title: The Kinetic Reaction Mechanism of the Vibrio cholerae Sodium-dependent NADH Dehydrogenase.
abstract: The sodium-dependent NADH dehydrogenase (Na(+)-NQR) is the main ion transporter in Vibrio cholerae. Its activity is linked to the operation of the respiratory chain and is essential for the development of the pathogenic phenotype. Previous studies have described different aspects of the enzyme, including the electron transfer pathways, sodium pumping structures, cofactor and subunit composition, among others. However, the mechanism of the enzyme remains to be completely elucidated. In this work, we have studied the kinetic mechanism of Na(+)-NQR with the use of steady state kinetics and stopped flow analysis. Na(+)-NQR follows a hexa-uni ping-pong mechanism, in which NADH acts as the first substrate, reacts with the enzyme, and the oxidized NAD leaves the catalytic site. In this conformation, the enzyme is able to capture two sodium ions and transport them to the external side of the membrane. In the last step, ubiquinone is bound and reduced, and ubiquinol is released. Our data also demonstrate that the catalytic cycle involves two redox states, the three- and five-electron reduced forms. A model that gathers all available information is proposed to explain the kinetic mechanism of Na(+)-NQR. This model provides a background to understand the current structural and functional information.
authors.affiliation: From the Department of Biological Sciences, Illinois Institute of Technology, Chicago, Illinois 60616 and.
authors.forename: Karina
authors.initials: K
authors.lastname: Tuz
authors.affiliation: the Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York 12180.
authors.forename: Katherine G
authors.initials: KG
authors.lastname: Mezic
authors.affiliation: From the Department of Biological Sciences, Illinois Institute of Technology, Chicago, Illinois 60616 and.
authors.forename: Tianhao
authors.initials: T
authors.lastname: Xu
authors.affiliation: the Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York 12180.
authors.forename: Blanca
authors.initials: B
authors.lastname: Barquera
authors.affiliation: From the Department of Biological Sciences, Illinois Institute of Technology, Chicago, Illinois 60616 and ojuarez@iit.edu.
authors.forename: Oscar
authors.initials: O
authors.lastname: Juárez
mesh_terms: D055162:Biocatalysis; D007700:Kinetics; D009245:NADH Dehydrogenase / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D013379:Substrate Specificity; D014734:Vibrio cholerae / Q000201:enzymology*
chemical_list: D012964:Sodium; D009245:NADH Dehydrogenase
keywords: Na+-NQR; electron transport; enzyme kinetics; enzyme mechanism; inhibition mechanism; membrane enzyme; membrane transport; ping pong mechanism; reaction mechanism; sodium transport