Characterization of purified cytochrome c1 from Rhodobacter sphaeroides R-26

Abstract

Cytochrome c 1 from a photosynthetic bacterium Rhodobacter sphaeroides R-26 has been purified to homogeneity. The purified protein contains 30 nmol heme per mg protein, has an isoelectric point of 5.7, and is soluble in aqueous solution in the absence of detergents. The apparent molecular weight of this protein is about 150 000, determined by Bio Gel A-0.5 m column chromatography; a minimum molecular weight of 30 000 is obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The absorption spectrum of this cytochrome is similar to that of mammalian cytochrome c 1, but the amino acid composition and circular dichroism spectral characteristics are different. The heme moiety of cytochrome c 1 is more exposed than is that of mammalian cytochrome c 1, but less exposed than that of cytochrome c 2. Ferricytochrome c 1 undergoes photoreduction upon illumination with light under anaerobic conditions. Such photoreduction is completely abolished when p-chloromercuriphenylsulfonate is added to ferricytochrome c 1, suggesting that the sulfhydryl groups of cytochrome c 1 are the electron donors for photoreduction. Purified cytochrome c 1 contains 3 ± 0.1 mol of the p-chloromercuriphenylsulfonate titratable sulfhydryl groups per mol of protein. In contrast to mammalian cytochrome c 1, the bacterial protein does not form a stable complex with cytochrome c 2 or with mammalian cytochrome c at low ionic strength. Electron transfer between bacterial ferrocytochrome c 1 and bacterial ferricytochrome c 2, and between bacterial ferrocytochrome c 1 and mammalian ferricytochrome c proceeds rapidly with equilibrium constants of 49 and 3.5, respectively. The midpoint potential of purified cytochrome c 1 is calculated to be 228 mV, which is identical to that of mammalian cytochrome c 1.