Superoxide production of mAOX1, mAOX3, mAOX4, and mAOX2

Superoxide production of enzymes was calculated by following the reduction of 100 μM cytochrome c at 550 nm in the presence of 50 μM substrate (p-DMAC or vanillin) in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) buffer. Substrate consumption was monitored directly by the decrease in UV absorbance at wavelengths of 398 and 347 nm for p-DMAC and vanillin, respectively, in the presence of oxygen as electron acceptor. Extinction coefficients used are 21,000 M–1cm−1 for cytochrome c, 30,500 M–1cm−1 for vanillin, and 25,100 M–1cm−1 for p-DMAC.

VanillinSubstrate consumption (U μmol−1 of enzyme)51.8529.729.175.78
Superoxide formation (U μmol−1 of enzyme)16.456.921.882.42
Ratio (%)31.723.320.441.8
p-DMACSubstrate consumption (U μmol−1 of enzyme)142.2827.4419.3715.83
Superoxide formation (U μmol−1 of enzyme)43.475.253.846.21
Ratio (%)30.619.119.939.3
  • One unit (U) is defined as the oxidation of 1 μmol substrate per minute under assay conditions.