Manny Semonis

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme capable of catalyzing the conversion of methane to methanol at ambient temperature and pressure. The enzyme is composed of a 245 kDa hydroxylase component (MMOH), a 37 kDa reductase component (MMOR), and a 16 kDa regulatory component (MMOB). Complex formation between MMOH and MMOB increases O2 reactivity 1000-fold, increases turnover 150-fold, and affects a 4000-fold increase in selectivity of methane over ethane. Previous studies have involved mutating MMOB amino acids present in the interface with MMOH, complemented by in-depth steady state and transient kinetics on the effect these mutants have on turnover and reaction cycle intermediates. The sMMO reaction cycle has been shown to have at least 8 intermediates termed Hox, Hred, O, P*, P, Q, R and T. The DBL2 (S109A/T111A) and DBL1 (N107G/S110A) MMOB mutants change the kinetics of the key intermediate Q as it reacts with alternative substrates larger than methane. The H5A and H33A mutants have effects on formation and/or decay rate constants of reaction cycles intermediates O, P*, and P. In this study, the MMOH complex with each MMOB mutant has been crystallized and the 3D structure determined using X-ray crystallography. These structures are used to rationalize the changes observed in the single turnover kinetics of reaction cycle intermediate formation and decay as a result of mutation.