Oxidative Phosphorylation and Functional Cytochromes in Skunk Cabbage Mitochondria.

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Abstract
A participate fraction (mitochondria) was isolated from homogenates of skunk cabbage (Symplocarpus foetidus) flowers by centrifugation at 10,000 X g. The isolation medium contained 0.5M sucrose and either 0.01 [image] Versene with 0.05 [image] Tris buffer (for P/O studies) or 0.05 [image] phosphate buffer (pH 7.0). Using a-ketoglutarate as substrate, phosphate incorporation/O2 uptake (P/O) ratios were usually greater than 3 and maximum values up to 3.8 were obtained. Low concentrations of 2,4-dinitrophenol reduced the P/O values to roughly 0.5. Azide and cyanide inhibited phosphorylation more than oxygen uptake, thus acting as "uncoupling" agents. These results suggest that there are 4 phosphorylation sites, one at substrate level and 3 associated with hydrogen (electron) transport to oxygen. Absorption spectra of the mitochondrial suspensions were measured in a Beckman DU with photo multiplier, using filter paper as the blank. Spectra were obtained after gassing the closed cuvette for 10 minutes with either nitrogen or oxygen; citrate was generally included as substrate. The difference spectrum (N2-O) showed large peaks at 426-428 and 443-447 m[mu]; smaller peaks were present at 480-485, 520-525, 553, 560-562 and 602-605 m[mu]. It is concluded that members of the cytochrome a, b, and c groups are present and involved in the reaction with O2. In the presence of cyanide, the difference spectrum is shifted, indicating a large inhibition of cyto-chromes a and c but relatively little inhibition of b (560-562). This suggests that cytochrome b can be oxidized by O2 via a cyanide-insensitive reaction that does not involve the c-a system. This alternate pathway may account for the "uncoupling" of phosphorylation and the cyanide insensitivity of the tissue respiration.