Reaction of Carbon Monoxide with the Reduced Active Site of Bacterial Nitric Oxide Reductase

Hendriks, Janneke; Prior, Louise; Baker, Adam; Thomson, Andrew J.; Saraste, Matti; Watmough, Nicholas J.

doi:10.1021/bi011428t

Cited by 38 publications

(37 citation statements)

References 49 publications

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“…The EPR spectra obtained at 0.5, 1.0, 3.0, CO Inhibition of Intermediate Formation-To characterize the reaction intermediate in more detail, we examined the CO inhibition of its formation. Previous studies on the reaction of fully reduced NOR with CO revealed that CO molecule bound to the heme b 3 iron was not replaced by NO within 100 ms (21,28,29), while CO could not bind to ferrous Fe B site (29). The CO-inhibited NOR was mixed with an NO buffer and freezetrapped at 0.5 ms.…”

Section: Resultsmentioning

confidence: 99%

“…Despite extensive studies using spectroscopic (18 -26), kinetic (27)(28)(29)(30), and mutagenetic methods (31), the molecular mechanism of the NO reduction reaction catalyzed by NOR has not yet been established. Thus far, two mechanisms have been proposed, each assuming a different structure of the binuclear center (heme b 3 and Fe B ) in the intermediate state of the NO reduction (Scheme 1).…”

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confidence: 99%

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NO Reduction by Nitric-oxide Reductase from Denitrifying Bacterium Pseudomonas aeruginosa

Kumita

Matsuura

Hino

et al. 2004

Journal of Biological Chemistry

100

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Nitric-oxide reductase (NOR) of a denitrifying bacterium catalyzes NO reduction to N 2 O at the binuclear catalytic center consisting of high spin heme b 3 and non-heme Fe B . The structures of the reaction intermediates in the single turnover of the NO reduction by NOR from Pseudomonas aeruginosa were investigated using optical absorption and EPR spectroscopies combined with an originally designed freeze-quench device. In the EPR spectrum of the sample, in which the fully reduced NOR was mixed with an NO solution and quenched at 0.5 ms after the mixing, two characteristic signals for the ferrous Fe B -NO and the penta-coordinated ferrous heme b 3 -NO species were observed. The CO inhibition of its formation indicated that two NO molecules were simultaneously distributed into the two irons of the same binuclear center of the enzyme in this state. The time-and temperature-dependent EPR spectral changes indicated that the species that appeared at 0.5 ms is a transient reaction intermediate prior to the N 2 O formation, in good agreement with the so-called "trans" mechanism. It was also found that the final state of the enzyme in the single turnover cycle is the fully oxidized state, in which the -oxo-bridged ligand is absent between the two irons of its binuclear center, unlike the resting form of NOR as isolated. On the basis of these present findings, we propose a newly developed mechanism for the NO reduction reaction conducted by NOR. Nitric-oxide reductase (NOR)1 is a metal-containing enzyme involved in biological denitrification processes. The enzyme catalyzes the two-electron reduction of nitric oxide (NO) to nitrous oxide (N 2 O) according to Equation 1 (1-4). Since the NO reduction reaction involves the N-O bond cleavage and the N-N bond formation, it is chemically interesting to elucidate the molecular mechanism of the NO reduction catalyzed by the metal enzymes.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

NO Reduction by Nitric-oxide Reductase from Denitrifying Bacterium Pseudomonas aeruginosa

Kumita

Matsuura

Hino

et al. 2004

Journal of Biological Chemistry

100

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“…The slow rebinding of this ligand, compared to CO, would explain the photolysis kinetics. 106 However, RR studies identify the ferrous heme b 3 as a 5-coordinate high-spin species, 11,107 thus, the kinetic data may be consistent with the presence of a weakly interacting group that can adopt variable configurations rather than with the displacement of a true distal ligand of heme b 3 (vide infra).…”

Section: The Diiron Site Can Accommodate Two Co Moleculesmentioning

confidence: 99%

Spectroscopic characterization of heme iron–nitrosyl species and their role in NO reductase mechanisms in diiron proteins

Moënne‐Loccoz

2007

Nat. Prod. Rep.

121

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Nitric oxide (NO) plays an important role in cell signalling and in the mammalian immune response to infection. On its own, NO is a relatively inert radical, and when it is used as a signalling molecule, its concentration remains within the picomolar range. However, at infection sites, the NO concentration can reach the micromolar range, and reactions with other radical species and transition metals lead to a broad toxicity. Under aerobic conditions, microorganisms cope with this nitrosative stress by oxidizing NO to nitrate (NO 3 − ). Microbial hemoglobins play an essential role in this NO-detoxifying process. Under anaerobic conditions, detoxification occurs via a 2-electron reduction of two NO molecules to N 2 O. In many bacteria and archaea, this NOreductase reaction is catalyzed by diiron proteins. Despite the importance of this reaction in providing microorganisms with a resistance to the mammalian immune response, its mechanism remains ill-defined. Because NO is an obligatory intermediate of the denitrification pathway, respiratory NO reductases also provide resistance to toxic concentrations of NO. This family of enzymes is the focus of this review. Respiratory NO reductases are integral membrane protein complexes that contain a norB subunit evolutionarily related to subunit I of cytochrome c oxidase (CcO). NorB anchors one high-spin heme b 3 and one non-heme iron known as Fe B , i.e., analogous to Cu B in CcO. A second group of diiron proteins with NO-reductase activity is comprised of the large family of soluble flavoprotein A found in strict and facultative anaerobic bacteria and archaea. These soluble detoxifying NO reductases contain a non-heme diiron cluster with a Fe-Fe distance of 3.4 Å and are only briefly mentioned here as a promising field of research. This article describes possible mechanisms of NO reduction to N 2 O in denitrifying NO-reductase (NOR) proteins and critically reviews recent experimental results. Relevant theoretical model calculations and spectroscopic studies of the NO-reductase reaction in heme/copper terminal oxidases are also overviewed.

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“…Because 0?1 mM NO { 2 alone did not activate the promoter, the high activity must be the result of the synergistic effect of CO and NO { 2 . CO is known to bind to the active site of NOR (Hendriks et al, 2001). It is probable that NOR was inhibited by the CO and that the NO produced from NO { 2 was not further metabolized as in the case of strain RM495.…”

Section: No-responding Induction Of the Nosr Promotermentioning

confidence: 99%

Transcriptional regulation of the nos genes for nitrous oxide reductase in Pseudomonas aeruginosa

2003

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The genes for nitrous oxide (N 2 O) reduction, nosRZDFYL, are clustered on the chromosome of Pseudomonas aeruginosa. Promoter assays using transcriptional fusions to lacZ revealed that the structural gene for nitrous oxide reductase, nosZ, is transcribed with the upstream nosR gene. The nosR gene product is not required for the activity of the nosR promoter. A sequence similar to the consensus FNR-binding motif was found 41?5 bp upstream from the major transcriptional start point of nosR. Mutation of the motif significantly reduced the promoter activity. DNR, an FNR-related transcriptional regulator required for the expression of denitrification genes in P. aeruginosa, is necessary for the transcription of nosR, indicating that the motif is recognized by DNR. Nitrite (NO 2 2 ), nitric oxide (NO) and NO-generating reagents induced nosR promoter activity, but N 2 O did not. The NO 2 2 -induced nosR promoter activity was reduced by mutation of the NO 2 2 reductase gene. However, a low concentration of NO 2 2 induced the promoter activity in a NO reductase mutant. These results indicate that NO is the inducer molecule for transcription of the nos genes.

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Reaction of Carbon Monoxide with the Reduced Active Site of Bacterial Nitric Oxide Reductase

Cited by 38 publications

References 49 publications

NO Reduction by Nitric-oxide Reductase from Denitrifying Bacterium Pseudomonas aeruginosa

NO Reduction by Nitric-oxide Reductase from Denitrifying Bacterium Pseudomonas aeruginosa

Spectroscopic characterization of heme iron–nitrosyl species and their role in NO reductase mechanisms in diiron proteins

Transcriptional regulation of the nos genes for nitrous oxide reductase in Pseudomonas aeruginosa

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