1. Kinetics of substrate inhibition of periplasmic nitrate reductase
- Author
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Bénédicte Burlat, Pascal Arnoux, Christophe Léger, Bruno Guigliarelli, Julien Jacques, David Pignol, Monique Sabaty, Vincent Fourmond, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Microbiologie Environnementale et Moléculaire (MEM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Stereochemistry ,Biophysics ,Reductase ,Nitrate reductase ,Photochemistry ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Nitrate ,[CHIM.ANAL]Chemical Sciences/Analytical chemistry ,Electrochemistry ,Enzyme kinetics ,Nitrites ,DMSO reductase ,[PHYS.PHYS]Physics [physics]/Physics [physics] ,Substrate (chemistry) ,Cell Biology ,Periplasmic space ,Kinetics ,chemistry ,Periplasm ,Thermodynamics ,Molybdenum cofactor ,Oxidation-Reduction ,EPR spectroscopy - Abstract
International audience; Periplasmic nitrate reductase catalyzes the reduction of nitrate into nitrite using a mononuclear molybdenum cofactor that has nearly the same structure in all enzymes of the DMSO reductase family. In previous electrochemical investigations, we found that the enzyme exists in several inactive states, some of which may have been previously isolated and mistaken for catalytic intermediates. In particular, the enzyme slowly and reversibly inactivates when exposed to high concentrations of nitrate. Here, we study the kinetics of substrate inhibition and its dependence on electrode potential and substrate concentration to learn about the properties of the active and inactive forms of the enzyme. We conclude that the substrate-inhibited enzyme never significantly accumulates in the EPR-active Mo(+ V) state. This conclusion is relevant to spectroscopic investigations where attempts are made to trap a Mo(+ V) catalytic intermediate using high concentrations of nitrate.
- Published
- 2014
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