NO reduction by nitric-oxide reductase from denitrifying bacterium Pseudomonas aeruginosa: Characterization of reaction intermediates that appear in the single turnover cycle

Hideyuki Kumita, Koji Matsuura, Tomoya Hino, Satoshi Takahashi, Hiroshi Hori, Yoshihiro Fukumori, Isao Morishima, Yoshitsugu Shiro

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82 Citations (Scopus)

Abstract

Nitric-oxide reductase (NOR) of a denitrifying bacterium catalyzes NO reduction to N2O at the binuclear catalytic center consisting of high spin heme b3 and non-heme FeB. The structures of the reaction intermediates in the single turnover of the NO reduction by NOR from Pseudomonas aeruginosa were investigated using optical absorption and EPR spectroscopies combined with an originally designed freeze-quench device. In the EPR spectrum of the sample, in which the fully reduced NOR was mixed with an NO solution and quenched at 0.5 ms after the mixing, two characteristic signals for the ferrous FeB-NO and the penta-coordinated ferrous heme b 3-NO species were observed. The CO inhibition of its formation indicated that two NO molecules were simultaneously distributed into the two irons of the same binuclear center of the enzyme in this state. The time- and temperature-dependent EPR spectral changes indicated that the species that appeared at 0.5 ms is a transient reaction intermediate prior to the N 2O formation, in good agreement with the so-called "irons" mechanism. It was also found that the final state of the enzyme in the single turnover cycle is the fully oxidized state, in which the μ-oxo-bridged ligand is absent between the two irons of its binuclear center, unlike the resting form of NOR as isolated. On the basis of these present findings, we propose a newly developed mechanism for the NO reduction reaction conducted by NOR.

Original languageEnglish
Pages (from-to)55247-55254
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number53
DOIs
Publication statusPublished - 2004 Dec 31

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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