The extracellular A-loop of dual oxidases affects the specificity of reactive oxygen species release

J Biol Chem. 2015 Mar 6;290(10):6495-506. doi: 10.1074/jbc.M114.592717. Epub 2015 Jan 13.


NADPH oxidase (Nox) family proteins produce superoxide (O2 (⨪)) directly by transferring an electron to molecular oxygen. Dual oxidases (Duoxes) also produce an O2 (⨪) intermediate, although the final species secreted by mature Duoxes is H2O2, suggesting that intramolecular O2 (⨪) dismutation or other mechanisms contribute to H2O2 release. We explored the structural determinants affecting reactive oxygen species formation by Duox enzymes. Duox2 showed O2 (⨪) leakage when mismatched with Duox activator 1 (DuoxA1). Duox2 released O2 (⨪) even in correctly matched combinations, including Duox2 + DuoxA2 and Duox2 + N-terminally tagged DuoxA2 regardless of the type or number of tags. Conversely, Duox1 did not release O2 (⨪) in any combination. Chimeric Duox2 possessing the A-loop of Duox1 showed no O2 (⨪) leakage; chimeric Duox1 possessing the A-loop of Duox2 released O2 (⨪). Moreover, Duox2 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA2 showed enhanced O2 (⨪) release, and Duox1 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA1 acquired O2 (⨪) leakage. Although we identified Duox1 A-loop residues (His(1071), His(1072), and Gly(1074)) important for reducing O2 (⨪) release, mutations of these residues to those of Duox2 failed to convert Duox1 to an O2 (⨪)-releasing enzyme. Using immunoprecipitation and endoglycosidase H sensitivity assays, we found that the A-loop of Duoxes binds to DuoxA N termini, creating more stable, mature Duox-DuoxA complexes. In conclusion, the A-loops of both Duoxes support H2O2 production through interaction with corresponding activators, but complex formation between the Duox1 A-loop and DuoxA1 results in tighter control of H2O2 release by the enzyme complex.

Keywords: Glycosylation; Hydrogen Peroxide; NADPH Oxidase; Superoxide Ion; Thyroid Hormone.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Membrane / enzymology
  • Dual Oxidases
  • Glycosylation
  • HEK293 Cells
  • Humans
  • Hydrogen Peroxide / chemistry*
  • Hydrogen Peroxide / metabolism
  • Mutation
  • NADPH Oxidase 1
  • NADPH Oxidases / chemistry
  • NADPH Oxidases / metabolism*
  • Oxygen / chemistry
  • Oxygen / metabolism
  • Reactive Oxygen Species / chemistry
  • Reactive Oxygen Species / metabolism*
  • Superoxides / chemistry
  • Superoxides / metabolism
  • Thyroid Hormones / metabolism


  • Reactive Oxygen Species
  • Thyroid Hormones
  • Superoxides
  • Hydrogen Peroxide
  • Dual Oxidases
  • NADPH Oxidase 1
  • NADPH Oxidases
  • NOX1 protein, human
  • DUOX1 protein, human
  • DUOX2 protein, human
  • Oxygen