FIG. 6.

Effect of GT on the difference spectrum of flavocytochrome b₅₅₈. Membranes (75 pmol of heme equivalent) were treated with 153 nmol of GT for 15 min at room temperature and washed by centrifugation before the determination of dithionite-reduced-minus-oxidized spectra of flavocytochrome b₅₅₈ (see Materials and Methods). One-third aliquots were transferred to black cuvettes for the spectral analysis. Representative difference spectra at least 5 min after the dithionite addition to untreated (−GT) and GT-treated (+GT) membranes are shown. The amounts of heme calculated from the Soret peaks at 426 nm were ca. 23 pmol in both cases and fit well with the expected theoretical value of 25 pmol. The GT-treated membranes were unable to reconstitute O₂⁻ generation when combined with recombinant cytosolic components in the presence of MA (see Table 2). The data are representative of three experiments.