Ratan Gachhui

Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase

Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca2+/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg1400, and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme.

The Ferrous-dioxy Complex of Neuronal Nitric Oxide Synthase DIVERGENT EFFECTS OF l-ARGININE AND TETRAHYDROBIOPTERIN ON ITS STABILITY

Nitric oxide synthases (NOS) are hemeproteins that catalyze oxidation of l-arginine to nitric oxide (NO) and citrulline. The NOS heme iron is expected to participate in oxygen activation during catalysis, but its interactions with O2 are not characterized. We utilized the heme-containing oxygenase domain of neuronal NOS (nNOSoxy) and stopped-flow methods to study formation and autooxidative decomposition of the nNOSoxy oxygenated complex at 10 °C. Mixing ferrous nNOSoxy with air-saturated buffer generated a transient species with absorption maxima at 427 and ∼560 nm. This species decayed within 1 s to form ferric nNOSoxy. Its formation was first order with respect to O2, monophasic, and gave rate constants fork on = 9 × 105 m −1 s−1 andk off = 108 s−1 for anl-arginine- and tetrahydrobiopterin (H4B)-saturated nNOSoxy. Omission of l-arginine and/or H4B did not greatly effect O2 binding and dissociation rates. Decomposition of the oxygenated intermediate was independent of O2 concentration and was either biphasic or monophasic depending on sample conditions. l-Arginine stabilized the oxygenated intermediate (decay rate = 0.14 s−1), while H4B accelerated its decay by a factor of 70 irrespective of l-arginine. The spectral and kinetic properties of the intermediate identify it as the FeIIO2 complex of nNOSoxy. Destabilization of a metallo-oxy species by H4B is unprecedented and may be important regarding the role of this cofactor in NO synthesis.

Characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast Pichia pastoris. Calmodulin response is complete within the reductase domain itself.

Rat neuronal NO synthase (nNOS) is comprised of a flavin-containing reductase domain and a heme-containing oxygenase domain. Calmodulin binding to nNOS increases the rate of electron transfer from NADPH into its flavins, triggers electron transfer from flavins to the heme, activates NO synthesis, and increases reduction of artificial electron acceptors such as cytochrome c. To investigate what role the reductase domain plays in calmodulins activation of these functions, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) in the yeast Pichia pastoris that for the first time exhibits a complete calmodulin response. The reductase domain was purified by 2′,5′-ADP affinity chromatography yielding 25 mg of pure protein per liter of culture. It contained 1 FAD and 0.8 FMN per molecule. Most of the protein as isolated contained an air-stable flavin semiquinone radical that was sensitive to FeCN6 oxidation. Anaerobic titration of the FeCN6-oxidized reductase domain with NADPH indicated the flavin semiquinone re-formed after addition of 1-electron equivalent and the flavins could accept up to 3 electrons from NADPH. Calmodulin binding to the recombinant reductase protein increased its rate of NADPH-dependent flavin reduction and its rate of electron transfer to cytochrome c, FeCN6, or dichlorophenolindophenol to fully match the rate increases achieved when calmodulin bound to native full-length nNOS. Calmodulins activation of the reductase protein was associated with an increase in domain tryptophan and flavin fluorescence. We conclude that many of calmodulins actions on native nNOS can be fully accounted for through its interaction with the nNOS reductase domain itself.

Inducible nitric oxide synthase: role of the N‐terminal β‐hairpin hook and pterin‐binding segment in dimerization and tetrahydrobiopterin interaction

The oxygenase domain of the inducible nitric oxide synthase (iNOSox; residues 1–498) is a dimer that binds heme, L‐arginine and tetrahydrobiopterin (H4B) and is the site for nitric oxide synthesis. We examined an N‐terminal segment that contains a β‐hairpin hook, a zinc ligation center and part of the H4B‐binding site for its role in dimerization, catalysis, and H4B and substrate interactions. Deletion mutagenesis identified the minimum catalytic core and indicated that an intact N‐terminal β‐hairpin hook is essential. Alanine screening mutagenesis of conserved residues in the hook revealed five positions (K82, N83, D92, T93 and H95) where native properties were perturbed. Mutants fell into two classes: (i) incorrigible mutants that disrupt side‐chain hydrogen bonds and packing interactions with the iNOSox C‐terminus (N83, D92 and H95) and cause permanent defects in homodimer formation, H4B binding and activity; and (ii) reformable mutants that destabilize interactions of the residue main chain (K82 and T93) with the C‐terminus and cause similar defects that were reversible with high concentrations of H4B. Heterodimers comprised of a hook‐defective iNOSox mutant subunit and a full‐length iNOS subunit were active in almost all cases. This suggests a mechanism whereby N‐terminal hooks exchange between subunits in solution to stabilize the dimer.