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, Reinhard Lange

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)3303-3309
Number of pages7
JournalBiophysical Journal
Volume85
Issue number5
DOIs
Publication statusPublished - 1 Nov 2003
Externally publishedYes

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