Spectral Properties of Bacterial Nitric-oxide Reductase

Field, Sarah J.; Prior, Louise; Roldán, María Dolores; Cheesman, Myles R.; Thomson, Andrew J.; Spiro, Stephen; Butt, Julea N.; Watmough, Nicholas J.; Richardson, David J.

doi:10.1074/jbc.m112202200

Cited by 39 publications

(27 citation statements)

References 33 publications

(29 reference statements)

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“…It was, thus, suggested that the four-electron reduced form 2 could not be thermodynamically achievable by the physiological electron-donor such as azurin and cytochrome c and that the three-electron reduced form, in which heme b 3 is oxidized, would be the physiologically accessible starting point for the NO reduction cycle in cells (22,24,26). However, our proposed mechanism in Fig.…”

Section: Discussionmentioning

confidence: 54%

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

Kumita

Matsuura

Hino

et al. 2004

Journal of Biological Chemistry

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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: Discussionmentioning

confidence: 54%

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

Kumita

Matsuura

Hino

et al. 2004

Journal of Biological Chemistry

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“…This feature, known as CT 2 , is one of a pair of ligand-to-metal charge-transfer bands that indicate the presence of high-spin ferric b-or o-type hemes at the active sites of cytochrome bo 3 (34,35) and bacterial nitric-oxide reductase (37). The energy and intensity of CT 2 depend on the nature of the distal (exogenous) ligand bound to the heme (35,37). A feature corresponding to CT 2 is clearly resolved in the RT-MCD spectrum of cytochrome cbb 3 oxidase (Fig.…”

Section: Fig 2 Multiple Sequence Alignments Of Ccoo and Ccopmentioning

confidence: 99%

Molecular and Spectroscopic Analysis of the Cytochromecbb3 Oxidase from Pseudomonas stutzeri

Pitcher¹,

Cheesman²,

Watmough³

2002

Journal of Biological Chemistry

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Cytochrome cbb 3 oxidase, a member of the heme-copper oxidase superfamily, is characterized by its high affinity for oxygen while retaining the ability to pump protons. These attributes are central to its proposed role in the microaerobic metabolism of proteobacteria. We have completed the first detailed spectroscopic characterization of a cytochrome cbb 3 oxidase, the enzyme purified from Pseudomonas stutzeri. A combination of UVvisible and magnetic CD spectroscopies clearly identified four low-spin hemes and the high-spin heme of the active site. This heme complement is in good agreement with our analysis of the primary sequence of the ccoNOPQ operon and biochemical analysis of the complex. Near-IR magnetic CD spectroscopy revealed the unexpected presence of a low-spin bishistidine-coordinated c-type heme in the complex. This was shown to be one of two c-type hemes in the CcoP subunit by separately expressing the subunit in Escherichia coli. Separate expression of CcoP also allowed us to unambiguously assign each of the signals associated with low-spin ferric hemes present in the X-band EPR spectrum of the oxidized enzyme. This work both underpins future mechanistic studies on this distinctive class of bacterial oxidases and raises questions concerning the role of CcoP in electron delivery to the catalytic subunit.

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“…65 From these observations, Thomson and coworkers proposed that the μ-oxo bridged diiron(III) cluster might represent a "closed" resting state of the enzyme, while the catalytic cycle may, in fact, be initiated by the binding of NO to the mixed valence heme b 3 (III)-Fe B (II) cluster. 64,65,78 Regardless of whether the diiron(III) μ-oxo bridge cluster is part of the catalytic cycle of NO reductases, this structure reveals a metal-metal distance that is 1.5 Å shorter that that measured in the crystal structures of terminal oxidases between the iron of heme a 3 and Cu B . [79][80][81][82][83] This significant distinction between the active sites of P.d.…”

Section: Spectroscopic Characterization Of Denitrifying No Reductasesmentioning

confidence: 99%

“…64 Room temperature absorption and MCD analyses of the threeelectron reduced enzyme at different pH values suggest that, upon reduction of Fe B , the μ-oxo bridge dissociates from Fe B (II) and the heme b 3 iron(III) rebinds the proximal histidine to form a 6-coordinate aquo/hydroxo complex with charge transfer bands at 635 and 605 nm, respectively. 78 Not surprisingly, cyanide was shown to bind much more readily to heme b 3 in the three-electron reduced form of the enzyme, where the displacement of the sixth aquo/ hydroxo ligand is facile (e.g., as in metmyoglobin), than in the fully oxidized enzyme, where the μ-oxo group bridge between the two iron(III) is quite stable. 65 From these observations, Thomson and coworkers proposed that the μ-oxo bridged diiron(III) cluster might represent a "closed" resting state of the enzyme, while the catalytic cycle may, in fact, be initiated by the binding of NO to the mixed valence heme b 3 (III)-Fe B (II) cluster.…”

Section: Spectroscopic Characterization Of Denitrifying No Reductasesmentioning

confidence: 99%

“…A lack of variation in both this absorption band and spectral features in the RR spectra suggest that the cluster is insensitive to pH changes between 6.0 and 9.0. 75,78 Mediated redox potentiometry has shown that the heme b 3 presents an unexpected low midpoint potential (E m = 60 mV), which allows for the formation of a three-electron reduced form of the enzyme where the heme b 3 remains as the sole iron(III) species. 64 Room temperature absorption and MCD analyses of the threeelectron reduced enzyme at different pH values suggest that, upon reduction of Fe B , the μ-oxo bridge dissociates from Fe B (II) and the heme b 3 iron(III) rebinds the proximal histidine to form a 6-coordinate aquo/hydroxo complex with charge transfer bands at 635 and 605 nm, respectively.…”

Section: Spectroscopic Characterization Of Denitrifying No Reductasesmentioning

confidence: 99%

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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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Spectral Properties of Bacterial Nitric-oxide Reductase

Cited by 39 publications

References 33 publications

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

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

Molecular and Spectroscopic Analysis of the Cytochromecbb3 Oxidase from Pseudomonas stutzeri

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

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