1. Light-Activated Electron Transfer and Catalytic Mechanism of Carnitine Oxidation by Rieske-Type Oxygenase from Human Microbiota.
- Author
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Shanmugam M, Quareshy M, Cameron AD, Bugg TDH, and Chen Y
- Subjects
- Acinetobacter baumannii enzymology, Acinetobacter baumannii isolation & purification, Bacterial Proteins genetics, Carnitine chemistry, Catalysis, Electron Spin Resonance Spectroscopy, Electron Transport, Humans, Iron-Sulfur Proteins chemistry, Iron-Sulfur Proteins genetics, Iron-Sulfur Proteins metabolism, Mutagenesis, Site-Directed, NAD chemistry, Oxidation-Reduction, Oxidoreductases genetics, Bacterial Proteins metabolism, Carnitine metabolism, Light, Microbiota, Oxidoreductases metabolism
- Abstract
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., (© 2020 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)- Published
- 2021
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