Direct Electrochemistry of the Hydroxylase of Soluble Methane Monooxygenase from <i>Methylococcus Capsulatus</i> (Bath)

Kazlauskaite, Jurate; Hill, H. Allen O.; Wilkins, Patricia C.; Dalton, Howard

doi:10.1111/j.1432-1033.1996.00552.x

Cited by 47 publications

(54 citation statements)

References 28 publications

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“…Proteolytically truncated MMOB binds to MMOH with only slightly lower affinity than the native protein, but loses its ability to tune the redox potential of the dinuclear iron centers of sMMO (26). The three sMMO regulatory protein sequences share high sequence conservation in their N termini, and our binding data suggest that D22 to A26 interact with the hydroxylase at 25°C.…”

Section: Discussionmentioning

confidence: 66%

Structure of the soluble methane monooxygenase regulatory protein B

Walters

Gassner

Lippard

et al. 1999

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

120

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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Section: Discussionmentioning

confidence: 66%

Structure of the soluble methane monooxygenase regulatory protein B

Walters

Gassner

Lippard

et al. 1999

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

120

148

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“…As judged from its gene content, this organism, named Methylomirabilis oxyfera, contains all the key enzymes that are also involved in aerobic methane oxidation [84][85][86][87][88][89][90][91][92], that is, methane monooxygenase (more specifically its membrane-bound version pMMO), methanol oxidase and the formaldehyde-activating enzyme (Fae). In contrast to ANMEs, where methane is transformed to methyl-CoM and then transferred to H 4 MPT for further oxidation, M. oxyfera uses methane via two consecutive 2e 2 oxidations yielding first methanol and then formaldehyde just as aerobic methanotrophs do (figure 3, left-hand side scheme).…”

Section: Denitrifying Methanotrophy: An Extant Energy Metabolism 'Reamentioning

confidence: 99%

Beating the acetyl coenzyme A-pathway to the origin of life

Nitschke

Russell

2013

Phil. Trans. R. Soc. B

118

174

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Attempts to draft plausible scenarios for the origin of life have in the past mainly built upon palaeogeochemical boundary conditions while, as detailed in a companion article in this issue, frequently neglecting to comply with fundamental thermodynamic laws. Even if demands from both palaeogeochemistry and thermodynamics are respected, then a plethora of strongly differing models are still conceivable. Although we have no guarantee that life at its origin necessarily resembled biology in extant organisms, we consider that the only empirical way to deduce how life may have emerged is by taking the stance of assuming continuity of biology from its inception to the present day. Building upon this conviction, we have assessed extant types of energy and carbon metabolism for their appropriateness to conditions probably pertaining in those settings of the Hadean planet that fulfil the thermodynamic requirements for life to come into being. Wood–Ljungdahl (WL) pathways leading to acetyl CoA formation are excellent candidates for such primordial metabolism. Based on a review of our present understanding of the biochemistry and biophysics of acetogenic, methanogenic and methanotrophic pathways and on a phylogenetic analysis of involved enzymes, we propose that a variant of modern methanotrophy is more likely than traditional WL systems to date back to the origin of life. The proposed model furthermore better fits basic thermodynamic demands and palaeogeochemical conditions suggested by recent results from extant alkaline hydrothermal seeps.

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“…After completion of the hydroxylation reaction, the regulatory protein may temporarily dissociate from the hydroxylase so that the reductase can bind and reinitiate the catalytic cycle. This scenario would explain why, in the sMMO system, binding of MMOB to MMOH shifts the redox potentials to disfavor reduction of the diiron center, whereas reductase binding shifts the potentials so that reduction is favored (65)(66)(67)(68). It is also possible that BMMs function via half-sites reactivity.…”

Section: Possible Consequences Of Regulatory Protein Binding On Transmentioning

confidence: 99%

X-ray Structure of a Hydroxylase−Regulatory Protein Complex from a Hydrocarbon-Oxidizing Multicomponent Monooxygenase, Pseudomonas sp. OX1 Phenol Hydroxylase^,

et al. 2006

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Phenol hydroxylase (PH) belongs to a family of bacterial multicomponent monooxygenases (BMMs) with carboxylate-bridged diiron active sites. Included are toluene/o-xylene (ToMO) and soluble methane (sMMO) monooxygenase. PH hydroxylates aromatic compounds, but unlike sMMO, it cannot oxidize alkanes despite having a similar dinuclear iron active site. Important for activity is formation of a complex between the hydroxylase and a regulatory protein component. To address how structural features of BMM hydroxylases and their component complexes may facilitate the catalytic mechanism and choice of substrate, we determined X-ray structures of native and SeMet forms of the PH hydroxylase (PHH) in complex with its regulatory protein (PHM) to 2.3 Å resolution. PHM binds in a canyon on one side of the (αβγ) 2 PHH dimer, contacting α-subunit helices A, E, and F ∼12 Å above the diiron core. The structure of the dinuclear iron center in PHH resembles that of mixed-valent MMOH, suggesting an Fe(II)Fe(III) oxidation state. Helix E, which comprises part of the iron-coordinating four-helix bundle, has more π-helical character than analogous E helices in MMOH and ToMOH lacking a bound regulatory protein. Consequently, conserved active site Thr and Asn residues translocate to the protein surface, and an ∼6 Å pore opens through the four-helix bundle. Of likely functional significance is a specific hydrogen bond formed between this Asn residue and a conserved Ser side chain on PHM. The PHM protein covers a putative docking site on PHH for the PH reductase, which transfers electrons to the PHH diiron center prior to O 2 activation, † This research was supported by National Institute of General Medical Sciences Grant GM32134 (S.J.L.) and the Italian Ministry of SUPPORTING INFORMATION AVAILABLE BMM hydroxylase and regulatory protein structures and their electrostatic surfaces (Figures S1 and S2), packing of the β-subunit Nterminus and a γ-subunit from an adjacent molecule in the PHH canyon ( Figure S3), comparison of known regulatory protein structures ( Figure S4), sequence alignments of the different regulatory proteins ( Figure S5), models of the PHH diiron center in electron density maps ( Figure S6), comparison of the hydroxylase zinc-binding sequences ( Figure S7), and electron density maps around helix F ( Figure S8). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2007 March 21. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptsuggesting that the regulatory component may function to block undesired reduction of oxygenated intermediates during the catalytic cycle. A series of hydrophobic cavities through the PHH α-subunit, analogous to those in MMOH, may facilitate movement of the substrate to and/or product from the active site pocket. Comparisons between the ToMOH and PHH structures provide insights into their substrate regiospecificities.Bacterial multicomponent monooxygenases...

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Direct Electrochemistry of the Hydroxylase of Soluble Methane Monooxygenase from Methylococcus Capsulatus (Bath)

Cited by 47 publications

References 28 publications

Structure of the soluble methane monooxygenase regulatory protein B

Structure of the soluble methane monooxygenase regulatory protein B

Beating the acetyl coenzyme A-pathway to the origin of life

X-ray Structure of a Hydroxylase−Regulatory Protein Complex from a Hydrocarbon-Oxidizing Multicomponent Monooxygenase, Pseudomonas sp. OX1 Phenol Hydroxylase^,

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Direct Electrochemistry of the Hydroxylase of Soluble Methane Monooxygenase from Methylococcus Capsulatus (Bath)

Cited by 47 publications

References 28 publications

Structure of the soluble methane monooxygenase regulatory protein B

Structure of the soluble methane monooxygenase regulatory protein B

Beating the acetyl coenzyme A-pathway to the origin of life

X-ray Structure of a Hydroxylase−Regulatory Protein Complex from a Hydrocarbon-Oxidizing Multicomponent Monooxygenase, Pseudomonas sp. OX1 Phenol Hydroxylase,

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Product

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X-ray Structure of a Hydroxylase−Regulatory Protein Complex from a Hydrocarbon-Oxidizing Multicomponent Monooxygenase, Pseudomonas sp. OX1 Phenol Hydroxylase^,