TY - JOUR
T1 - Rapid kinetic studies of electron transfer in the three isoforms of nitric oxide synthase
AU - Miller, R. Timothy
AU - Martásek, Pavel
AU - Omura, Tsuneo
AU - Siler Masters, Bettie Sue
PY - 1999/11/11
Y1 - 1999/11/11
N2 - 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 mono-oxygenation 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 Ca2+ concentrations. Thus, modulation of electron transfer rates appears to be regulated at the level of the flavoprotein domains of the NOS isoforms.
AB - 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 mono-oxygenation 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 Ca2+ concentrations. Thus, modulation of electron transfer rates appears to be regulated at the level of the flavoprotein domains of the NOS isoforms.
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U2 - 10.1006/bbrc.1999.1643
DO - 10.1006/bbrc.1999.1643
M3 - Article
C2 - 10548511
AN - SCOPUS:0344240118
VL - 265
SP - 184
EP - 188
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 1
ER -