Characterization of a New Dihemic c₄-Type Cytochrome Isolated from Thiobacillus ferrooxidans

Abstract

A new soluble c-type cytochrome has been purified to homogeneity from the acidophilic proteobacterium Thiobacillus ferrooxidans BRGM. It is characterized by an alpha-peak wavelength of 552 nm, a molecular mass of 26 567 Da (as determined by mass spectroscopy) and a pI value of 8. Optical redox titrations 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 two 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 cytochrome were observed on complex formation with the blue copper protein rusticyanin, another soluble electron carrier in the genus Thiobacillus. N-Terminal sequencing yielded the polypeptide sequence up to the 50th residue. The determined sequence was found to be present (at 100% amino acid identity) in the (unfinished) genome of T. ferrooxidans ATCC 23270, and the corresponding full-length protein turned out to be surprisingly similar (34.5% amino 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 of which is also present (at 97% amino acid identity) in the T. ferrooxidans ATCC 23270 genome. The physicochemical properties and sequence characteristics of both c(4) cytochromes present in the same bacteria are compared, and the functional role of this new diheme protein in the iron(II)-oxidizing electron transport chain in the genus Thiobacillus is discussed.

Quinol-cytochrome c Oxidoreductase and Cytochrome c4 Mediate Electron Transfer during Selenate Respiration in Thauera selenatis

“…A database search with the translated sequence of this gene produced a large number of similar proteins, most of which are classified as cytochromes c 4 , a group of di-heme cytochromes c characterized by high redox potentials, a low intensity asymmetrical ␣ band around 550 nm and a low ␣/␤ peak ratio (37,38). A number of members of this group have been characterized, including members from Acidithiobacillus ferrooxidans (39,40), Pseudomonas stutzeri (41), Thiocapsa roseopersicina (42), and Azotobacter vinelandii (43). Cytochromes c 4 have been proposed to be electron donors to oxidases due to the presence of a cytochrome c 4 in the aerobe A. vinelandii (43), but the discovery of a cytochrome c 4 in T. roseopersicina, a facultative anaerobic photosynthetic bacterium, suggests that their roles are much more varied (42,44).…”

The Multicenter Aerobic Iron Respiratory Chain of Acidithiobacillus ferrooxidans Functions as an Ensemble with a Single Macroscopic Rate Constant

“…A number of laboratories have described redox-active proteins expressed by At. ferrooxidans that might participate in the iron respiratory chain (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Perhaps the most generally accepted model for the iron respiratory chain of At.…”

Respiratory Isozyme, Two Types of Rusticyanin ofAcidithiobacillus ferrooxidans

“…On the other hand, several diŠerent C-type cytochromes have been discovered in several strains of the bacterium. 9,12,[29][30][31][32][33][34][35] These results together, rus is present with C-type cytochrome in an apparent operon in strain ATCC 33020, and, multiple C-type cytochromes exist in some strains of the bacterium, suggested the possibility that some strains might also synthesize additional isoforms of rusticyanin. In this study, therefore, we isolated rus from six strains of A. ferrooxidans and identiˆed its copy number, sequence, and some of the physical characteristics of its gene product.…”