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 m otif in its reductase domain. We mutated a target residue in that site (Ser -1412 to Asp) to mimic phosphorylation and then characterized the mutant us ing conventional and stopped-flow spectroscopies. Compared with wild-type, S1412D nNOS catalyzed faster cytochrome c and ferricyanide reduction but di splayed slower steady-state NO synthesis with greater uncoupling of NADPH o xidation, Paradoxically, the mutant had faster heme reduction, faster heme- NO complex formation, and greater heme-NO complex accumulation at steady st ate. 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, Re ductase activity and two catalytic parameters (speed and amount of heme-NO complex formation) related directly to the speed of flavin and heme reducti on. In contrast, steady-state NO synthesis increased, reached a plateau, an d then fell at the highest rate of heme reduction that was obtained with S1 412D nNOS + CaM. Substituting with soybean CaM slowed heme reduction and in creased steady-state NO synthesis by the mutant. We conclude the following. 1) The S1412D mutation speeds electron transfer out of the reductase domai n. 2) Faster heme reduction speeds intrinsic NO synthesis but diminishes NO release in the steady state. 3) Heme reduction displays an optimum regardi ng NO release during steady state. The unique behavior of S1412D nNOS revea ls the importance of heme reduction rate in controlling steady-state activi ty and suggests that nNOS already has a near-optimal rate of heme reduction .