| The soluble methane monooxygenase system (sMMO) from Methylosinus trichosporium OB3b catalyzes the O2-dependent conversion of methane to methanol and consists of three proteins: a hydroxylase (MMOH), a reductase (MMOR), and an effector protein (MMOB). The active site of MMOH contains a binuclear iron cluster that reacts with oxygen to form an Fe(IV) 2-bis-μ-oxo species, termed compound Q (Q), which reacts, in turn, with substrates. The goal of this thesis is to elucidate novel aspects of the mechanism of the reaction of Q with substrate and the role of MMOB in this reaction.; Transient kinetic techniques are used to provide support for a two-step mechanism of the Q reaction with substrates: nominally, substrate binding followed by C-H bond cleavage. This process occurs with a large deuterium KIE when methane is the substrate indicative of rate-limiting C-H bond cleavage. In contrast, ethane oxidation shows no KIE, presumably because binding is rate limiting. Using a mutant form of MMOB (N107G/S109A/S110A/T111A; “Quad” mutant), a KIE is observed for ethane oxidation suggesting that access into the MMOH is facilitated, shifting the rate-limiting step to C-H bond cleavage. In contrast, the KIE for methane oxidation decreases substantially suggesting that the mechanism of C-H bond cleavage depends on the structure of the MMOH-MMOB complex. Further evaluation of the Quad mutant kinetics shows that Thr111 is primarily responsible for the changes in sMMO catalysis and active site structure. Protease mapping in combination with MALDI-TOF mass spectrometry is used to probe the binding surface between MMOB and MMOH. The first description the MMOH surface participating in the interface is provided. A model of the MMOB:MMOH complex is proposed that provides the first structural rationale for the functional effects of MMOB and the Quad mutant. The molecular mechanism of sMMO was probed using the diagnostic substrate norcarane. The resulting product distribution suggests that an intermediate substrate radical is formed the radical intermediate by the one electron reduced form of Q (intermediate R) prior to the normal rebound chemistry. |
