Role of Asp1393 in catalysis, flavin reduction, NADP(H) binding, FAD thermodynamics, and regulation of the nNOS flavoprotein.
Nitric oxide synthases (NOS) are flavoheme enzymes with important roles in biology. The reductase domain of neuronal NOS (nNOSr) contains a widely conserved acidic residue (Asp(1393)) that is thought to facilitate hydride transfer between NADPH and FAD. Previously we found that the D1393V and D1393N mutations lowered the NO synthesis activity and the rates of heme and flavin reduction in full-length nNOS. To examine the mechanisms for these results in greater detail, we incorporated D1393V and D1393N substitutions into nNOSr along with a truncated NADPH-FAD domain construct (FNR) and characterized the mutants. D1393V nNOSr had markedly lower (<or=1000x) cytochrome c reductase, ferricyanide reductase, and NADPH oxidase activities than the wild type. D1393N nNOSr also had lower reductase activities (<or=10x) but had greater NADPH oxidase activity than that of the wild type, as did its FNR fragment. Both mutants had an altered interaction between FAD and the nicotinamide ring of NADP(+), slower flavin reduction by NADPH, altered FAD midpoint potentials, a normal CaM response, and, in one case (D1393N), faster flavin oxidation by O(2) and a lack of FMN shielding in response to NADPH binding. The results suggest that the two mutants have compromised catalysis for two different reasons. In D1393V nNOSr, hydride transfer from NADPH to FAD is so slow that it compromises all downstream electron-transfer events. In D1393N nNOSr, the increased oxidation of reduced flavins by O(2) and thermodynamic destabilization of the FAD semiquinone uncouples or limits electron transfer to an extent that it inhibits downstream catalysis. These effects are due in part to the mutations eliminating (D1393V) or altering (D1393N) the native side-chain hydrogen-bonding properties of Asp(1393) as well as removing its negative charge.
Konas
David W
DW
Department of Pathobiology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Takaya
Naoki
N
Sharma
Manisha
M
Stuehr
Dennis J
DJ
D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004579:Electron Transport; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D017931:DNA Primers; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D001224:Aspartic Acid; D009249:NADP; D052248:Nitric Oxide Synthase Type I