Figure 5.
Cytochrome c prevents Erv1-dependent generation of hydrogen peroxide. (A) Production of hydrogen peroxide (H2O2) was assayed in a fluorescence-based assay using Amplex red. 2 μM of purified Erv1 was incubated with 50 mM Amplex red and 1 U/ml horseradish peroxidase in 600 μl of 100 mM potassium phosphate, pH 7.4. Upon addition of DTT, fluorescence emission at 610 nm was recorded at an excitation wavelength of 550 nm. Incubation with 150 and 300 nmol cytochrome c counteracted the production of hydrogen peroxide linearly with time (arrows). (top, inset) The generation of hydrogen peroxide represents the same measurement at a larger scale of the y axis. (B) 1 nmol hydrogen peroxide was preincubated with or without a twofold excess of oxidized cytochrome c for 1 min at 25°C before fluorescence was analyzed in the Amplex red assay. Note that the presence of cytochrome c did not quench the fluorescence signal. (C) Model for the interaction of the disulfide relay system and the mitochondrial respiratory chain. The electron flow from the imported proteins to the final electron acceptor oxygen is indicated. The cytochrome c–independent side reaction of Erv1 with oxygen is shown in light gray. Cytochrome c reductase and oxidase complexes are indicated as complexes III and IV, respectively. Q indicates the ubiquinone pool.