Mechanistic Studies of Atypical Reactions and Uncommon Substrates Native to Bacterial Cytochrome P450 Enzymes OleT\u3csub\u3eJE\u3c/sub\u3e and NikQ

Cytochrome P450 (CYP) enzymes typically perform hydroxylation chemistry on small molecule substrates using atmospheric oxygen and electrons from redox partner proteins. This reactivity is dependent on a thiolate-ligated heme cofactor that also allows for spectroscopic detection of ligation, oxidation, and environmental changes to the iron metal. The onset of the genomics era has resulted in the discovery of CYPs that deviate from this paradigm in terms of reaction performed, cosubstrate requirements, and substrate-scope. This work focuses on two of these unusual P450s – OleT and NikQ. OleT performs an unconventional H2O2-dependent decarboxylation reaction on a standard small molecule fatty acid substrate to yield 1-olefins and CO2. A desire to maximize the biosynthetic potential of this enzyme while addressing limitations imposed by the peroxide requirement has resulted in numerous reports that OleT may have the capacity to utilize biological redox donors and O2 in a canonical P450 reaction mechanism. Chapter 2 centers on transient kinetics, cryoradiolysis, and turnover studies used to investigate the precise origins of OleT alkene production using surrogate redox systems and dioxygen. While results show that this enzyme is ultimately incapable of performing true O2-driven catalysis, conclusions do illuminate several strategies for improving OleT for downstream biocatalytic applications. Chapters 3 and 4 focus on NikQ, which carries out classical P450 β-hydroxylation chemistry using O2 and reducing equivalents from redox donors on a ʟ-histidine substrate requisitely appended to NikP1, a ~75 kDa nonribosomal peptide synthetase protein. The first half of the CYP catalytic cycle is typically regulated at multiple steps by the binding of substrate; however, this mechanistic gating in P450s acting on protein-tethered substrates is not well-elucidated. We use various spectroscopic techniques, transient kinetics, and spectroelectrochemical studies to investigate the influence of this complex substrate on early NikQ catalysis. Results indicate that NikP1-binding does not substantially regulate NikQ, which is highly atypical of P450s. Possible molecular determinants and functional significance of this mechanistic peculiarity will also be discussed