The Second Step of the Nitric Oxide Synthase Reaction: Evidence for Ferric-Peroxo as the Active Oxidant

Abstract

Nitric oxide synthase (NOS) is a P450 mono-oxygenase that catalyzes the oxidation of l-arginine to citrulline and NO through the stable intermediate N(G)-hydroxy-l-arginine (NHA). The oxidation of NHA by NOS is unique. There is little direct evidence in support of the nature of the heme bound oxidant [i.e., ferric-peroxo vs Fe(IV)O(por(*+))] responsible for this transformation. Previous work characterizing the H(2)O(2)-driven oxidation of NHA by NOS showed the formation of citrulline and the side product N(delta)-cyanoornithine (CN-orn). This led to the proposed involvement of a ferric-peroxo intermediate in the oxidation of NHA to citrulline. To test this hypothesis we used this model reaction to study the effects of pH, heme substitution, active site mutagenesis, and a fluorinated substrate analogue on the product distribution. Further, the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) by H(2)O(2) and iNOS(heme) was used to probe the protein-catalyzed breakdown of peroxide to the Fe(IV)O(por(*+)) intermediate. At pH 6.5, 7.5, and 8.5 the peroxide shunt reaction forms 26 +/- 2, 36 +/- 1, and 51 +/- 1% citrulline, respectively. The rate of peroxidase activity, however, was negatively correlated to pH, with a peroxide breakdown rate of 13.1 +/- 0.3, 8.3 +/- 0.2, and 4.2 +/- 0.1 M(-1) s(-1) at pH 6.5, 7.5, and 8.5, respectively. Mutation of active site valine 346 to an alanine shifted the product distribution to 5.2 +/- 0.5% citrulline while enhancing the peroxide cleavage rate to 14.3 +/- 0.7 M(-1) s(-1). Substitution of the heme cofactor with iron mesoporphyrin IX (Fe-MPIX) alters the product distribution from 36 +/- 1% citrulline to 22 +/- 3% citrulline. Metal substitution with Mn results in the formation of 64.7 +/- 0.8% citrulline. Conversely, the electrophilic 4,4-difluoro-N(G)-hydroxy-l-arginine substrate analogue shifted the product distribution to 68.6 +/- 0.6% 4,4-difluorocitrulline. The peroxidase data provide insight into the chemical features of NOS that control the processing of the ferric-peroxo species to the Fe(IV)O(por(*+)) intermediate and help interpret the product distributions observed for the peroxide shunt under various conditions. In all cases, the ability of the protein to break down peroxide is negatively correlated with the formation of citrulline by the peroxide shunt. These results support the high valent Fe(IV)O(por(*+)) intermediate as the species responsible for CN-orn formation and are consistent with the involvement of the ferric-peroxo intermediate in the oxidation of NHA to citrulline.

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

“…The first step of NOS chemistry, Arg 3 NOHA, is generally believed to follow a typical P450-like mono-oxygenation reaction based on the double protonation of the Fe III -OO Ϫ peroxo intermediate, the heterolytic cleavage of the peroxo O-O bond leading to the formation of a Compound I species, followed by a typical P450-like "radical rebound" mechanism (Scheme 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).…”

“…ϩ -Fe IV ϭO) (25) believed to be responsible for the hydroxylation of the guanidine moiety of L-Arg to NOHA (24 -26). The second catalytic step (oxidation of NOHA) is believed to also involve the formation of the ferric-peroxo Fe III -OO Ϫ species (27, 28), as described above, but at this point there ensues a nucleophilic attack of the peroxo group upon the NOHA hydroxyguanidinium carbon atom followed by a rearrangement of the resulting tetrahedral complex, ultimately leading to the release of NO (24,29). Although this analogous P450 model has been the working paradigm for the NOS mechanism, alternative models have been proposed (30 -34) to address serious deficiencies.…”

“…However, whereas low pK a arylguanidines failed to become hydroxylated, the corresponding N-hydroxyguanidines still led to significant production of NO (Table 2) (61,86,89). This suggests that the Fe III -peroxo complex might be sufficient to achieve NOHA oxidation (29); without the participation of an additional water molecule, low pK a substrates, such as NOHA, will stabilize the Fe III -peroxo complex and favor its direct reaction on the guanidinium moiety (Scheme 4, step 2).…”

NO formation by a catalytically self-sufficient bacterial nitric oxide synthase from Sorangium cellulosum

“…The second reaction, NHA conversion to citrulline and NO, is unique to NOS. While the mechanism is still under debate, one widely accepted proposal involves a tetrahedral intermediate formed via the nucleophilic attack of a Fe III -hydroperoxo species to NHA (17,18). In both steps, the tetrahydrobiopterin cofactor is involved in electron transfer (17,19).…”

Development and evaluation of kinetic spectrophotometric assays for horseradish Peroxidase by Catalytic Coupling of Paraphenylenediamine and mequinol

“…Spectrophotometers are economical, easy to handle and the reagents used in the assay are inexpensive. Some of the common co-substrates employed in the colorimetric method include guaiacol, 11 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonicacid), 12 o-dianisidine, benzidine, p-phenylenediamine, 13 pyrogallol, 14 3,3′,5,5′-tetramethylbenzidine (TMB), 15 o-phenylenediamine, 16 4-aminoantipyrine-phenol, 17 pyrocatechol-aniline, 18 10-Nmethylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine and bis[3-bis(4-chlorophenyl)methyl-4-dimethylaminophenyl]amine, 19 but these reagents have some limitations like the carcinogenicity of o-dianisidine and benzidine and the broader linearity range of pyrogallol and guaiacol due to their autoxidation. TMB has initial problems like stability and poor solubility in aqueous buffer solutions.…”