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

Abstract

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 (His1071, His1072, and Gly1074) 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.