bi0c00459_si_002.mpg (19.65 MB)
Structural Studies of the Methylosinus trichosporium OB3b Soluble Methane Monooxygenase Hydroxylase and Regulatory Component Complex Reveal a Transient Substrate Tunnel
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posted on 2020-07-31, 11:46 authored by Jason
C. Jones, Rahul Banerjee, Ke Shi, Hideki Aihara, John D. LipscombThe 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.
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crystal structures showRegulatory Component Complex RevealX-ray crystal structuresoxygen-bridged diferrous metal clusterMethylosinus trichosporium OB 3b So...sMMOHTransient Substrate TunnelO 2 conduitW 308-tunnelCHO 2Methylococcus capsulatus BathO 2 conduitsO 2 access routesMethylosinus trichosporium OB 3bMMOB V 41R variant decreases
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