416 |
| Popis: | TxtE is a cytochrome P450 (CYP) homologue that mediates the nitric oxide (NO)-dependent direct nitration of l-tryptophan (Trp) to form 4-nitro-l-tryptophan (4-NO(2)-Trp). A recent report showed evidence that TxtE activity requires NO to react with a ferric-superoxo intermediate. Given this minimal mechanism, it is not clear how TxtE avoids Trp hydroxylation, a mechanism that also traverses the ferric-superoxo intermediate. To provide insight into canonical CYP intermediates that TxtE can access, electron coupling efficiencies to form 4-NO(2)-Trp under single-or limited-turnover conditions were measured and compared to steady-state efficiencies. As previously reported, Trp nitration by TxtE is supported by the engineered self-sufficient variant, TB14, as well as by reduced putidaredoxin. Ferrous (Fe(II)) TxtE exhibits excellent electron coupling (70%), which is 50-fold higher than that observed under turnover conditions. In addition, two- or four-electron reduced TB14 exhibits electron coupling (∼6%) that is 2-fold higher than that of one-electron reduced TB14 (3%). The combined results suggest (1) autoxidation is the sole TxtE uncoupling pathway and (2) the TxtE ferric-superoxo intermediate cannot be reduced by these electron transfer partners. The latter conclusion is further supported by ultraviolet−visible absorption spectral time courses showing neither spectral nor kinetic evidence for reduction of the ferric-superoxo intermediate. We conclude that resistance of the ferric-superoxo intermediate to reduction is a key feature of TxtE that increases the lifetime of the intermediate and enables its reaction with NO and efficient nitration activity. |
| Databáze: | OpenAIRE |
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