title: Regulation of methane monooxygenase catalysis based on size exclusion and quantum tunneling.
abstract: The hydroxylase component (MMOH) of the soluble form of methane monooxygenase (sMMO) isolated from Methylosinus trichosporium OB3b catalyzes both the O2 activation and the CH4 oxidation reactions at the oxygen-bridged dinuclear iron cluster present in its buried active site. During the reaction cycle, the diiron cluster forms a bis-mu-oxo-(Fe(IV))2 intermediate termed compound Q (Q) that reacts directly with methane. Many adventitious substrates also react with Q, most at a relatively slow rate. We have proposed that Q reacts preferentially with CH4 because the sMMO regulatory component MMOB induces a size selective pore into the MMOH active site as the two components form a complex. Support for this proposal has come through the observation of a nonlinear Arrhenius plot for the CH4 oxidation, presumably due to a shift in rate-limiting step from substrate binding at low temperature to C-H bond cleavage at high temperature. Reactions of all substrates other than CH4 fail to exhibit a break in the Arrhenius plot because binding is always rate limiting in the temperature range explored. Here we show that it is possible to induce a break in the Arrhenius plot for the ethane reaction with Q by using an MMOB mutant termed DBL2 (S109A/T111A) in which residues at the MMOH-MMOB interface are reduced in size. We hypothesize that this increases the ethane binding rate and shifts the Arrhenius breakpoint into the observable temperature range. As a result of this shift, the kinetic and activation parameters of the C-H bond breaking reaction for both methane and ethane can be observed using the DBL2 mutant. A 2H-KIE is observed for both substrate oxidation reactions when using DBL2, whereas only CH4 oxidation exhibits an effect when using wild type MMOB, consistent with the C-H bond cleaving reaction becoming at least partially rate limiting for ethane. Analysis of the temperature dependence of the 2H-KIE for ethane and methane for reactions using both mutant and wild type forms of MMOB suggests that quantum tunneling plays a significant role in methane oxidation but not ethane oxidation.
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics and Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Zheng
authors.forename: John D
authors.initials: JD
authors.lastname: Lipscomb
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D004980:Ethane / Q000737:chemistry; D058085:Iron Compounds / Q000737:chemistry; D007700:Kinetics; D008697:Methane / Q000737:chemistry; D020602:Methylosinus trichosporium / Q000201:enzymology*; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016062:Porosity; D011789:Quantum Theory
chemical_list: D058085:Iron Compounds; D010105:Oxygenases; C021555:methane monooxygenase; D004980:Ethane; D008697:Methane