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Short-lived intermediate in N₂O generation by P450 NO reductase captured by time-resolved IR spectroscopy and XFEL crystallography.

ABSTRACT: Nitric oxide (NO) reductase from the fungus Fusarium oxysporum is a P450-type enzyme (P450nor) that catalyzes the reduction of NO to nitrous oxide (N₂O) in the global nitrogen cycle. In this enzymatic reaction, the heme-bound NO is activated by the direct hydride transfer from NADH to generate a short-lived intermediate ( I ), a key state to promote N-N bond formation and N-O bond cleavage. This study applied time-resolved (TR) techniques in conjunction with photolabile-caged NO to gain direct experimental results for the characterization of the coordination and electronic structures of I TR freeze-trap crystallography using an X-ray free electron laser (XFEL) reveals highly bent Fe-NO coordination in I , with an elongated Fe-NO bond length (Fe-NO = 1.91 Å, Fe-N-O = 138°) in the absence of NAD⁺ TR-infrared (IR) spectroscopy detects the formation of I with an N-O stretching frequency of 1,290 cm^-1 upon hydride transfer from NADH to the Fe³⁺-NO enzyme via the dissociation of NAD⁺ from a transient state, with an N-O stretching of 1,330 cm^-1 and a lifetime of ca. 16 ms. Quantum mechanics/molecular mechanics calculations, based on these crystallographic and IR spectroscopic results, demonstrate that the electronic structure of I is characterized by a singly protonated Fe³⁺-NHO^•- radical. The current findings provide conclusive evidence for the N₂O generation mechanism via a radical-radical coupling of the heme nitroxyl complex with the second NO molecule.

SUBMITTER: Nomura T

PROVIDER: S-EPMC8166195 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate.

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XFEL serial crystallography reveals the room temperature structure of methyl-coenzyme M reductase.

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