Structural studies of constitutive nitric oxide synthases with diatomic ligands bound

Abstract

Crystal structures are reported for the endothelial nitric oxide synthase (eNOS)-arginine-CO ternary complex as well as the neuronal nitric oxide synthase (nNOS) heme domain complexed with L: -arginine and diatomic ligands, CO or NO, in the presence of the native cofactor, tetrahydrobiopterin, or its oxidized analogs, dihydrobiopterin and 4-aminobiopterin. The nature of the biopterin has no influence on the diatomic ligand binding. The binding geometries of diatomic ligands to nitric oxide synthase (NOS) follow the {MXY}(n) formalism developed from the inorganic diatomic-metal complexes. The structures reveal some subtle structural differences between eNOS and nNOS when CO is bound to the heme which correlate well with the differences in CO stretching frequencies observed by resonance Raman techniques. The detailed hydrogen-bonding geometries depicted in the active site of nNOS structures indicate that it is the ordered active-site water molecule rather than the substrate itself that would most likely serve as a direct proton donor to the diatomic ligands (CO, NO, as well as O(2)) bound to the heme. This has important implications for the oxygen activation mechanism critical to NOS catalysis.

Role of Arginine Guanidinium Moiety in Nitric-oxide Synthase Mechanism of Oxygen Activation

“…Our analysis of the structural and electrostatic influences of the guanidinium pK a , combined with the characterization of NOS-catalyzed oxidation of the series analogues studied (Table 2), leads us to propose a model for the mechanism of Fe II -O 2 activation (Scheme 4) that completes the ones proposed by Rousseau and co-workers (69) and Poulos and co-workers (39). High pK a alkylguanidines (including L-Arg) that favor the formation of the second H-bond between the distal water molecule and the terminal oxygen atom of the Fe III -peroxo complex prove to be good substrates of iNOS despite slightly higher autoxidation rates (Table 2).…”

“…The second step, NOHA 3 citrulline ϩ NO, is supposed to involve the direct reaction of the (hydro)peroxo species on the NOHA hydroxyguanidinium moiety, followed by the rearrangement of the resulting tetrahedral complex and the release of citrulline and NO (26,29). These models are supported by crystallographic, spectroscopic, and cryogenic experimental evidences (39,69,80,83,87), but recently serious questions have been raised (30,32). In the absence of the definitive isolation, characterization, and identification of key reaction intermediates, an unequivocal NOS mechanism cannot be advanced and therefore several alternative mechanisms have been proposed for each catalytic step.…”

“…The mechanistic difference between the first and second steps of NOS catalytic activity (Scheme 1) is commonly explained by the difference in pK a values of the guanidinium proton of L-Arg and that of NOHA (37)(38)(39). However, until now, no clear picture of specific proton and electron transfer events has emerged to clarify this aspect of the NOS mechanism (24,25,30,32).…”

“…Failed electron and proton transfer can also directly generate RNS by tunneling NOS catalytic cycle toward an unproductive reaction intermediate such as the ferrous heme-nitric oxide complex, whose oxidation can lead to peroxynitrite production (36). The differences in the pK a values of the N (H) guanidinium proton of L-Arg and NOHA could modify the characteristics of the proton transfer processes, which might in turn account for the catalytic differences between the first and second steps (37)(38)(39) and regulate the nature of NOS catalytic production. However, despite the crucial role of proton transfer in NOS catalysis, little is known about the role of the guanidinium moiety of the NOS substrates, and about the H-bond network surrounding the dioxygen ligand.…”

“…Structural Model of Interaction between Fe II -CO and NOS Substrate-The crystallographic structures of NOS-hemeFe II -CO (39) and ferrous heme-nitric oxide complex (39,83), in the presence of L-Arg and NOHA, suggest the existence of an H-bond between the L-Arg-guanidinium and the distal ligand (39,83). Additionally, in the presence of L-Arg, all crystallographic structures reveal the presence of a structural water molecule involved in H-bonding interactions with both the ligand (CO or NO) and the guanidinium moiety of L-Arg (Fig.…”