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Cohn's Crenothrix is a filamentous methane oxidizer with an unusual methane monooxygenase.

ABSTRACT: 135 years ago Ferdinand Cohn, the founder of bacteriology, microscopically observed a conspicuous filamentous bacterium with a complex life cycle and described it as Crenothrix polyspora. This uncultured bacterium is infamous for mass developments in drinking water systems, but its phylogeny and physiology remained unknown. We show that C. polyspora is a gammaproteobacterium closely related to methanotrophs and capable of oxidizing methane. We discovered that C. polyspora encodes a phylogenetically very unusual particulate methane monooxygenase whose expression is strongly increased in the presence of methane. Our findings demonstrate a previously unrecognized complexity of the evolutionary history and cell biology of methane-oxidizing bacteria.

SUBMITTER: Stoecker K

PROVIDER: S-EPMC1413686 | biostudies-literature | 2006 Feb

REPOSITORIES: biostudies-literature

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Cohn's Crenothrix is a filamentous methane oxidizer with an unusual methane monooxygenase.

Stoecker Kilian K Bendinger Bernd B Schöning Björn B Nielsen Per H PH Nielsen Jeppe L JL Baranyi Christian C Toenshoff Elena R ER Daims Holger H Wagner Michael M

Proceedings of the National Academy of Sciences of the United States of America 20060201 7

135 years ago Ferdinand Cohn, the founder of bacteriology, microscopically observed a conspicuous filamentous bacterium with a complex life cycle and described it as Crenothrix polyspora. This uncultured bacterium is infamous for mass developments in drinking water systems, but its phylogeny and physiology remained unknown. We show that C. polyspora is a gammaproteobacterium closely related to methanotrophs and capable of oxidizing methane. We discovered that C. polyspora encodes a phylogenetica ...[more]

PMID: 16452171

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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

Cohn's Crenothrix is a filamentous methane oxidizer with an unusual methane monooxygenase.

Publications

Cohn's Crenothrix is a filamentous methane oxidizer with an unusual methane monooxygenase.

Similar Datasets

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