Spectroscopic studies of the coupled binuclear non-heme iron active site in the fully reduced hydroxylase component of methane monooxygenase: comparison to deoxy and deoxy-azide hemerythrin

Pulver, Sabine Coates; Froland, Wayne A.; Fox, Brian G.; Lipscomb, John D.; Solomon, Edward I.

doi:10.1021/ja00079a024

Cited by 94 publications

(149 citation statements)

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“…For example, interactions of regulatory subunits such as MMOB with the hydroxylase protein of methane monooxygenase and acyl carrier protein with Δ 9 -desaturase affect the spectroscopic characteristics of the diiron(II) cluster at the active site. [117][118][119] These observations led to the concept of carboxylate shifts controlling the number of open coordination sites and the range of Fe-Fe distances during catalytic turnover. 120 Thus, in NOR, where glutamate side chains in the vicinity of coordinating histidine residues are known to be conserved, it is conceivable that carboxylate shifts might control the coordination of diatomic molecules at the heme/non-heme diiron site.…”

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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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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“…The dinuclear iron centers of the hydroxylase reside just 12 Å below the canyon floor, and it has been suggested that the binding sites of MMOR and MMOB may be located in these deep recesses (8). The results of spectroscopic (9,10), chemical crosslinking (11), and steady-state kinetic and isothermal calorimetric (3) studies support this proposal.…”

mentioning

confidence: 96%

Structure of the soluble methane monooxygenase regulatory protein B

Walters

Gassner

Lippard

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

119

148

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The soluble methane monooxygenase (sMMO; EC 1.14.13.25) from the pseudothermophile Methylococcus capsulatus (Bath) is a three-component enzyme system that catalyzes the selective oxidation of methane to methanol. We have used NMR spectroscopy to produce a highly refined structure of MMOB, the 16-kDa regulatory protein of this system. This structure has a unique and intricate fold containing seven ␤-strands forming two ␤-sheets oriented perpendicular to each other and bridged by three ␣-helices. The rate and efficiency of the methane hydroxylation by sMMO depend on dynamic binding interactions of the hydroxylase with the reductase and regulatory protein components during catalysis. We have monitored by NMR the binding of MMOB to the hydroxylase in the presence and absence of the reductase. The results of these studies provide structural insight into how the regulatory protein interacts with the hydroxylase.Methanotrophic bacteria abound at the interface of aerobic and anaerobic sediments, where they rely on methane as their primary source of carbon and energy. In the first step of metabolism, these organisms employ either soluble or membrane-bound methane monooxygenase (MMO; EC 1.14.13.25) systems to oxidize methane selectively to methanol (1, 2):The soluble methane monooxygenase system (sMMO) isolated from the pseudothermophile Methylococcus capsulatus (Bath) functions optimally at 45°C. It is composed of a 251-kDa dinuclear iron-containing dimeric hydroxylase (MMOH), a 39-kDa NADH-dependent [2Fe-2S]-and FAD-containing reductase (MMOR), and a 16-kDa regulatory protein (MMOB). The regulatory protein (MMOB) exists in solution as a monomer (3, 4) and binds to the hydroxylase dimer at two weakly interacting sites (3). When present at low concentration, MMOB converts MMOH from an oxidase to a hydroxylase (3, 5) and stabilizes intermediates required for the activation of dioxygen (6, 7). As the MMOB concentration is increased to saturate both binding sites, the hydroxylase activity is attenuated (3, 7).The crystal structure of MMOH reveals two juxtaposed canyon regions formed by its ␣ and ␤ subunits (8). The dinuclear iron centers of the hydroxylase reside just 12 Å below the canyon floor, and it has been suggested that the binding sites of MMOR and MMOB may be located in these deep recesses (8). The results of spectroscopic (9, 10), chemical crosslinking (11), and steady-state kinetic and isothermal calorimetric (3) studies support this proposal.We have used high-resolution NMR spectroscopy to determine the solution structure of the regulatory protein and to identify residues on its surface involved in hydroxylase binding. The effects of reductase on the MMOB͞H interaction were also investigated. MATERIALS AND METHODSSample Preparation. MMOB was prepared from a recombinant expression system in Escherichia coli (ref. 12; D. Coufal, J. L. Blazyk, and S.J.L., unpublished work) and purified as described previously (3). NMR experiments were typically run at 25°C with 1.0 mM samples in 50 mM sodium phosphate buffer...

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“…The soluble MMO (sMMO) is a complex threecomponent system consisting of a hydroxylase, a reductase, and a small regulatory protein (4). The sMMO has been investigated extensively by several research groups (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). The xray crystal structure of the sMMO hydroxylase isolated from Methylococcus capsulatus (Bath) has been solved (22,23).…”

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confidence: 99%

The Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath) Is a Novel Copper-containing Three-subunit Enzyme

Nguyen

Elliott

Yip

et al. 1998

Journal of Biological Chemistry

219

240

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The particulate methane monooxygenase (pMMO) is known to be very difficult to study mainly due to its unusual activity instability in vitro. By cultivating Methylococcus capsulatus (Bath) under methane stress conditions and high copper levels in the growth medium, membranes highly enriched in the pMMO with exceptionally stable activity can be isolated from these cells. Purified and active pMMO can be subsequently obtained from these membrane preparations using protocols in which an excess of reductants and anaerobic conditions were maintained during membrane solubilization by dodecyl ␤-D-maltoside and purification by chromatography. The pMMO was found to be the major constituent in these membranes, constituting 60 -80% of total membrane proteins. The dominant species of the pMMO was found to consist of three subunits, ␣, ␤, and ␥, with an apparent molecular mass of 45, 26, and 23 kDa, respectively. A second species of the pMMO, a proteolytically processed version of the enzyme, was found to be composed of three subunits, ␣, ␤, and ␥, with an apparent molecular mass of 35, 26, and 23 kDa, respectively. The ␣ and ␣ subunits from these two forms of the pMMO contain identical N-terminal sequences. The ␥ subunit, however, exhibits variation in its N-terminal sequence. The pMMO is a copper-containing protein only and shows a requirement for Cu(I) ions. Approximately 12-15 Cu ions per 94-kDa monomeric unit were observed. The pMMO is sensitive to dioxygen tension. On the basis of dioxygen sensitivity, three kinetically distinct forms of the enzyme can be distinguished. A slow but air-stable form, which is converted into a "pulsed" state upon direct exposure to atmospheric oxygen pressure, is considered as type I pMMO. This form was the subject of our pMMO isolation effort. Other forms (types II and III) are deactivated to various extents upon exposure to atmospheric dioxygen pressure. Under inactivating conditions, these unstable forms release protons to the buffer (ϳ10 H ؉ /94-kDa monomeric unit) and eventually become completely inactive.The enzyme methane monooxygenase, found in methanotrophic bacteria, catalyzes the conversion of methane to methanol using dioxygen as a co-substrate at ambient temperatures and pressures (1, 2). This system has attracted considerable attention, since it provides an ideal natural model to study methane activation and functionalization, a subject of significant current interest (3). Two distinct species of methane monooxygenase (MMO) 1 are known to exist at different cellular locations, a cytoplasmic (soluble) MMO and a membrane-bound (particulate) MMO (4). The soluble MMO (sMMO) is a complex threecomponent system consisting of a hydroxylase, a reductase, and a small regulatory protein (4). The sMMO has been investigated extensively by several research groups (5-21). The xray crystal structure of the sMMO hydroxylase isolated from Methylococcus capsulatus (Bath) has been solved (22, 23). The hydroxylase active site contains a non-heme binuclear iron cluster. In contrast, the particulat...

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Spectroscopic studies of the coupled binuclear non-heme iron active site in the fully reduced hydroxylase component of methane monooxygenase: comparison to deoxy and deoxy-azide hemerythrin

Cited by 94 publications

References 9 publications

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

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

Structure of the soluble methane monooxygenase regulatory protein B

The Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath) Is a Novel Copper-containing Three-subunit Enzyme

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