Structural Studies of the Methylosinus trichosporium OB3b Soluble Methane Monooxygenase Hydroxylase and Regulatory Component Complex Reveal a Transient Substrate Tunnel

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Abstract

The metalloenzyme soluble methane monooxygenase (sMMO) consists of hydroxylase (sMMOH), regulatory (MMOB), and reductase components. When sMMOH forms a complex with MMOB, the rate constants are greatly increased for the sequential access of O2, protons, and CH4 to an oxygen-bridged diferrous metal cluster located in the buried active site. Here, we report high-resolution X-ray crystal structures of the diferric and diferrous states of both sMMOH and the sMMOH:MMOB complex using the components from Methylosinus trichosporium OB3b. These structures are analyzed for O2 access routes enhanced when the complex forms. Previously reported, lower-resolution structures of the sMMOH:MMOB complex from the sMMO of Methylococcus capsulatus Bath revealed a series of cavities through sMMOH postulated to serve as the O2 conduit. This potential role is evaluated in greater detail using the current structures. Additionally, a search for other potential O2 conduits in the M. trichosporium OB3b sMMOH:MMOB complex revealed a narrow molecular tunnel, termed the W308-tunnel. This tunnel is sized appropriately for O2 and traverses the sMMOH-MMOB interface before accessing the active site. The kinetics of reaction of O2 with the diferrous sMMOH:MMOB complex in solution show that use of the MMOB V41R variant decreases the rate constant for O2 binding >25000-fold without altering the component affinity. The location of Val41 near the entrance to the W308-tunnel is consistent with the tunnel serving as the primary route for the transfer of O2 into the active site. Accordingly, the crystal structures show that formation of the diferrous sMMOH:MMOB complex restricts access through the chain of cavities while opening the W308-tunnel.

Original languageEnglish (US)
Pages (from-to)2946-2961
Number of pages16
JournalBiochemistry
Volume59
Issue number32
DOIs
StatePublished - Aug 18 2020

Bibliographical note

Funding Information:
This work is based upon research conducted at the Northeastern Collaborative Access Team (NE-CAT) beamlines, which are funded by U.S National Institutes of Health (NIH) Grant P30 GM124165. The Pilatus 6M detector on beamline 24-ID-C is funded by NIH-ORIP HEI Grant S10 RR029205. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357. The authors thank the staff at the NE-CAT beamlines, Advanced Photon Source, Argonne National Laboratory, for assisting with data collection.

Funding Information:
The authors acknowledge the financial support of this work from National Institutes of Health Grant GM118030 (to J.D.L.), Grant GM118047 (to H.A.), and Training Grant GM08347 (to J.C.J.).

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