The E-loop Is Involved in Hydrogen Peroxide Formation by the NADPH Oxidase Nox4

TY - JOUR

T1 - The E-loop Is Involved in Hydrogen Peroxide Formation by the NADPH Oxidase Nox4

AU - Takac, Ina

AU - Schroeder, Katrin

AU - Zhang, Leilei

AU - Lardy, Bernard

AU - Anilkumar, Narayana

AU - Lambeth, J. David

AU - Shah, Ajay M.

AU - Morel, Francoise

AU - Brandes, Ralf P.

PY - 2011/4/15

Y1 - 2011/4/15

N2 - In contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O-2(radical anion)), Nox4 produces hydrogen peroxide (H2O2). We constructed chimeric proteins and mutants to address the protein region that specifies which reactive oxygen species is produced. Reactive oxygen species were measured with luminol/horseradish peroxidase and Amplex Red for H2O2 versus L-012 and cytochrome c for O-2(radical anion). The third extracytosolic loop (E-loop) of Nox4 is 28 amino acids longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in Nox4 or exchange of the two cysteines in these stretches switched Nox4 from H2O2 to O-2(radical anion) generation while preserving expression and intracellular localization. In the presence of an NO donor, the O-2(radical anion)-producing Nox4 mutants, but not wild-type Nox4, generated peroxynitrite, excluding artifacts of the detection system as the apparent origin of O-2(radical anion). In Cos7 cells, in which Nox4 partially localizes to the plasma membrane, an antibody directed against the E-loop decreased H2O2, but increased O-2(radical anion), formation by Nox4 without affecting Nox1-dependent O-2(radical anion) formation. The E-loop of Nox4 but not Nox1 and Nox2 contains a highly conserved histidine that could serve as a source for protons to accelerate spontaneous dismutation of superoxide to form H2O2. Mutation of this but not of four other conserved histidines also switched Nox4 from H2O2, to O-2(radical anion) formation, Thus, H2O2, formation is an intrinsic property of Nox4 that involves its E-loop. The structure of the E-loop may hinder O-2(radical anion) egress and/or provide a source for protons, allowing dismutation to form H2O2.

AB - In contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O-2(radical anion)), Nox4 produces hydrogen peroxide (H2O2). We constructed chimeric proteins and mutants to address the protein region that specifies which reactive oxygen species is produced. Reactive oxygen species were measured with luminol/horseradish peroxidase and Amplex Red for H2O2 versus L-012 and cytochrome c for O-2(radical anion). The third extracytosolic loop (E-loop) of Nox4 is 28 amino acids longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in Nox4 or exchange of the two cysteines in these stretches switched Nox4 from H2O2 to O-2(radical anion) generation while preserving expression and intracellular localization. In the presence of an NO donor, the O-2(radical anion)-producing Nox4 mutants, but not wild-type Nox4, generated peroxynitrite, excluding artifacts of the detection system as the apparent origin of O-2(radical anion). In Cos7 cells, in which Nox4 partially localizes to the plasma membrane, an antibody directed against the E-loop decreased H2O2, but increased O-2(radical anion), formation by Nox4 without affecting Nox1-dependent O-2(radical anion) formation. The E-loop of Nox4 but not Nox1 and Nox2 contains a highly conserved histidine that could serve as a source for protons to accelerate spontaneous dismutation of superoxide to form H2O2. Mutation of this but not of four other conserved histidines also switched Nox4 from H2O2, to O-2(radical anion) formation, Thus, H2O2, formation is an intrinsic property of Nox4 that involves its E-loop. The structure of the E-loop may hinder O-2(radical anion) egress and/or provide a source for protons, allowing dismutation to form H2O2.

U2 - 10.1074/jbc.M110.192138

DO - 10.1074/jbc.M110.192138

M3 - Article

SN - 1083-351X

VL - 286

SP - 13304

EP - 13313

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 15

ER -