Electron Transfer by Neuronal Nitric-oxide Synthase Is Regulated by Concerted Interaction of Calmodulin and Two Intrinsic Regulatory Elements

Abstract

The nitric-oxide synthases (NOSs) are modular, cofactorcontaining enzymes, divided into a heme-containing oxygenase domain and an FMN-and FAD-containing reductase domain. The domains are connected by a calmodulin (CaM)-binding sequence, occupancy of which is required for nitric oxide (NO) production. Two additional CaM-modulated regulatory elements are present in the reductase domains of the constitutive isoforms, the autoregulatory region (AR) and the C-terminal tail region. Deletion of the AR reduces CaM stimulation of electron flow through the reductase domain from 10-fold in wild-type nNOS to 2-fold in the mutant. Deletion of the C terminus yields an enzyme with greatly enhanced reductase activity in the absence of CaM but with activity equivalent to that of wild-type enzyme in its presence. A mutant in which both the AR and C terminus were deleted completely loses CaM modulation through the reductase domain. Thus, transduction of the CaM effect through the reductase domain of nNOS is dependent on these elements. Formation of nitric oxide is, however, still stimulated by CaM in all three mutants. A CaM molecule in which the N-terminal lobe was replaced by the C-terminal lobe (CaM-CC) supported NO synthesis by the deletion mutants but not by wild-type nNOS. We propose a model in which the AR, the C-terminal tail, and CaM interact directly to regulate the conformational state of the reductase domain of nNOS.The nitric-oxide synthases (NOSs) 2 comprise a family of enzymes that catalyze the formation of nitric oxide and L-citrulline from L-arginine through a series of monooxygenation steps using NADPH as the electron donor. There are three different isoforms encoded by different genes, neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). Nitric oxide has been implicated in hemodynamic control, neurotransmission, and the immune response, depending on the cell type in which it is being expressed (for review see Refs. 1-8).All three NOS isoforms are modular, cofactor-containing enzymes. They can be roughly divided into an oxygenase domain, containing heme, tetrahydrobiopterin, and the arginine-binding site, and a reductase domain, containing the flavins FMN and FAD, as well as the NADPH-binding site. These two domains are connected by a calmodulin (CaM)-binding site, which is always occupied under physiological conditions by CaM in iNOS, whereas CaM binding to nNOS and eNOS requires an increase in intracellular calcium.CaM controls NOS activity by regulating the rate of electron flux, by enhancing electron flow through the flavin domain as well as switching on transfer of electrons from the flavin to the heme domain. The flow of electrons is in the form of a hydride ion from NADPH to FAD, from FAD to FMN, and as an electron from FMN to the final acceptor, i.e. the heme domain of the adjacent monomer in the NOS dimer (9 -11) or exogenously added cytochrome c. Recent models (12, 13) propose that bound NADPH locks the enzyme into a state in which the FMN domain is closely associated with the FAD and NADPH bindin...

Architecture of the Nitric-oxide Synthase Holoenzyme Reveals Large Conformational Changes and a Calmodulin-driven Release of the FMN Domain

“…Finally, although the core mechanism of electron transfer is highly conserved, the NO synthesis rates vary significantly among the isoforms and have been attributed to differences in sequence elements between the domains (45,46). In particular, the 40-residue autoinhibitory loop found in nNOS and eNOS affects the Ca 2ϩ response in CaM binding, whereas the C-terminal tail segment has been shown to regulate multiple steps in electron transfer (47).…”

Catalytic Reduction of a Tetrahydrobiopterin Radical within Nitric-oxide Synthase

“…Regarding NOS flavin-to-heme electron transfer, a variety of data (35)(36)(37)(38)(39)(40) suggest the FMN module undergoes a relatively large-scale movement to contact-shuttle between the NADPH-FAD module (to receive electrons) and the oxygenase domain (to donate electrons) during catalysis (Fig. 7).…”

A Bridging Interaction Allows Calmodulin to Activate NO Synthase through a Bi-modal Mechanism

“…The CT, AI, and charge pairing interactions all stabilize the FMN-shielded state of equilibrium A in the CaM-free NOS. CaM binding changes equilibrium A by disabling the influence of the CT and perhaps also the AI control element (27), but the molecular details of the various actions of CaM remain a matter of conjecture (10,25).…”