title: Use of high pressure to study elementary steps in P450 and nitric oxide synthase.
abstract: Chemical reactions are often highly pressure-dependent. A perturbation of the elementary steps by pressure therefore offers the possibility of a detailed characterization of enzyme mechanisms. We used this method to study distinct steps in the reaction of nitric-oxide synthase (NOS), and compared them to analogous steps in the reaction of cytochrome P450 BM3 (BM3). Our results indicate that, in BM3, electron transfer depends on electrostatic interactions. In NOS, pressure, similarly to chemical denaturants, can mimic the structural effects of Ca/calmodulin. This helps to better understand the structural basis of the regulatory effect of Ca/calmodulin. Furthermore, stopped-flow kinetics under high pressure show that CO binding to the heme iron is hindered by substrate in NOS, but not in BM3. This indicates a relatively large or flexible substrate binding site in BM3, and a more narrow and rigid binding site in NOS.
authors.affiliation: INSERM U128, IFR24, 1919 Route de Mende, 34293, Montpellier, France. lange@crbm.cnrs-mop.fr
authors.forename: R
authors.initials: R
authors.lastname: Lange
authors.forename: N
authors.initials: N
authors.lastname: Bec
authors.forename: P
authors.initials: P
authors.lastname: Anzenbacher
authors.forename: A W
authors.initials: AW
authors.lastname: Munro
authors.forename: A C
authors.initials: AC
authors.lastname: Gorren
authors.forename: B
authors.initials: B
authors.lastname: Mayer
mesh_terms: D001426:Bacterial Proteins; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000737:chemistry; D009251:NADPH-Ferrihemoprotein Reductase; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D010084:Oxidation-Reduction; D011312:Pressure
chemical_list: D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019001:Nitric Oxide Synthase; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases