Purification, redox and spectroscopic properties of the tetraheme cytochrome c isolated from Rubrivivax gelatinosus

The tetraheme cytochrome c subunit of the Rubrivivax gelatinosus reaction c enter was isolated in the presence of octyl beta-D-thioglucoside by ammoniu m sulfate precipitation and solubilization at pH 9 in a solution of Deripha t 160. Several biochemical properties of this purified cytochrome were char acterized. In particular, it forms small oligomers and its N-terminal amino acid is blocked. In the presence or absence of diaminodurene, ascorbate an d dithionite, different oxidation/reduction states of the isolated cytochro me were studied by absorption, EPR and resonance Raman spectroscopies; All the data show two hemes quickly reduced by ascorbate, one heme slowly reduc ed by ascorbate and one heme only reduced by dithionite. The quickly ascorbate-reduced hemes have paramagnetic propel ties very simi lar to those of the two low-potential hemes of the reaction center-bound cy tochrome (g(z) = 3.34), but their alpha band is split with two components p eaking at 552 nm and 554 nm in the reduced state. Their axial ligands did n ot change, being His/Met and His/His, as indicated by the resonance Raman s pectra. The slowly ascorbate-reduced heme and the dithionite-reduced heme a re assigned to the two high-potential hemes of the bound cytochrome. Their alpha band was blue-shifted at 551 nm and the g(z) values decreased to 2.96 , although the axial ligations (His/Met) were conserved. It was concluded t hat the estimated 300 mV potential drop of these hemes reflected changes in their solvent accessibility, while the reduction in g(z) indicates an incr eased symmetry of their cooordination spheres. These structural modificatio ns impaired the cytochrome's essential function as the electron donor to th e photooxidized bacteriochlorophyll dimer of the reaction center. In contra st to its native state, the isolated cytochrome was unable to reduce effici ently the reaction center purified from a Rubrivivax gelatinosus mutant in which the tetraheme was absent. Despite the conformational changes of the c ytochrome, its four hemes are still divided into two groups with a pair of low-potential hemes and a pair of high-potential hemes.