Purification, some properties and the specific biological activity of cytochromes c4 and c5 from Azotobacter vinelandii

Abstract

Two cytochromes of the "c" type, cytochromes C4 and C5, were extracted from A. vinelandii and purified. The absorption spectra of cytochrome c4 and cytochrome C5 resemble that of cytochrome c. The [alpha]-, [beta]- and gamma-bands of the reduced cytochrome C4 lie at 551,552, and 416 m[mu]. respectively. Those of cytochrome C5 lie 4 m[mu] nearer the red end of the spectrum. Pure cytochrome C4 contains 0.46% of Fe. Its molecular weight, calculated on the basis of one atom of Fe per molecule, is about 12,000. The [alpha]-absorption bands of the pyridine hemochromogens derived from both cytochromes C4 and c5 lie at about 550 m[mu]. The isoelectric points of both pigments are in the acid range. The oxidation-reduction potentials, E''o, were found to be +0.30 v for cytochrome C4 and +0.32 v for cytochrome c5, between pH 6.0 and 7.5. Two particlate fractions were isolated from cell-free extracts: fraction L, composed of large particles, and fraction SP, consisting of the smallest particles. Fraction SP had the highest activity in the oxidation of succinate. Both particulate fractions contain the same cytochrome pigments as are found in the intact cells. Cytochromes C4 and C5 were found to react only with the oxidase from A. vinelandii; these bacterial oxidase preparations did not oxidise heart-muscle cytochrome c, Rhodospirilium rubrum cytochrome c, or cytochrome f.