Abstract:

<p>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 O, protons, and CH 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 OB3b. These structures are analyzed for O access routes enhanced when the complex forms. Previously reported, lower resolution structures of the sMMOH:MMOB complex from the sMMO of Bath revealed a series of cavities through sMMOH postulated to serve as the O conduit. This potential role is evaluated in greater detail using the current structures. Additionally, a search for other potential O conduits in OB3b sMMOH:MMOB revealed a narrow molecular tunnel, termed the W308-Tunnel. This tunnel is sized appropriately for O and traverses the sMMOH-MMOB interface before accessing the active site. Kinetics of O reaction with diferrous sMMOH:MMOB in solution show that use of the MMOB V41R variant decreases the rate constant for O binding &gt;25,000-fold without altering component affinity. The location of Val41 near the entrance to the W308-Tunnel is consistent with the tunnel serving as the primary route for O transfer 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.</p>