Nitric oxide binding at the mononuclear active site of reduced Pyrococcus furiosus superoxide reductase
AUTOR(ES)
Clay, Michael D.
FONTE
The National Academy of Sciences
RESUMO
Nitric oxide (NO) has been used as a substrate analog to explore the structural and electronic determinants of enzymatic superoxide reduction at the mononuclear iron active site of Pyrococcus furiosus superoxide reductase (SOR) through the use of EPR, resonance Raman, Fourier transform IR, UV-visible absorption, and variable-temperature variable-field magnetic CD spectroscopies. The NO adduct of reduced SOR is shown to have a near-axial S = 3/2 ground state with E/D = 0.06 and D = 12 ± 2 cm−1 (where D and E are the axial and rhombic zero-field splitting parameters, respectively) and the UV-visible absorption and magnetic CD spectra are dominated by an out-of-plane NO−(π*)-to-Fe3+(dπ) charge–transfer transition, polarized along the zero-field splitting axis. Resonance Raman studies indicate that the NO adduct is six-coordinate with NO ligated in a bent conformation trans to the cysteinyl S, as evidenced by the identification of ν(N–O) at 1,721 cm−1, ν(Fe–NO) at 475 cm−1, and ν(Fe–S(Cys), at 291 cm−1, via 34S and 15NO isotope shifts. The electronic and vibrational properties of the S = 3/2 {FeNO}7 unit are rationalized in terms of a limiting formulation involving a high-spin (S = 5/2) Fe3+ center antiferromagnetically coupled to a (S = 1) NO− anion, with a highly covalent Fe3+−NO− interaction. The results support a catalytic mechanism for SOR, with the first step involving oxidative addition of superoxide to form a ferric-peroxo intermediate, and indicate the important roles that the Fe spin state and the trans cysteinate ligand play in effecting superoxide reduction and peroxide release.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=153001Documentos Relacionados
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