%0 Journal Article
%T Dioxygen Activation by the Biofuel-Generating Cytochrome P450 OleT
%J -
%D 2018
%R https://doi.org/10.1021/acscatal.8b02631
%X OleT, a recently discovered member of the CYP152 family of cytochrome P450s, catalyzes a unique decarboxylation reaction, converting free fatty acids into 1-olefins and carbon dioxide using H2O2 as an oxidant. The C¨CC cleavage reaction proceeds through hydrogen atom abstraction by an iron(IV)-oxo intermediate known as Compound I. The capacity of the enzyme for generating important commodity chemicals and liquid biofuels has inspired a flurry of investigations seeking to maximize its biosynthetic potential. One common approach has sought to address the limitations imposed by the H2O2 cosubstrate, particularly for in vivo applications. Numerous reports have shown relatively efficient decarboxylation activity with various combinations of the enzyme with pyridine nucleotides, biological redox donors, and dioxygen, implicating a mechanism whereby OleT can generate Compound I via a canonical P450 O2 dependent reaction scheme. Here, we have applied transient kinetics, cryoradiolysis, and steady state turnover studies to probe the precise origins of OleT turnover from surrogate redox systems. Electron transfer from several redox donors is prohibitively sluggish, and the enzyme is unable to form the hydroperoxo-ferric adduct that serves as a critical precursor to Compound I. Despite the ability for OleT to readily bind O2 once it is reduced, autoxidation of the enzyme and redox partners leads to the generation of H2O2, which is ultimately responsible for the vast majority of turnover. These results illuminate several strategies for improving OleT for downstream biocatalytic applications
%U https://pubs.acs.org/doi/10.1021/acscatal.8b02631