Light‐Activated Electron Transfer and Catalytic Mechanism of Carnitine Oxidation by Rieske‐Type Oxygenase from Human Microbiota

Oxidation of quaternary ammonium substrate, carnitine by non‐heme iron containing Acinetobacter baumannii (Ab) oxygenase CntA/reductase CntB is implicated in the onset of human cardiovascular disease. Herein, we develop a blue‐light (365 nm) activation of NADH coupled to electron paramagnetic resonance (EPR) measurements to study electron transfer from the excited state of NADH to the oxidized, Rieske‐type, [2Fe‐2S](2+) cluster in the AbCntA oxygenase domain with and without the substrate, carnitine. Further electron transfer from one‐electron reduced, Rieske‐type [2Fe‐2S](1+) center in AbCntA‐WT to the mono‐nuclear, non‐heme iron center through the bridging glutamate E205 and subsequent catalysis occurs only in the presence of carnitine. The electron transfer process in the AbCntA‐E205A mutant is severely affected, which likely accounts for the significant loss of catalytic activity in the AbCntA‐E205A mutant. The NADH photo‐activation coupled with EPR is broadly applicable to trap reactive intermediates at low temperature and creates a new method to characterize elusive intermediates in multiple redox‐centre containing proteins.