Dataset Information

Differential Transcriptional Activation of Genes Encoding Soluble Methane Monooxygenase in a Facultative Versus an Obligate Methanotroph.

ABSTRACT: Methanotrophs are a specialized group of bacteria that can utilize methane (CH?) as a sole energy source. A key enzyme responsible for methane oxidation is methane monooxygenase (MMO), of either a soluble, cytoplasmic type (sMMO), or a particulate, membrane-bound type (pMMO). Methylocellasilvestris BL2 and Methyloferulastellata AR4 are closely related methanotroph species that oxidize methane via sMMO only. However, Methyloferulastellata is an obligate methanotroph, while Methylocellasilvestris is a facultative methanotroph able to grow on several multicarbon substrates in addition to methane. We constructed transcriptional fusions of the mmo promoters of Methyloferulastellata and Methylocellasilvestris to a promoterless gfp in order to compare their transcriptional regulation in response to different growth substrates, in the genetic background of both organisms. The following patterns were observed: (1) The mmo promoter of the facultative methanotroph Methylocella silvestris was either transcriptionally downregulated or repressed by any growth substrate other than methane in the genetic background of Methylocellasilvetris; (2) Growth on methane alone upregulated the mmo promoter of Methylocellasilvetris in its native background but not in the obligate methanotroph Methyloferulastellata; (3) The mmo promoter of Methyloferulastellata was constitutive in both organisms regardless of the growth substrate, but with much lower promoter activity than the mmo promoter of Methylocellasilvetris. These results support a conclusion that a different mode of transcriptional regulation of sMMO contributes to the facultative lifestyle of Methylocellasilvetris compared to the obligate methanotroph Methyloferulastellata.

SUBMITTER: Smirnova AV

PROVIDER: S-EPMC5874634 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Differential Transcriptional Activation of Genes Encoding Soluble Methane Monooxygenase in a Facultative Versus an Obligate Methanotroph.

Smirnova Angela V AV Dunfield Peter F PF

Microorganisms 20180306 1

Methanotrophs are a specialized group of bacteria that can utilize methane (CH₄) as a sole energy source. A key enzyme responsible for methane oxidation is methane monooxygenase (MMO), of either a soluble, cytoplasmic type (sMMO), or a particulate, membrane-bound type (pMMO). Methylocellasilvestris BL2 and Methyloferulastellata AR4 are closely related methanotroph species that oxidize methane via sMMO only. However, Methyloferulastellata is an obligate m ...[more]

PMID: 29509697

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Project description:The controlled oxidation of methane to methanol is a chemical transformation of great value, particularly in the pursuit of alternative fuels, but the reaction remains underutilized industrially because of inefficient and costly synthetic procedures. In contrast, methane monooxygenase enzymes (MMOs) from methanotrophic bacteria achieve this chemistry efficiently under ambient conditions. In this Account, we discuss the first observable step in the oxidation of methane at the carboxylate-bridged diiron active site of the soluble MMO (sMMO), namely, the reductive activation of atmospheric O(2). The results provide benchmarks against which the dioxygen activation mechanisms of other bacterial multicomponent monooxygenases can be measured. Molecular oxygen reacts rapidly with the reduced diiron(II) cen-ter of the hydroxylase component of sMMO (MMOH). The first spectroscopically characterized intermediate that results from this process is a peroxodiiron(III) species, P*, in which the iron atoms have identical environments. P* converts to a second peroxodiiron(III) unit, H(peroxo), in a process accompanied by the transfer of a proton, probably with the assistance of a residue near the active site. Proton-promoted O-O bond scission and rearrangement of the diiron core then leads to a diiron(IV) unit, termed Q, that is directly responsible for the oxidation of methane to methanol. In one section of this Account, we provide a detailed discussion of these processes, with particular emphasis on possible structures of the intermediates. The geometries of P* and H(peroxo) are currently unknown, and recent synthetic modeling chemistry has highlighted the need for further structural characterization of Q, currently assigned as a di(μ-oxo)diiron(IV) "diamond core." In another section of the Account, we discuss in detail proton transfer during the O(2) activation events. The role of protons in promoting O-O bond cleavage, thereby initiating the conversion of H(peroxo) to Q, was previously a controversial topic. Recent studies of the mechanism, covering a range of pH values and in D(2)O instead of H(2)O, confirmed conclusively that the transfer of protons, possibly at or near the active site, is necessary for both P*-to-H(peroxo) and H(peroxo)-to-Q conversions. Specific mechanistic insights into these processes are provided. In the final section of the Account, we present our view of experiments that need to be done to further define crucial aspects of sMMO chemistry. Here our goal is to detail the challenges that we and others face in this research, particularly with respect to some long-standing questions about the system, as well as approaches that might be used to solve them.

Project description:During a single turnover of the hydroxylase component (MMOH) of soluble methane monooxygenase from Methylosinus trichosporium OB3b, several discrete intermediates are formed. The diiron cluster of MMOH is first reduced to the Fe(II)Fe(II) state (H(red)). O₂ binds rapidly at a site away from the cluster to form the Fe(II)Fe(II) intermediate O, which converts to an Fe(III)Fe(III)-peroxo intermediate P and finally to the Fe(IV)Fe(IV) intermediate Q. Q binds and reacts with methane to yield methanol and water. The rate constants for these steps are increased by a regulatory protein, MMOB. Previously reported transient kinetic studies have suggested that an intermediate P* forms between O and P in which the g = 16 EPR signal characteristic of the reduced diiron cluster of H(red) and O is lost. This was interpreted as signaling oxidation of the cluster, but a low level of accumulation of P* prevented further characterization. In this study, three methods for directly detecting and trapping P* are applied together to allow its spectroscopic and kinetic characterization. First, the MMOB mutant His33Ala is used to specifically slow the decay of P* without affecting its formation rate, leading to its nearly quantitative accumulation. Second, spectra-kinetic data collection is used to provide a sensitive measure of the formation and decay rate constants of intermediates as well as their optical spectra. Finally, the substrate furan is included to react with Q and quench its strong chromophore. The optical spectrum of P* closely mimics those of H(red) and O, but it is distinctly different from that of P. The reaction cycle rate constants allowed prediction of the times for maximal accumulation of the intermediates. Mössbauer spectra of rapid freeze-quench samples at these times show that the intermediates are formed at almost exactly the predicted levels. The Mössbauer spectra show that the diiron cluster of P*, quite unexpectedly, is in the Fe(II)Fe(II) state. Thus, the loss of the g = 16 EPR signal results from a change in the electronic structure of the Fe(II)Fe(II) center rather than oxidation. The similarity of the optical and Mössbauer spectra of H(red), O, and P* suggests that only subtle changes occur in the electronic and physical structure of the diiron cluster as P* forms. Nevertheless, the changes that do occur are necessary for O₂ to be activated for hydrocarbon oxidation.

Dataset Information

Differential Transcriptional Activation of Genes Encoding Soluble Methane Monooxygenase in a Facultative Versus an Obligate Methanotroph.

Publications

Differential Transcriptional Activation of Genes Encoding Soluble Methane Monooxygenase in a Facultative Versus an Obligate Methanotroph.

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