Rapid kinetic studies of electron transfer in the three isoforms of nitric oxide synthase.
The nitric oxide synthases (NOSs) consist of a flavin-containing reductase domain, linked to a heme-containing oxygenase domain, by a calmodulin (CaM) binding sequence. The flavin-containing reductase domains of the NOS isoforms possess close sequence homology to NADPH-cytochrome P450 reductase (CPR). Additionally, the oxygenase domains catalyze monooxygenation of L-arginine through a cytochrome P450-like cysteine thiolate-liganded heme bound in the active site. With these considerations in mind, we conducted studies in an attempt to gain insight into the intermediates involved in flavoprotein-to-heme electron transfer in the NOSs. Static, steady-state, and stopped-flow kinetic studies indicated that nNOS must be reduced to a more than one-electron-reduced intermediate before efficient electron transfer can occur. Therefore, the possibility exists that the oxygenase domains of the NOS isoforms may receive their electrons from the reductase domains by a mechanism resembling the CPR-P450 interaction. Furthermore, the rate-limiting step in electron transfer appears to be the transfer of electrons from the flavoprotein to the oxygenase domain facilitated by the binding of CaM at increased intracellular Ca(2+) concentrations. Thus, modulation of electron transfer rates appears to be regulated at the level of the flavoprotein domains of the NOS isoforms.
Miller
R T
RT
Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229-3900, USA.
Martásek
P
P
Omura
T
T
Siler Masters
B S
BS
D000818:Animals; D001921:Brain / Q000201:enzymology*; D004579:Electron Transport; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D051381:Rats; D013053:Spectrophotometry; D013997:Time Factors
D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496322:Nos1 protein, rat; C496318:Nos2 protein, rat; C496316:Nos3 protein, rat; D009251:NADPH-Ferrihemoprotein Reductase