FMN fluorescence in inducible NOS constructs reveals a series of conformational states involved in the reductase catalytic cycle.
Nitric oxide synthases (NOSs) produce NO as a molecular signal in the nervous and cardiovascular systems and as a cytotoxin in the immune response. NO production in the constitutive isoforms is controlled by calmodulin regulation of electron transfer. In the tethered shuttle model for NOS reductase function, the FMN domain moves between NADPH dehydrogenase and oxygenase catalytic centers. Crystal structures of neuronal NOS reductase domain and homologs correspond to an 'input state', with FMN in close contact with FAD. We recently produced two domain 'output state' (oxyFMN) constructs showing calmodulin dependent FMN domain association with the oxygenase domain. FMN fluorescence is sensitive to enzyme conformation and calmodulin binding. The inducible NOS (iNOS) oxyFMN construct is more fluorescent than iNOS holoenzyme. The difference in steady state fluorescence is rationalized by the observation of a series of characteristic states in the two constructs, which we assign to FMN in different environments. OxyFMN and holoenzyme share open conformations with an average lifetime of ~4.3 ns. The majority state in holoenzyme has a short lifetime of ~90 ps, probably because of FAD-FMN interactions. In oxyFMN about 25-30% of the FMN is in a state with a lifetime of 0.9 ns, which we attribute to quenching by heme in the output state. Occupancy of the output state together with our previous kinetic results yields a heme edge to FMN distance estimate of 12-15 Å. These results indicate that FMN fluorescence is a valuable tool to study conformational states involved in the NOS reductase catalytic cycle.
Department of Medicine, Duke University, VA Medical Centers, Durham, NC, USA. dghosh036@gmail.com
Dipak K
DK
Ghosh
Krishanu
K
Ray
Albert J
AJ
Rogers
Nicholas J
NJ
Nahm
John C
JC
Salerno
D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D005486:Flavin Mononucleotide / Q000737:chemistry* / Q000378:metabolism; D008968:Molecular Conformation; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
D002147:Calmodulin; D005486:Flavin Mononucleotide; D010088:Oxidoreductases; D052247:Nitric Oxide Synthase Type II