Fig. 3. Production of H₂O₂ during Fe(II) oxidation in mYfh1p.

A, reactions containing 96 μM mYfh1p and 24 or 48 αM Fe(II) were incubated in 50 mM HEPES-KOH, pH 7.0, for 30 min at 30 °C in the presence of Amplex Red/HRP reagent. Immediately afterward, fluorescence intensity was recorded from 570 to 610 nm. The fluorescence intensity curves before background correction are shown (bottom graph). For the standard curve (top graph), H₂O₂ standards (50 – 600 nM) were incubated in 50 mM HEPES-KOH, pH 7.0, for 30 min at 30 °C in the presence of Amplex Red/HRP reagent. The fluorescence intensity curves were recorded, and samples containing buffer plus Amplex Red/HRP were used for background corrections. The corrected fluorescence intensity curves were integrated, and a standard curve was constructed. The correlation coefficient of the fitted line to the data is 0.999. To determine the concentration of H₂O₂ produced in the presence of mYfh1p (see “Results”), samples containing buffer plus 24 or 48 μM Fe(II) and Amplex Red/HRP were used as blanks for background corrections. The corrected fluorescence intensity curves for mYfh1p were integrated, and the H₂O₂ concentration was calculated from the standard curve. A.U., arbitrary units. B, reactions containing 96 μM mYfh1p were incubated for 30 min at 30 °C in the absence or presence of 48 μM Fe(II) under the experimental conditions used in the Amplex Red/HRP assays described above. Following incubation with 2,4-dinitrophenylhy-drazine (DNPH) to derivatize carbonyl groups to DNP, the samples (7.5 μg of total protein) were analyzed by SDS/PAGE and Western blotting using a polyclonal anti-DNP antiserum. C, 2 μg of the purified mYfh1p monomer used in the experiments described above was analyzed by SDS/PAGE and Coomassie Blue staining (lane 5). D, following immunodetection, the membrane was subjected to SYPRO Ruby protein blot staining. Arrows d, degradation products of mYfh1p; lane MW, molecular mass standards. E, a mixture of 48 μM Fe(II), 24 μM H₂O₂, and 5 mM 2-deoxyribose (DOR) was incubated in 10 mM HEPES-KOH, pH 7.0, in the absence or presence of 96 μM mYfh1p for 30 min at 30 °C, and production of malondialdehyde-thiobarbituric acid (MDA-TBA) (ε₅₃₂ = 1.54 × 10⁵ M⁻¹ cm⁻¹) was measured (31). The indicated controls were analyzed at the same time and treated identically. The bars represent the means ± S.D. of four independent measurements.