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

Vivek Srinivas, Rahul Banerjee, Hugo Lebrette, Jason C. Jones, Oskar Aurelius, In Sik Kim, Cindy C. Pham, Sheraz Gul, Kyle D. Sutherlin, Asmit Bhowmick, Juliane John, Esra Bozkurt, Thomas Fransson, Pierre Aller, Agata Butryn, Isabel Bogacz, Philipp Simon, Stephen Keable, Alexander Britz, Kensuke TonoKyung Sook Kim, Sang Youn Park, Sang Jae Lee, Jaehyun Park, Roberto Alonso-Mori, Franklin D. Fuller, Alexander Batyuk, Aaron S. Brewster, Uwe Bergmann, Nicholas K. Sauter, Allen M. Orville, Vittal K. Yachandra, Junko Yano, John D. Lipscomb, Jan Kern, Martin Högbom

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29 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)14249-14266
Number of pages18
JournalJournal of the American Chemical Society
Issue number33
StatePublished - Aug 19 2020

Bibliographical note

Funding Information:
The authors acknowledge the financial support of this work from Grants NIH GM118030 (to J.D.L.), training grant GM08347 (to J.C.J.), training grant GM133081 (to K.D.S.), GM117126 (to N.K.S.), GM55302 (to V.K.Y.), GM110501 (to J.Y.), GM126289 (to J.K.), the Knut and Alice Wallenberg Foundation, and the Swedish Research Council 2017-04018, and the European Research Council HIGH-GEAR 724394 (to M.H.), Biotechnology and Biological Sciences Research Council Grant 102593 (to A.M.O.); Wellcome Investigator Award in Science 210734/Z/18/Z (to A.M.O.); Royal Society Wolfson Fellowship RSWF\R2\182017 (to A.M.O.) and by the Director, Office of Science, Office of Basic Energy Sciences (OBES), Division of Chemical Sciences, Geosciences, and Biosciences of the Department of Energy (DOE) (to J.K., J.Y., and V.K.Y.). We thank the synchrotron facilities at the European Synchrotron Radiation facility (Proposal MX-1948) and Swiss Light Source (Proposal 20182304). This research used resources of the National Energy Research Scientific Computing Center, a User Facility supported by the Office of Science, DOE, under contract DE-AC02-05CH11231. The structure refinements were performed using the PReSTO software stack on resources provided by the Swedish National Infrastructure for Computing (SNIC) at LUNARC Aurora, Lund University; Proposal SNIC 2018/3-251 and SNIC 2019/35-61. XFEL data were collected under proposal LU50 at LCLS/SLAC, Stanford, USA, under Proposal 2017B8085 at SACLA, Japan, and under Proposal 2019-2nd-NCI-029 at PAL-XFEL, Korea. The Rayonix detector used at LCLS was supported by the NIH grant S10 OD023453. LCLS, SLAC National Accelerator Laboratory, is supported by DOE, OBES under Contract DE-AC02-76SF00515. We thank the staff at LCLS, SACLA, and PAL-XFEL.

Publisher Copyright:
Copyright © 2020 American Chemical Society.


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