Protein engineering of a cytochrome P450 decarboxylase from Jeotgalicoccus sp. ATCC 8456: structural, spectroscopic and kinetic studies

The bacterial cytochrome P450 peroxygenase family (CYP152) represents a unique class of heme-containing monooxygenases that utilise hydrogen peroxide as an oxidant, bypassing the canonical P450 redox partner system. Members of this family, particularly OleTJE, originally isolated from Jeotgalicoccus sp. ATCC 8456, are capable of catalysing oxidative decarboxylation of fatty acids to produce terminal alkenes (olefins). These olefins are of growing industrial interest as potential drop-in biofuels. The key reactive intermediate in this catalytic cycle is the ferryl-oxo porphyrin π-cation radical, commonly referred to as Compound I. However, due to its high reactivity and transient nature, direct characterisation of Compound I remains a significant challenge. This study focuses on engineering the hydrogen-bonding network surrounding the heme-b prosthetic group in OleTJE through site-directed mutagenesis, with the aim of extending the lifetime of Compound I. A combination of structural, kinetic, and spectroscopic techniques was employed to characterise the impact of these mutations. Fatty acid binding titrations revealed altered binding affinities through dissociation constants, stopped-flow UV/Vis spectrophotometry indicated a significant reduction in Compound I decay rate, and high-resolution macromolecular crystallography provided structural insights into the modified active site architecture. Future studies will incorporate techniques such as electron paramagnetic resonance (EPR) spectroscopy to investigate changes in the heme iron spin state equilibrium, offering deeper mechanistic insight into how these mutations influence the electronic environment of the active site as well as GC-MS analysis to confirm product formation and fatty acid presence post-purification. Collectively, these results demonstrate the potential of rational protein engineering to stabilise reactive intermediates and enhance enzymatic performance for biotechnological applications.