Neuronal nitric-oxide synthase mutant (Ser-1412 --> Asp) demonstrates surprising connections between heme reduction, NO complex formation, and catalysis.
Rat neuronal NO synthase (nNOS) contains an Akt-dependent phosphorylation motif in its reductase domain. We mutated a target residue in that site (Ser-1412 to Asp) to mimic phosphorylation and then characterized the mutant using conventional and stopped-flow spectroscopies. Compared with wild-type, S1412D nNOS catalyzed faster cytochrome c and ferricyanide reduction but displayed slower steady-state NO synthesis with greater uncoupling of NADPH oxidation. Paradoxically, the mutant had faster heme reduction, faster heme-NO complex formation, and greater heme-NO complex accumulation at steady state. To understand how these behaviors related to flavin and heme reduction rates, we utilized three soybean calmodulins (CaMs) that supported a range of slower flavin and heme reduction rates in mutant and wild-type nNOS. Reductase activity and two catalytic parameters (speed and amount of heme-NO complex formation) related directly to the speed of flavin and heme reduction. In contrast, steady-state NO synthesis increased, reached a plateau, and then fell at the highest rate of heme reduction that was obtained with S1412D nNOS + CaM. Substituting with soybean CaM slowed heme reduction and increased steady-state NO synthesis by the mutant. We conclude the following. 1) The S1412D mutation speeds electron transfer out of the reductase domain. 2) Faster heme reduction speeds intrinsic NO synthesis but diminishes NO release in the steady state. 3) Heme reduction displays an optimum regarding NO release during steady state. The unique behavior of S1412D nNOS reveals the importance of heme reduction rate in controlling steady-state activity and suggests that nNOS already has a near-optimal rate of heme reduction.
Adak
S
S
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA. adaks@ccf.org
Santolini
J
J
Tikunova
S
S
Wang
Q
Q
Johnson
J D
JD
Stuehr
D J
DJ
D019943:Amino Acid Substitution; D001224:Aspartic Acid; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis; D003574:Cytochrome c Group / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D013025:Glycine max
D002147:Calmodulin; D003574:Cytochrome c Group; D011994:Recombinant Proteins; D001224:Aspartic Acid; D009569:Nitric Oxide; D006418:Heme; D012694:Serine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I