Interaction of Nitric Oxide with Cytochrome P450 BM3

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• 作者：Luca G. Quaroni ; Harriet E. Seward ; Kirsty J. McLean ; Hazel M. Girvan ; Tobias W. B. Ost ; Michael A. Noble ; Sharon M. Kelly ; Nicholas C. Price ; Myles R. Cheesman ; W. Ewen Smith ; and Andrew W. Munro
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：December 28, 2004
• 年：2004
• 卷：43
• 期：51
• 页码：16416 - 16431
• 全文大小：177K
• 年卷期：v.43,no.51(December 28, 2004)
• ISSN：1520-4995

文摘

The interaction of nitric oxide with cytochrome P450 BM3 from Bacillus megaterium hasbeen analyzed by spectroscopic techniques and enzyme assays. Nitric oxide ligates tightly to the ferricheme iron, inducing large changes in each of the main visible bands of the heme and inhibiting the fattyacid hydroxylase function of the protein. However, the ferrous adduct is unstable under aerobic conditions,and activity recovers rapidly after addition of NADPH to the flavocytochrome due to reduction of theheme via the reductase domain and displacement of the ligand. The visible spectral properties revert tothat of the oxidized resting form. Aerobic reduction of the nitrosyl complex of the BM3 holoenzyme orheme domain by sodium dithionite also displaces the ligand. A single electron reduction destabilizes theferric-nitrosyl complex such that nitric oxide is released directly, as shown by the trapping of releasednitric oxide. Aerobically and in the absence of exogenous reductant, nitric oxide dissociates completelyfrom the P450 over periods of several minutes. However, recovery of the nativelike visible spectrum isaccompanied by alterations in the catalytic activity of the enzyme and changes in the resonance Ramanspectrum. Specifically, resonance Raman spectroscopy identifies the presence of internally located nitratedtyrosine residue(s) following treatment with nitric oxide. Analysis of a Y51F mutant indicates that this isthe major nitration target under these conditions. While wild-type P450 BM3 does not form an aerobicallystable ferrous-nitrosyl complex, a site-directed mutant of P450 BM3 (F393H) does form an isolatableferrous-nitrosyl complex, providing strong evidence for the role of this residue in controlling the electronicproperties of the heme iron. We report here the spectroscopic characterization of the ferric- and ferrous-nitrosyl complexes of P450 BM3 and describe the use of resonance Raman spectroscopy to identify nitratedtyrosine residue(s) in the enzyme. Nitration of tyrosine in P450 BM3 may exemplify a typical mechanismby which the ubiquitous messenger molecule nitric oxide exerts a regulatory function over the cytochromesP450.