The Extracellular A-loop of Dual Oxidases Affects the Specificity of Reactive Oxygen Species Release [Molecular Bases of Disease]

January 13th, 2015 by Ueyama, T., Sakuma, M., Ninoyu, Y., Hamada, T., Dupuy, C., Geiszt, M., Leto, T. L., Saito, N.

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 (H1071, H1072, and G1074) 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.