Reactions of spinach nitrite reductase with its substrate, nitrite, and a putative intermediate, hydroxylamine.
Plant nitrite reductase (NiR) catalyzes the reduction of nitrite (NO(2)(-)) to ammonia, using reduced ferredoxin as the electron donor. NiR contains a [4Fe-4S] cluster and an Fe-siroheme, which is the nitrite binding site. In the enzyme's as-isolated form ([4Fe-4S](2+)/Fe(3+)), resonance Raman spectroscopy indicated that the siroheme is in the high-spin ferric hexacoordinated state with a weak sixth axial ligand. Kinetic and spectroscopic experiments showed that the reaction of NiR with NO(2)(-) results in an unexpectedly EPR-silent complex formed in a single step with a rate constant of 0.45 +/- 0.01 s(-)(1). This binding rate is slow compared to that expected from the NiR turnover rates reported in the literature, suggesting that binding of NO(2)(-) to the as-isolated form of NiR is not the predominant type of substrate binding during enzyme turnover. Resonance Raman spectroscopic characterization of this complex indicated that (i) the siroheme iron is low-spin hexacoordinated ferric, (ii) the ligand coordination is unusually heterogeneous, and (iii) the ligand is not nitric oxide, most likely NO(2)(-). The reaction of oxidized NiR with hydroxylamine (NH(2)OH), a putative intermediate, results in a ferrous siroheme-NO complex that is spectroscopically identical to the one observed during NiR turnover. Resonance Raman and absorption spectroscopy data show that the reaction of oxidized NiR ([4Fe-4S](2+)/Fe(3+)) with hydroxylamine is binding-limited, while the NH(2)OH conversion to nitric oxide is much faster.
Kuznetsova
Sofya
S
Service de Bioénergétique and CNRS URA 2096, Département de Biologie Joliot Curie, CEA Saclay, 91191 Gif-sur-Yvette Cedex, France.
Knaff
David B
DB
Hirasawa
Masakazu
M
Sétif
Pierre
P
Mattioli
Tony A
TA
D019811:Hydroxylamine / Q000737:chemistry* / Q000378:metabolism; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D009572:Nitrite Reductases / Q000737:chemistry* / Q000378:metabolism; D009573:Nitrites / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D013057:Spectrum Analysis; D013059:Spectrum Analysis, Raman; D018724:Spinacia oleracea / Q000201:enzymology
D008024:Ligands; D009573:Nitrites; D019811:Hydroxylamine; D007501:Iron; D009572:Nitrite Reductases