Mechanism of spinach chloroplast ferredoxin-dependent nitrite reductase: spectroscopic evidence for intermediate states.
Nitrite reductases found in plants, algae, and cyanobacteria catalyze the six-electron reduction of nitrite to ammonia with reduced ferredoxin serving as the electron donor. They contain one siroheme and one [4Fe-4S] cluster, acting as separate one-electron carriers. Nitrite is thought to bind to the siroheme and to remain bound until its complete reduction to ammonia. In the present work the enzyme catalytic cycle, with ferredoxin reduced by photosystem 1 as an electron donor, has been studied by EPR and laser flash absorption spectroscopy. Substrate depletion during enzyme turnover, driven by a series of laser flashes, has been demonstrated. A complex of ferrous siroheme with NO, formed by two-electron reduction of the enzyme complex with nitrite, has been shown to be an intermediate in the enzyme catalytic cycle. The same complex can be formed by incubation of free oxidized nitrite reductase with an excess of nitrite and ascorbate. Hydroxylamine, another putative intermediate in the reduction of nitrite catalyzed by nitrite reductase, was found to react with oxidized nitrite reductase to produce the same ferrous siroheme-NO complex, with a characteristic formation time of about 13 min. The rate-limiting step for this reaction is probably hydroxylamine binding to the enzyme, with the conversion of hydroxylamine to NO at the enzyme active site likely being 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
Lagoutte
Bernard
B
Sétif
Pierre
P
D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins / Q000737:chemistry* / Q000378:metabolism; D005296:Ferrous Compounds / Q000737:chemistry; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry; D019811:Hydroxylamine / Q000737:chemistry; D007700:Kinetics; D009572:Nitrite Reductases / Q000737:chemistry* / Q000378:metabolism; D009573:Nitrites / Q000737:chemistry; D010084:Oxidation-Reduction; D010782:Photolysis; D013053:Spectrophotometry; D018724:Spinacia oleracea / Q000201:enzymology; D013379:Substrate Specificity
D005288:Ferredoxins; D005296:Ferrous Compounds; D009573:Nitrites; D019811:Hydroxylamine; D006418:Heme; C009272:siroheme; D009572:Nitrite Reductases