Abstract
Nitric oxide (NO) synthases (NOSs) catalyze the formation of NO from l-arginine. We have shown previously that the NOS enzyme catalytic cycle involves a large number of reactions but can be characterized by a global model with three main rate-limiting steps. These are the rate of heme reduction by the flavin domain (kr ), of dissociation of NO from the ferric heme-NO complex (kd ), and of oxidation of the ferrous heme-NO complex (kox). The reaction of oxygen with the ferrous heme-NO species is part of a futile cycle that does not directly contribute to NO synthesis but allows a population of inactive enzyme molecules to return to the catalytic cycle, and thus, enables a steady-state NO synthesis rate. Previously, we have reported that this reaction does involve the reaction of oxygen with the NO-bound ferrous heme complex, but the mechanistic details of the reaction, that could proceed via either an inner-sphere or an outer-sphere mechanism, remained unclear. Here, we present additional experiments with neuronal NOS (nNOS) and inducible NOS (iNOS) variants (nNOS W409F and iNOS K82A and V346I) and computational methods to study how changes in heme access and electronics affect the reaction. Our results support an inner-sphere mechanism and indicate that the particular heme-thiolate environment of the NOS enzymes can stabilize an N-bound FeIII-N(O)OO- intermediate species and thereby catalyze this reaction, which otherwise is not observed or favorable in proteins like globins that contain a histidine-coordinated heme.
Highlights
Nitric oxide (NO) synthases (NOSs) catalyze the formation of NO from L-arginine
Our results suggest that bound H4B retards kox in iNOSoxy, whereas bound L-Arg does not, and that O2 access or reactivity remains rate-limiting under all three conditions
Our results indicate that the reactivity of the FeII-NO complex can be finely tuned by the NO synthases (NOSs) heme environment
Summary
We have shown previously that the NOS enzyme catalytic cycle involves a large number of reactions but can be characterized by a global model with three main ratelimiting steps These are the rate of heme reduction by the flavin domain (kr), of dissociation of NO from the ferric heme-NO complex (kd), and of oxidation of the ferrous heme-NO complex (kox). The kinetics of NO synthesis are complicated by the fact that not all of the NO synthesized by NOS is effectively released out of the enzyme, but instead can remain bound to the heme and be oxidized to nitrate To describe this process, we have proposed a global mechanism that includes the NO biosynthetic reactions, the NO release step, and a futile cycling of NOS through its ferrous heme-NO complex without net NO release (Fig. 1A) (6 –8). It has received somewhat less attention than the other kinetic parameters of NOS catalysis, the rate of heme reduction by the reductase domain (kr in Fig. 1A), or the rate of NO dishydro-L-biopterin; kr, reduction rate of the heme by the reductase (flavoprotein) domain of NOS; kd, dissociation rate of NO from the ferric heme-NO complex of NOS; kox, oxidation rate of the ferrous heme-NO complex of NOS; kon, association rate for the binding of O2 to the ferrous heme-NO complex; koff, dissociation rate for the ferrous heme-NO-O2 complex; nNOS, neuronal nitric-oxide synthase; NOSoxy, oxygenase domain of nitric-oxide synthase; nNOSoxy, oxygenase domain of the neuronal nitricoxide synthase; iNOS, inducible nitric-oxide synthase; iNOSoxy, oxygenase domain of the inducible nitric-oxide synthase; eNOS, endothelial nitricoxide synthase; NOD, nitric oxide dioxygenation; DFT, density functional theory; PES, potential energy surface; PDB, Protein Data Bank
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