Electron-accepting properties of cytochrome o purified from Vitreoscilla

The reduction of cytochrome o which contains two hemes/molecule required two electrons/molecule when titrated with dithionite under anaerobic conditions. Two types of spectral transitions occurred during this reduction, first a decrease in absorption bands for the oxidized protein at 540 and 405 nm and then a shift in the absorption maxima to 555 and 425 nm, respectively. Each of these two transitions required approximately one electron, evidence that the two hemes reduced separately. Preliminary estimates of the midpoint potentials of the two hemes enabled the selection of two suitable oxidation-reduction dyes, toluylene blue (E'o = +0.115 V) and indigo carmine (E'o = -0.125 V) which were used to estimate more accurately the midpoint potentials of the high and low potential hemes, respectively, using equilibrium photochemical titrations with EDTA and flavin mononucleotide. The midpoint potentials of the two hemes of cytochrome o determined by this technique were +0.118 and -0.122 V.