Soluble methane monooxygenase (sMMO) catalyzes the cleavage of the strong C-H bond of methane during the O2-dependent conversion to methanol.1 sMMO generates nature’s most powerful oxidizing agent, a Fe2-(mu-oxo)2 species termed Q, for this reaction. The sMMO catalytic cycle is strictly regulated to ensure that methane is afforded preferential access to Q, as Q is capable of oxidizing any molecule with a C-H or C=C bond that gains access to the active site. This methane selection process is ascribed to a small-molecule tunnel to the active site that discriminates based upon the molecular size of the substrate.2 The external transfer of electrons required to reset the dinuclear iron cluster for consecutive catalytic cycles is also temporally regulated to ensure that Q is not quenched by its own reductase partner protein. This regulation is proposed to be orchestrated by the presence of competitive protein complexes in the multiprotein sMMO complex.3,4 The experimental validation of these regulatory models is held back by the inability to mutate the hydroxylase protein (MMOH), which harbors the active site, in a site-specific manner. We have overcome this obstacle by recombinantly expressing MMOH in E.coli through co-expression with two other proteins, MMOG and MMOD, from the sMMO operon. This results in a large yield of a fully functional MMOH protein for sMMO structure-function studies. We have used this newfound capability to create site-directed mutants of the small-molecule tunnel for hydrocarbon substrate access within MMOH as well as the proposed route for electron transfer to the diiron cluster. Transient kinetic studies of the mutant MMOH protein has enabled us to delineate how the MMOH protein structure regulates the selection and temporal delivery of the hydrocarbon and electron substrates.