Mechanism and regulation of ferrous heme-nitric oxide (NO) oxidation in NO synthases

Abstract

Edited by F. Peter Guengerich 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 ratelimiting steps. These are the rate of heme reduction by the flavin domain (k r), of dissociation of NO from the ferric heme-NO complex (k d), and of oxidation of the ferrous heme-NO complex (k ox). 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 outersphere 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 Fe III-N(O)OO ؊ intermediate species and thereby catalyze this reaction, which otherwise is not observed or favorable in proteins like globins that contain a histidinecoordinated heme. This article contains Tables S1-S3 and Figs. S1-S7.

Coordination Chemistry of Nitric Oxide and Biological Signaling

“…By using variants of n-and i-NOS isoforms, the rate constants for dioxygenation reactions of bound NO, k O2 , were found in the range 10 2 -10 4 M -1 s -1 , depending on the presence/absence of substrate (L-arg), cofactor (BH 4 ) and/or H-bonding interactions either at the distal-or proximal heme sites. Oxygen binding to the NOS ferrous heme is extremely fast (ill-defined k O2 , reported to be higher than 10 5 M -1 s -1 ), [101] and thus the corresponding point might be well located up-and left-wards (out of the plot) in Figure 14. Thus, the mechanisms appear to be similar for the heme-and non heme systems (both affording N-bound peroxynitrite initial intermediates), by using the values of k O2 por pure NOS and E NO+/NO (~ −0.3 V).…”

“…However, the smaller denitrosylation rates for the NOS-surrogates lead to strong deviations. This is not a surprise, given that in contrast to the diffusional encounter of reactants for the non heme nitrosyls, a restricted access of O 2 to the distal pocket has been demonstrated for the NOS isozymes; [101] thus, the rates are not controlled solely by k O2 (direct reactivity of O 2 to nitrosyl), and therefore the observed denitrosylation rates become much lower for the enzymatic systems.…”

“…In the NOS-catalyzed endogenous production of NO by oxidation of L-arginine with O 2 , a 6C Fe II -NO intermediate (containing cysteinate as a trans-ligand to NO) played a crucial role in the global catalytic/regulatory cycle by reacting with O 2 and giving NO 3 -, which was rapidly released. [101] In this way, regeneration of the labile aqua-site (5C high-spin Fe III ) allowed performing the catalytic turnover through subsequent reduction to Fe(II) and O 2 -coordination for reacting with the arginine substrate. By using variants of n-and i-NOS isoforms, the rate constants for dioxygenation reactions of bound NO, k O2 , were found in the range 10 2 -10 4 M -1 s -1 , depending on the presence/absence of substrate (L-arg), cofactor (BH 4 ) and/or H-bonding interactions either at the distal-or proximal heme sites.…”

Electronic structure and mechanism for the uptake of nitric oxide by the Ru(iii) antitumor complex NAMI-A

“…This result is also consistent with some experimental data in the literature. 34,71,72 Fig . S1 and S2 † shows the SA-CASSCF active orbitals containing the largest contribution from the p orbitals from NO and the d orbitals from ruthenium for the reactants and TS points located along the triplet potential energy surface.…”

Glutathione and Nitric Oxide: Key Team Players in Use and Disuse of Skeletal Muscle

“…Among the heme-proteins, soluble guanylyl cyclase (sGC) is the principal and most studied receptor for NO: This enzyme plays a central role in blood pressure regulation and several other physiological actions. The binding of NO to a ferrous heme in its β subunit increases both catalytic rate and active site affinity for the substrate [37].…”