Formation of transient oxygen complexes of cytochrome p450 BM3 and nitric oxide synthase under high pressure.
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 (ABH(4)), an analog to the natural second electron donor tetrahydrobiopterin (BH(4)), 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 ABH(4) was replaced by ABH(2), 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.
Marchal
Stéphane
S
Institut National de la Santé et de la Recherche Médicale, Montpellier, France.
Girvan
Hazel Mary
HM
Gorren
Antonius C F
AC
Mayer
Bernd
B
Munro
Andrew William
AW
Balny
Claude
C
Lange
Reinhard
R
D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D011312:Pressure; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019001:Nitric Oxide Synthase; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D010100:Oxygen