Intense interest has focused on the development of enzymes as next-generation catalysts for the production of molecules with biotechnological potential. OleTJE, a member of the cytochrome P450 CYP152 family is an excellent candidate for the generation of advanced biofuels. OleT JE catalyzes the hydrogen peroxide-dependent decarboxylation of C n fatty acids to produce Cn-1 terminal alkenes. Despite the ability of this enzyme to yield long chain alkenes with nearly exclusive chemoselectivity, its reactivity towards shorter chain length substrates results in undesired levels of hydroxylated side-products, limiting its industrial potential. Previous transient kinetic studies have demonstrated that, despite its unusual chemistry, the reaction is initiated by canonical substrate hydrogen-atom abstraction by an iron(IV)-oxo intermediate (compound I) followed by the formation of a hyperstable iron(IV)-hydroxide species (compound II). The origin of chemoselectivity in OleTJE, and CYP152s in general, has been probed by a coordinated study of several CYP152 family members, including OleTJE, P450-BSbeta, CYP-MP, and the newly characterized OleTSA. Spectroscopically labeled substrates, in concert with UV-Vis spectroscopy, EPR spectroscopy and transient kinetic studies show that substrate constriction is necessary for alkene production, but compromises the rate of product egress. Moreover, alteration of the environment surrounding the axial thiolate ligand in OleTSA via mutagenesis has allowed the establishment of a platform to extend research towards the electronic characterization of the catalytically relevant intermediates, compound I and compound II.