The key role of glutamate 172 in the mechanism of type II NADH: quinone oxidoreductase of Staphylococcus aureus

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Abstract

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane bound enzymes that deliver electrons to the respiratory chain by oxidation of NADH and reduction of quinones. In this way, these enzymes also contribute to the regeneration of NAD+, allowing several metabolic pathways to proceed. As for the other members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, the enzymatic mechanism of NDH-2s is still little explored and elusive. In this work we addressed the role of the conserved glutamate 172 (E172) residue in the enzymatic mechanism of NDH-2 from Staphylococcus aureus. We aimed to test our earlier hypothesis that E172 plays a key role in proton transfer to allow the protonation of the quinone. For this we performed a complete biochemical characterization of the enzyme's variants E172A, E172Q and E172S. Our steady state kinetic measurements show a clear decrease in the overall reaction rate, and our substrate interaction studies indicate the binding of the two substrates is also affected by these mutations. Interestingly our fast kinetic results show quinone reduction is more affected than NADH oxidation. We have also determined the X-ray crystal structure of the E172S mutant (2.55 Ǻ) and compared it with the structure of the wild type (2.32 Ǻ). Together these results support our hypothesis for E172 being of central importance in the catalytic mechanism of NDH-2, which may be extended to other members of the tDBDF superfamily.

Original languageEnglish
Pages (from-to)823-832
Number of pages10
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume1858
Issue number10
DOIs
Publication statusPublished - 1 Oct 2017

Keywords

  • Alternative NADH dehydrogenase
  • Charge transfer complex
  • Disulfide reductases
  • Electron transfer chain
  • Energetic metabolism
  • Respiratory chain

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