Enzymology
Identification of the binding sites for ubiquinone and inhibitors in the Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae by photoaffinity labeling
The Na+-pumping NADH-quinone oxidoreductase (Na+-NQR) is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, including Vibrio cholerae. The V. cholerae Na+-NQR has been extensively studied, but its binding sites for ubiquinone and inhibitors remain controversial. Here, using a photoreactive ubiquinone PUQ-3 as well as two aurachin-type inhibitors [125I]PAD-1 and [125I]PAD-2 and photoaffinity labeling experiments on the isolated enzyme, we demonstrate that the ubiquinone ring binds to the NqrA subunit in the regions Leu-32–Met-39 and Phe-131–Lys-138, encompassing the rear wall of a predicted ubiquinone-binding cavity.The Kinetic Reaction Mechanism of the Vibrio cholerae Sodium-dependent NADH Dehydrogenase
Background: Na+-NQR is the main sodium pump in Vibrio cholerae.Results: Na+-NQR follows a hexa-uni ping-pong mechanism, in which two redox states participate in the catalytic cycle.Conclusion: A comprehensive kinetic model of the reaction catalyzed by Na+-NQR is proposed, covering pre-steady and steady conditions.Significance: The kinetic model proposed provides a background to understand the structural and functional data.
