Structure of Nitric Oxide Synthase Oxygenase Dimer with Pterin and Substrate

Abstract

Crystal structures of the murine cytokine-inducible nitric oxide synthase oxygenase dimer with active-center water molecules, the substrate L-arginine (L-Arg), or product analog thiocitrulline reveal how dimerization, cofactor tetrahydrobiopterin, and L-Arg binding complete the catalytic center for synthesis of the essential biological signal and cytotoxin nitric oxide. Pterin binding refolds the central interface region, recruits new structural elements, creates a 30 angstrom deep active-center channel, and causes a 35 degrees helical tilt to expose a heme edge and the adjacent residue tryptophan-366 for likely reductase domain interactions and caveolin inhibition. Heme propionate interactions with pterin and L-Arg suggest that pterin has electronic influences on heme-bound oxygen. L-Arginine binds to glutamic acid-371 and stacks with heme in an otherwise hydrophobic pocket to aid activation of heme-bound oxygen by direct proton donation and thereby differentiate the two chemical steps of nitric oxide synthesis.

1,5-Disubstituted indole derivatives as selective human neuronal nitric oxide synthase inhibitors

“…5 Investigation into the synthesis and chemistry of novel isoform-selective NOS inhibitors has been an ongoing challenge, even though the general pharmacophore requirements are well established. [6][7][8][9][10][11][12] Synthesis of substrate (L-arginine) based peptidomimetic non-selective as well as selective nNOS inhibitors have been extensively reported in the literature. 13 In an effort to improve PK/PD properties by decreasing their peptidic nature, various small molecule selective nNOS inhibitors have also been reported.…”

Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide

“…Consistent with this was the observation that radioligand binding with [$H]H % Bip and [$H]NO # Arg revealed a dual allosteric interaction between the H % Bip-binding site and -arginine-binding site of NOS-I [35], whereas for NOS-II this has still remained controversial [37,38]. However, at the crystal level H % Bip was not found to affect substrate binding [34], in direct disagreement with an earlier report [16]. Moreover, in enzyme kinetic studies, H % Bip did not alter the IC &!…”

“…In a radioligand binding study, Mayer and co-workers demonstrated a 6-fold increase in the affinity of NOS-I for [$H]H % Bip in the presence of -arginine [35]. For NOS-II, both positive [37] and negative [38] [34] as had previously been demonstrated [16]. Consequently, it remains to be established whether this binding-site co-operativity is a general phenomenon for all NOS isoforms and of relevance during catalysis.…”

“…as an oxygen acceptor and electron donor catalysing the initial N-hydroxylation of -arginine. However, the current consensus in the literature does not support such a classical catalytic role [13][14][15][16][17], although some studies can be found to the contrary [18]. Alternatively, protective and stabilizing effects of H % Bip have been described, including (1) the stabilization of the NOS mRNA product [19], (2) protection of the NOS catalytic centre from oxidative damage [16,20], (3) prevention of the formation of superoxide (O # − d) [21] and H # O # [22] by coupling reductive oxygen activation to -arginine oxidation, and (4) promotion of subunit assembly from monomers and stabilization of dimers in the presence of chemical denaturants [23,24] or under native conditions during -arginine turnover [17,25].…”

“…Interestingly, the stabilizing effects of H % Bip might be isoform-dependent. For NOS-I, H % Bip is required for dimer stabilization [17,24,25] but not for subunit assembly [26,27] ; for NOS-II and III, the current evidence that supports a role for H % Bip in subunit assembly is inconsistent [23,[28][29][30][31][32][33] even when the enzymes have been solved at the crystal level [16,34].…”