Dataset Information

High-Resolution XFEL Structure of the Soluble Methane Monooxygenase Hydroxylase Complex with its Regulatory Component at Ambient Temperature in Two Oxidation States.

ABSTRACT: Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O₂ activation and substrate hydroxylation. Structural studies of metalloenzymes using traditional synchrotron-based X-ray crystallography are often complicated by partial X-ray-induced photoreduction of the metal center, thereby obviating determination of the structure of the enzyme in pure oxidation states. Here, microcrystals of the sMMOH:MMOB complex from Methylosinus trichosporium OB3b were serially exposed to X-ray free electron laser (XFEL) pulses, where the ≤35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Merging diffraction patterns obtained from thousands of crystals generates radiation damage-free, 1.95 Å resolution crystal structures for the fully oxidized and fully reduced states of the sMMOH:MMOB complex for the first time. The results provide new insight into the manner by which the diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. This study also emphasizes the value of XFEL and serial femtosecond crystallography (SFX) methods for investigating the structures of metalloenzymes with radiation sensitive metal active sites.

SUBMITTER: Srinivas V

PROVIDER: S-EPMC7457426 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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High-Resolution XFEL Structure of the Soluble Methane Monooxygenase Hydroxylase Complex with its Regulatory Component at Ambient Temperature in Two Oxidation States.

Srinivas Vivek V Banerjee Rahul R Lebrette Hugo H Jones Jason C JC Aurelius Oskar O Kim In-Sik IS Pham Cindy C CC Gul Sheraz S Sutherlin Kyle D KD Bhowmick Asmit A John Juliane J Bozkurt Esra E Fransson Thomas T Aller Pierre P Butryn Agata A Bogacz Isabel I Simon Philipp P Keable Stephen S Britz Alexander A Tono Kensuke K Kim Kyung Sook KS Park Sang-Youn SY Lee Sang Jae SJ Park Jaehyun J Alonso-Mori Roberto R Fuller Franklin D FD Batyuk Alexander A Brewster Aaron S AS Bergmann Uwe U Sauter Nicholas K NK Orville Allen M AM Yachandra Vittal K VK Yano Junko J Lipscomb John D JD Kern Jan J Högbom Martin M

Journal of the American Chemical Society 20200805 33

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O<sub>2</sub> activation and substrate hydroxylation. Structural studie ...[more]

PMID: 32683863

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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.

Dataset Information

High-Resolution XFEL Structure of the Soluble Methane Monooxygenase Hydroxylase Complex with its Regulatory Component at Ambient Temperature in Two Oxidation States.

Publications

High-Resolution XFEL Structure of the Soluble Methane Monooxygenase Hydroxylase Complex with its Regulatory Component at Ambient Temperature in Two Oxidation States.

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