Characterization of a new dihemic c(4)-type cytochrome isolated from Thiobacillus ferrooxidans

A new soluble c-type cytochrome has been purified to homogeneity from the a cidophilic proteobacterium Thiobacillus ferrooxidans BRGM. It is characteri zed by an alpha-peak wavelength of 552 nm, a molecular mass of 26 567 Da (a s determined by mass spectroscopy) and a pI value of 8. Optical redox titra tions at pH 4.0 revealed the presence of two distinguishable redox species with an E-m of 510 mV and an E-m of 430 +/- 20 mV. EPR spectra recorded for this heme protein demonstrated the presence of stoichiometric amounts of t wo low-spin hemes with a g(z) of 3.08 (510 mV species) and a g(z) of 3.22 ( 430 mV species). Modifications of the physicochemical properties of the cyt ochrome were observed on complex formation with the blue copper protein rus ticyanin, another soluble electron carrier in the genus Thiobacillus. N-Ter minal sequencing yielded the polypeptide sequence up to the 50th residue. T he determined sequence was found to be present (at 100% amino acid identity ) in the (unfinished) genome of T. ferrooxidans ATCC 23270, and the corresp onding full-length protein turned out to be surprisingly similar (34.5% ami no acid identity) to another c(4)-type diheme protein from T. ferrooxidans BRGM [Cavazza, C., et al, (1996) Eur. J, Biochem. 242, 308-314], the gene o f which is also present (at 97% amino acid identity) in the T. ferrooxidans ATCC 23270 genome. The physicochemical properties and sequence characteris tics of both c(4) cytochromes present in the same bacteria are compared, an d the functional role of this new diheme protein in the iron(II)-oxidizing electron transport chain in the genus Thiobacillus is discussed.