A functional nitric oxide reductase model

Abstract

A functional heme/nonheme nitric oxide reductase (NOR) model is presented. The fully reduced diiron compound reacts with two equivalents of NO leading to the formation of one equivalent of N 2O and the bis-ferric product. NO binds to both heme Fe and nonheme Fe complexes forming individual ferrous nitrosyl species. The mixed-valence species with an oxidized heme and a reduced nonheme Fe B does not show NO reduction activity. These results are consistent with a so-called ''trans'' mechanism for the reduction of NO by bacterial NOR.functional model ͉ NO reduction ͉ N2O ͉ "trans" mechanism N itric oxide reductase (NOR) is a membrane-bound enzyme that catalyzes the 2e Ϫ reduction of nitric oxide (NO) to nitrous oxide (N 2 O), an obligatory step involved in the sequential reduction of nitrate to dinitrogen known as bacterial denitrification. The active site of NOR consists of a monohistidine ligated five-coordinate heme and a trisimidazole ligated nonheme Fe B . This structure strongly resembles the active site of oxygen reduction enzyme-cytochrome c oxidase (CcO), which possesses a heme-a 3 /Cu B center ( Fig. 1) (1, 2). Essentially, the distal metal Cu B in CcO is replaced by a nonheme Fe metal in NOR; NOR and CcO are thought to be distant relatives.The dinuclear iron active site in NOR was confirmed a decade ago by spectroscopic studies (3). Presumably, two NO molecules are turned over to give one molecule of N 2 O and one molecule of H 2 O at the diiron center with the consumption of two electrons and two protons. Although many enzyme studies of NOR have been focused on the intermediate trapping and elucidation of the reaction mechanism (4-15), the details of the catalytic cycle are still unresolved because of the lack of structural information and uncertainty regarding short-lived intermediates.In contrast to enzyme studies, synthetic biomimetic model complexes provide a straightforward and controlled method to understand how this chemical transformation proceeds at the enzyme active site. However, only a few synthetic models have been developed that mimic the active site of NOR; moreover, these compounds either lack a proximal imidazole ligand (16,17) or use pyridine as a replacement for the histidine ligands (18)(19)(20). No functional NOR models have been reported to date. Our CcO model complexes have proved to be functionally active for oxygen reduction reaction with minimal reactive oxygen species (ROS) formation (21-24). These appear to be promising NOR model candidates if the distal Cu metal is replaced by an iron because the resulting diiron compound has almost all of the key components in NOR: a heme Fe with a proximal imidazole ligand and a trisimidazole ligated nonheme Fe center.In this report, we disclose the first synthetic functional NOR model LFe II /Fe II (Fig. 2), which reacts with two equivalents of NO to give one equivalent of N 2 O and the bis-ferric product. We have shown that NO binds to both heme Fe and Fe B to form a possible bis-nitrosyl intermediate; subsequently, the two bound NO molec...

Continuous measurements of nitrous oxide isotopomers during incubation experiments

“…Nitric oxide reductase is the primary enzyme in a chain of catalytic reactions leading to the production of N 2 O (Hino et al, 2010;Hendriks et al, 2000). The catalytic cycle involving production of N 2 O from NO has yet to be completely understood with respect to the formation of the N-N double bond, the complexity of the structural information of nitric oxide reductase, the proton transfer pathway into nitric oxide reductase (Tosha and Shiro, 2013), and the very short lifetime of the intermediate states of the molecules (Collman et al, 2008). We hypothesized that the difference in the bulk observed during incubation of our two bacterial species was due to different nitric oxide reductases produced by the two species.…”

Understanding the Role of Hyponitrite in Nitric Oxide Reduction

“…In biological systems, this reaction is performed by NO reductase (NOR) and flavodiiron NO reductase (FDP), [1][2][3][4][5][6][7][8][9][10][11][12] which convert two equivalents of nitric oxide, two reducing equivalents, and two protons, into N 2 O and water. The exact order of proton and electron additions during their catalytic cycles is not known, nor is the mechanism by which the N-N bond of N 2 O is formed.…”

Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin

“…Therefore, studies of these synthetic models have offered many insights. For example, Collman et al showed that a fully reduced heme/nonheme Fe B compound can react with two equivalents of NO leading to the formation of one equivalent of N 2 O and a bis-ferric product (41). On the other hand, Karlin and coworkers showed that a small heme/Cu complex can efficiently lead to reductive coupling of NO to N 2 O (43).…”

“…Despite these successes, the roles of the conserved glutamates and metal ions still remain to be fully elucidated, partly because of the difficulty in obtaining native NOR in high yield and the lack of a 3D structure. Even if these problems are resolved, it is still difficult to replace iron in the native Fe B site with other metal ions, and spectroscopic studies of native NOR are often complicated by the presence of other metal cofactors (e.g., low-spin heme).To overcome these limitations, a number of synthetic models of NOR using small organic molecules as ligands, have been made in which the nonheme Fe B site can be replaced by a copper ion (17,(38)(39)(40)(41)(42)(43)(44)(45). In addition, since these model systems lack additional metal-binding sites, spectroscopic studies are often simplified.…”

“…To overcome these limitations, a number of synthetic models of NOR using small organic molecules as ligands, have been made in which the nonheme Fe B site can be replaced by a copper ion (17,(38)(39)(40)(41)(42)(43)(44)(45). In addition, since these model systems lack additional metal-binding sites, spectroscopic studies are often simplified.…”