Regulation of FMN subdomain interactions and function in neuronal nitric oxide synthase.
Nitric oxide synthases (NOS) are modular, calmodulin- (CaM-) dependent, flavoheme enzymes that catalyze oxidation of l-arginine to generate nitric oxide (NO) and citrulline. During catalysis, the FMN subdomain cycles between interaction with an NADPH-FAD subdomain to receive electrons and interaction with an oxygenase domain to deliver electrons to the NOS heme. This process can be described by a three-state, two-equilibrium model for the conformation of the FMN subdomain, in which it exists in two distinct bound states (FMN-shielded) and one common unbound state (FMN-deshielded). We studied how each partner subdomain, the FMN redox state, and CaM binding may regulate the conformational equilibria of the FMN module in rat neuronal NOS (nNOS). We utilized four nNOS protein constructs of different subdomain composition, including the isolated FMN subdomain, and determined changes in the conformational state by measuring the degree of FMN shielding by fluorescence, electron paramagnetic resonance, or stopped-flow spectroscopic techniques. Our results suggest the following: (i) The NADPH-FAD subdomain has a far greater capacity to interact with the FMN subdomain than does the oxygenase domain. (ii) CaM binding has no direct effects on the FMN subdomain. (iii) CaM destabilizes interaction of the FMN subdomain with the NADPH-FAD subdomain but does not measurably increase its interaction with the oxygenase domain. Our results imply that a different set point and CaM regulation exists for either conformational equilibrium of the FMN subdomain. This helps to explain the unique electron transfer and catalytic behaviors of nNOS, relative to other dual-flavin enzymes.
Ilagan
Robielyn P
RP
Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Tejero
Jesús
J
Aulak
Kulwant S
KS
Ray
Sougata Sinha
SS
Hemann
Craig
C
Wang
Zhi-Qiang
ZQ
Gangoda
Mahinda
M
Zweier
Jay L
JL
Stuehr
Dennis J
DJ
D000818:Animals; D001483:Base Sequence; D002853:Chromatography, Liquid; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D011199:Potentiometry; D011487:Protein Conformation; D051381:Rats
D017931:DNA Primers; D005486:Flavin Mononucleotide; D052248:Nitric Oxide Synthase Type I