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Formation of Transient Oxygen Complexes of Cytochrome P450 BM3 and Nitric Oxide Synthase under High Pressure

Stéphane Marchal ^*, Hazel Mary Girvan , Antonius C. F. Gorren , Bernd Mayer , Andrew William Munro , Claude Balny ^* and Reinhard Lange ^*

^* Institut National de la Santé et de la Recherche Médicale, Montpellier, France; Department of Biochemistry, University of Leicester, Leicester, UK; and Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität, Graz, Austria

Correspondence: Address reprint requests to Reinhard Lange, INSERM U128, 1919 route de Mende, campus du CNRS, F-34293 Montpellier Cedex, France. Tel.: 33-46-761-3365; Fax: 33-46-752-3681; E-mail: lange{at}montp.inserm.fr.

The kinetics of formation and transformation of oxygen complexes of two heme-thiolate proteins (the F393H mutant of cytochrome P450 BM3 and the oxygenase domain of endothelial nitric oxide synthase, eNOS) were studied under high pressure. For BM3, oxygen-binding characteristics (rate and activation volume) matched those measured for CO-binding. In contrast, pressure revealed a different CO- and oxygen-binding mechanism for eNOS, suggesting that it is hazardous to take CO-binding as a model for oxygen-binding. With eNOS, a ferric NO complex is formed as an intermediate in the second reaction cycle. Here we report the pressure stability of this compound. Furthermore, in the presence of 4-amino-tetrahydrobiopterin (ABH₄), an analog to the natural second electron donor tetrahydrobiopterin (BH₄), biphasic pressure profiles of the oxygen-binding rates were observed, both in the first and the second reaction cycles, indicative of the formation of an additional reaction intermediate. This was confirmed by experiments where ABH₄ was replaced by ABH₂, a cofactor which cannot deliver an electron. Altogether, high pressure appears to be a useful tool to characterize elementary steps in the reaction cycle of heme-thiolate proteins.

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