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

Proc Natl Acad Sci U S A. 2021 May 25;118(21):e2101481118. doi: 10.1073/pnas.2101481118.

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.

Keywords: NOR; XFEL-based crystallography; in crystallo microspectroscopy; nitric oxide; time-resolved IR spectroscopy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Crystallography, X-Ray / methods
  • Cytochrome P-450 Enzyme System / chemistry*
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism
  • Electrons
  • Fungal Proteins / chemistry*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Fusarium / chemistry*
  • Fusarium / enzymology
  • Fusarium / genetics
  • Gene Expression
  • Heme / chemistry
  • Heme / metabolism
  • Iron / chemistry
  • Iron / metabolism
  • NAD / chemistry
  • NAD / metabolism
  • Nitric Oxide / chemistry*
  • Nitric Oxide / metabolism
  • Nitrogen Oxides / chemistry
  • Nitrogen Oxides / metabolism
  • Nitrous Oxide / chemistry*
  • Nitrous Oxide / metabolism
  • Oxidation-Reduction
  • Oxidoreductases / chemistry*
  • Oxidoreductases / genetics
  • Oxidoreductases / metabolism
  • Protons

Substances

  • Fungal Proteins
  • Nitrogen Oxides
  • Protons
  • NAD
  • Nitric Oxide
  • Heme
  • Cytochrome P-450 Enzyme System
  • Iron
  • Oxidoreductases
  • nitric-oxide reductase
  • nitroxyl
  • Nitrous Oxide

Supplementary concepts

  • Fusarium oxysporum