Isotope effects and intermediates in the reduction of NO by P450NOR

Daiber, A.; Nauser, T.; Takaya, N.; Kudo, T.; Weber, P.; Hultschig, C.; Shoun, H.; Ullrich, V.

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Isotope effects and intermediates in the reduction of NO by P450NOR

Daiber, A., Nauser, T., Takaya, N., Kudo, T., Weber, P., Hultschig, C., et al. (2001). Isotope effects and intermediates in the reduction of NO by P450NOR. Journal of Inorganic Biochemistry, 88(3 - 4), 343-352.

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Item Permalink: https://hdl.handle.net/11858/00-001M-0000-0010-8CD5-D Version Permalink: https://hdl.handle.net/11858/00-001M-0000-0010-8CD6-B

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Daiber, A., Author
Nauser, T., Author
Takaya, N., Author
Kudo, T., Author
Weber, P., Author
Hultschig, C.¹, Author
Shoun, H., Author
Ullrich, V., Author

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1Max Planck Society, ou_persistent13

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Free keywords: Nitric oxide; P450 enzymes; Hydride transfer; Fusarium oxysporum; Heme-thiolate catalysis

Abstract: The mechanism of the heme-thiolate-dependent NADH-NO reductase (P450NOR) from Fusarium oxysporum was investigated by kinetic isotope effects including protio, [4S-2H]-, [4R-2H]-, [4,42H2]-NADH and stopped-flow measurements. The respective kinetic isotope effects were measured at high NO concentrations and were found to be 1.7, 2.3 and 3.8 indicating a rate-limitation at the reduction step and a moderate stereoselectivity in binding of the cofactor NADH. In a different approach the kinetic isotope effects were determined directly for the reaction of the FeIII–NO complex with [4R-2H]- and [4S-2H]-NADH by stopped-flow spectroscopy. The resulting isotope effects were 2.7±0.4 for the R-form and 1.1±0.1 for the S-form. In addition the 444 nm intermediate could be chemically generated by addition of an ethanolic borohydride solution to the ferric–NO complex at -10°C. In pulse radiolysis experiments a similar absorbing species could be observed when hydroxylamine radicals were generated in the presence of Fe (III) P450NOR. Based on these results we postulate hydride transfer from NADH to the ferric P450–NO complex resulting in a ferric hydroxylamine-radical or ferryl hydroxylamine-complex and this step, as indicated by the kinetic isotope effects, to be rate-limiting at high concentrations of NO. However, at low concentrations of NO the decay of the 444 nm species becomes the rate-limiting step as envisaged by stopped-flow and optical kinetic measurements in a system in which NO was continuously generated. The last step in the catalytic cycle may proceed by a direct addition of the NO radical to the Fe–hydroxylamine complex or by electron transfer from the NO radical to the ferric–thiyl moiety in analogy to the postulated mechanisms of prostacyclin and thromboxane biosynthesis by the corresponding P450 enzymes. The latter process of electron transfer could then constitute a common step in all heme-thiolate catalyzed reactions.

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Language(s): eng - English

Dates: Date issued: 2001-11-20

Publication Status: Issued

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Identifiers: eDoc: 27109

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Title: Journal of Inorganic Biochemistry

Source Genre: Journal

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Pages: - Volume / Issue: 88 (3 - 4) Sequence Number: - Start / End Page: 343 - 352 Identifier: -