Neuroglobin (Ngb) is a globin expressed in the nervous system of humans and other organisms that is involved in the protection of the brain from ischemic damage. Despite considerable interest, however, the in vivo function of Ngb is still a conundrum. In this paper we report a number of kinetic experiments with O 2 and NO that we have interpreted on the basis of the 3D structure of Ngb, now available for human and murine metNgb and murine NgbCO. kinetics ͉ neuron protection ͉ nitric oxide reduction ͉ physiological role ͉ structural changes N euroglobin (Ngb) is a heme protein belonging to the extended globin family, which was discovered by Burmester et al. (1) in the brain of humans and mice. Interestingly, Ngb is predominantly expressed in specific areas of the brain, notably the frontal lobe, the subthalamic nucleus, and the thalamus (1, 2), although at a fairly low concentration (approximately micromolar) compared with, for example, myoglobin (Mb) in the heart (Ϸ0.2 mM) (3, 4). The unexpected discovery of Ngb raised considerable curiosity from the standpoints of evolutionary biology and pathophysiology alike. The interest in the structure and function of Ngb intensified considerably after the recent findings by Greenberg and collaborators (5, 6) that (i) its expression in neurons is up-regulated under conditions of hypoxia and (ii) the extent of ischemic damage after experimental stroke in the rat is reduced when the amount of Ngb expressed in the brain is enhanced and vice versa.Ngb was found to bind O 2 reversibly at the ferrous heme iron, with an affinity [P 50 Ϸ 2 torr (1 torr ϭ 133 Pa)] (1) comparable with that of Mb (P 50 Ϸ 1 torr) (7). This observation suggested that its function in the brain would mimic that of Mb in red muscles, i.e., storage, transport, and facilitated intracellular diffusion of O 2 . This hypothesis, however, is hardly tenable given the low average concentration of Ngb (8). Like other heme proteins, ferrous Ngb also binds CO and NO (9-13).The 3D structures of metNgb from humans (14) and mice (15) show the typical three-over-three ␣-helical globin fold.Over and above a number of interesting features that emerged from examination of the high resolution 3D structures, two findings stand out: namely (i) the ferric heme iron is hexacoordinated, with the distal His(E7)64 and the proximal His(F8)96 directly bound to the metal ion, and (ii) the protein contains a very large (Ϸ290

We show that the heme-copper terminal oxidases of Thermus thermophilus (called ba 3 and caa3) are able to catalyze the reduction of nitric oxide (NO) to nitrous oxide (N 2O) under reducing anaerobic conditions. The rate of NO consumption and N 2O production were found to be linearly dependent on enzyme concentration, and activity was abolished by enzyme denaturation. Thus, contrary to the eukaryotic enzyme, both T. thermophilus oxidases display a NO reductase activity (3.0 ؎ 0.7 mol NO͞mol ba 3 ؋ min and 32 ؎ 8 mol NO͞mol caa 3 ؋ min at [NO] Ϸ 50 M and 20°C) that, though considerably lower than that of bona fide NO reductases (300 -4,500 mol NO͞mol enzyme ؋ min), is definitely significant. We also show that for ba 3 oxidase, NO reduction is associated to oxidation of cytochrome b at a rate compatible with turnover, suggesting a mechanism consistent with the stoichiometry of the overall reaction. We propose that the NO reductase activity of T. thermophilus oxidases may depend on a peculiar Cu B ؉ coordination, which may be revealed by the forthcoming three-dimensional structure. These findings support the hypothesis of a common phylogeny of aerobic respiration and bacterial denitrification, which was proposed on the basis of structural similarities between the Pseudomonas stutzeri NO reductase and the cbb 3 terminal oxidases. Our findings represent functional evidence in support of this hypothesis. Heme-copper terminal oxidases and bacterial NO reductases (NOR) were suggested to have originated during evolution from a common ancestor (1-3). The common phylogeny was proposed because of structural similarities between these enzymes (see ref. 4 for a review), notably in the large catalytic subunit, which displays significant sequence homology and conservation of crucial residues (including the six metal-binding histidines). The topology of the catalytic subunit of NOR (NorB) is predicted to comprise 12 transmembrane helices, as shown for subunit I of heme-copper oxidases (5, 6). Finally, the active site is, in both cases, a bimetallic center, consisting of a heme-iron and a second metal, which is Cu in oxidases and Fe in NOR (7,8).On the basis of these structural similarities, it was presumed that the mechanisms of O 2 and NO reduction may share common features and, possibly, that O 2 and NO may be used as alternative substrates by both enzyme families. The mechanism of NO reduction by NOR is, at present, largely hypothetical, which makes any comparison with the mechanism of O 2 reduction by oxidases difficult. It is interesting, however, that a bacterial NOR with O 2 reductase activity was found in Paracoccus denitrificans ATCC 35512 (9); in contrast, there is no unequivocal experimental evidence in support of the hypothesis that heme-copper oxidases catalyze the reduction of NO to N 2 O (2NO ϩ 2e Ϫ ϩ 2H ϩ 3 N 2 O ϩ H 2 O). Brudwig et al. (10) reported that beef heart cytochrome c oxidase enhances (by a factor of 2) the reduction of NO by ascorbate and N,N,NЈ,NЈ-tetramethyl-p-phenylenediamine (TMPD), but on a time...

The flavodiiron proteins (FDP) are widespread among strict or facultative anaerobic prokaryotes, where they are involved in the response to nitrosative and/or oxidative stress. Unexpectedly, FDPs were fairly recently identified in a restricted group of microaerobic protozoa, including Giardia intestinalis, the causative agent of the human infectious disease giardiasis. The FDP from Giardia was expressed, purified, and extensively characterized by x-ray crystallography, stopped-flow spectroscopy, respirometry, and NO amperometry. Contrary to flavorubredoxin, the FDP from Escherichia coli, the enzyme from Giardia has high O 2 -reductase activity (>40 s ؊1 ), but very low NO-reductase activity (ϳ0.2 s ؊1 ); O 2 reacts with the reduced protein quite rapidly (milliseconds) and with high affinity (K m < 2 M), producing H 2 O. The three-dimensional structure of the oxidized protein determined at 1.9 Å resolution shows remarkable similarities with prokaryotic FDPs. Consistent with HPLC analysis, the enzyme is a dimer of dimers with FMN and the nonheme di-iron site topologically close at the monomer-monomer interface. Unlike the FDP from Desulfovibrio gigas, the residue His-90 is a ligand of the di-iron site, in contrast with the proposal that ligation of this histidine is crucial for a preferential specificity for NO. We propose that in G. intestinalis the primary function of FDP is to efficiently scavenge O 2 , allowing this microaerobic parasite to survive in the human small intestine, thus promoting its pathogenicity.The flavodiiron proteins (FDP, 2 originally named A-type flavoproteins (1)) are widespread among Bacteria and Archaea, either strict or facultative anaerobes, where they have been proposed to play a role in the response to nitrosative and/or oxidative stress (2, 3). A few prokaryotic FDPs have been characterized to date, namely those from the bacteria Desulfovibrio gigas (originally named rubredoxin:oxygen oxidoreductase, ROO (4 -7), and hereafter denoted FDP Dg ), Escherichia coli (named flavorubredoxin, FlRd, 3 Refs. 2, 8 -11), Desulfovibrio vulgaris (12), Moorella thermoacetica (FDP Mt , (13, 14)), and the homologous enzyme from the methanogenic archaeon Methanothermobacter marburgensis (FDP Mm , Refs. 15, 16). The FDPs contain two redox centers: a FMN, the electron entry site into the enzyme, and a non-heme Fe-Fe center, the active site (13). They are cyanide-insensitive enzymes able to catalyze the reduction of O 2 (to H 2 O) and/or NO (to N 2 O). Some of these enzymes are almost exclusively reactive toward NO (such as E. coli FlRd, Refs. 2, 9), 4 others toward O 2 (such as the M. marburgensis enzyme, (15)), whereas some FDPs catalyze the reduction of both gases, though with different efficiency (7,12,13). These enzymes are expected to play a protective role in anaerobic or microaerobic microorganisms that need to survive under O 2 and cope with NO produced by the host defense system to counteract infection (17,18).Surprisingly, a few years ago, genes coding for FDPs were identified also in the geno...