Real-time capture of reactive intermediates in an enzymatic reaction: insights into a P450-catalyzed oxidation

Enzymes are indispensable for a myriad of biochemical reactions in nature. Although rapid spectroscopic techniques and <i>operando</i> methods allow for real-time assessment of enzyme kinetics, isolating and identifying the reactive intermediates in enzymatic reactions remains a significant challenge. These intermediates often possess fleeting lifespans, prohibiting their direct detection. This study harnessed online mass spectrometric techniques and microfluidic sampling methods to monitor the formation of reactive intermediates in real time during enzymatic catalysis <i>in vitro</i>. Using CYP175A1, a thermophilic P450 enzyme known for its dual-function capabilities in peroxygenase and peroxidase activities, we investigated the oxidative dimerization of 1-methoxynaphthalene. This biocatalysis involves multiple intermediates, including resonating radical forms. By transposing the enzyme reaction into a 500 mM ammonium acetate buffer and spraying it into a mass spectrometer, we could detect various transient intermediate species, suggesting their preservation in buffer droplets during their flight. The resonance-like radical intermediates were distinguished and temporally evaluated using a parallel reaction monitoring approach in tandem mass spectrometry after being trapped with TEMPO, a radical marker. This comprehensive analysis successfully elucidated the complete catalytic cycle of the enzyme, offering a robust method for gaining deeper insights into enzyme action by tracking multiple intermediates in real time.