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Reactions of Spinach Nitrite Reductase with Its Substrate, Nitrite, and a Putative Intermediate, Hydroxylamine

2004; American Chemical Society; Volume: 43; Issue: 33 Linguagem: Inglês

10.1021/bi048826r

1943-295X

С. А. Кузнецова, David B. Knaff, Masakazu Hirasawa, Pièrre Sétif, Tony A. Mattioli,

Enzyme Structure and Function

Plant nitrite reductase (NiR) catalyzes the reduction of nitrite (NO2-) 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+/Fe3+), 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 NO2- 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 NO2- 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 NO2-. The reaction of oxidized NiR with hydroxylamine (NH2OH), 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+/Fe3+) with hydroxylamine is binding-limited, while the NH2OH conversion to nitric oxide is much faster.