Substrate-Protein Interactions of Type II NADH:Quinone Oxidoreductase from Escherichia coli

Biochemistry. 2016 May 17;55(19):2722-34. doi: 10.1021/acs.biochem.6b00070. Epub 2016 May 5.

Abstract

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains and responsible for the maintenance of NADH/NAD(+) balance in cells. NDH-2s are the only enzymes with NADH dehydrogenase activity present in the respiratory chain of many pathogens, and thus, they were proposed as suitable targets for antimicrobial therapies. In addition, NDH-2s were also considered key players for the treatment of complex I-related neurodegenerative disorders. In this work, we explored substrate-protein interaction in NDH-2 from Escherichia coli (EcNDH-2) combining surface-enhanced infrared absorption spectroscopic studies with electrochemical experiments, fluorescence spectroscopy assays, and quantum chemical calculations. Because of the specific stabilization of substrate complexes of EcNDH-2 immobilized on electrodes, it was possible to demonstrate the presence of two distinct substrate binding sites for NADH and the quinone and to identify a bound semiprotonated quinol as a catalytic intermediate.

MeSH terms

  • Benzoquinones / chemistry*
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • NAD / chemistry*
  • NADH Dehydrogenase / chemistry*
  • NADH Dehydrogenase / genetics
  • NADH Dehydrogenase / metabolism
  • Substrate Specificity

Substances

  • Benzoquinones
  • Escherichia coli Proteins
  • NAD
  • quinone
  • NADH dehydrogenase II
  • NADH Dehydrogenase