Biomedical subjects

Dagmar Rother

Publications and source records attributed to Dagmar Rother.

5 recordsLinked to original sources

PubMed2005

Structure of the cytochrome complex SoxXA of Paracoccus pantotrophus, a heme enzyme initiating chemotrophic sulfur oxidation.

The sulfur-oxidizing enzyme system (Sox) of the chemotroph Paracoccus pantotrophus is composed of several proteins, which together oxidize hydrogen sulfide, sulfur, thiosulfate or sulfite and transfers the gained electrons to the respiratory chain. The hetero-dimeric cytochrome c complex SoxXA functions as heme enzyme and links covalently the sulfur substrate to the thiol of the cysteine-138 residue of the SoxY protein of the SoxYZ complex. Here, we report the crystal structure of the c-type cytochrome complex SoxXA. The structure could be solved by molecular replacement and refined to a resolution of 1.9A identifying the axial heme-iron coordination involving an unusual Cys-251 thiolate of heme2. Distance measurements between the three heme groups provide deeper insight into the electron transport inside SoxXA and merge in a better understanding of the initial step of the aerobic sulfur oxidation process in chemotrophic bacteria.

Amino Acid Sequence↗

PubMed2005

Sulfur dehydrogenase of Paracoccus pantotrophus: the heme-2 domain of the molybdoprotein cytochrome c complex is dispensable for catalytic activity.

Sulfur dehydrogenase, Sox(CD)(2), is an essential part of the sulfur-oxidizing enzyme system of the chemotrophic bacterium Paracoccus pantotrophus. Sox(CD)(2) is a alpha(2)beta(2) complex composed of the molybdoprotein SoxC (43 442 Da) and the hybrid diheme c-type cytochrome SoxD (37 637 Da). Sox(CD)(2) catalyzes the oxidation of protein-bound sulfur to sulfate with a unique six-electron transfer. Amino acid sequence analysis identified the heme-1 domain of SoxD proteins to be specific for sulfur dehydrogenases and to contain a novel ProCysMetXaaAspCys motif, while the heme-2 domain is related to various cytochromes c(2). Purification of sulfur dehydrogenase without protease inhibitor yielded a dimeric SoxCD(1) complex consisting of SoxC and SoxD(1) of 30 kDa, which contained only the heme-1 domain. The heme-2 domain was isolated as a new cytochrome SoxD(2) of about 13 kDa. Both hemes of SoxD in Sox(CD)(2) are redox-active with midpoint potentials at E(m)1 = 218 +/- 10 mV and E(m)2 = 268 +/- 10 mV, while SoxCD(1) and SoxD(2) both exhibit a midpoint potential of E(m) = 278 +/- 10 mV. Electrochemically induced FTIR difference spectra of Sox(CD)(2), SoxCD(1), and SoxD(2) were distinct. A carboxy group is protonated upon reduction of the SoxD(1) heme but not for SoxD(2). The specific activity of SoxCD(1) and Sox(CD)(2) was identical as was the yield of electrons with thiosulfate in the reconstituted Sox enzyme system. To examine the physiological significance of the heme-2 domain, a mutant was constructed that was deleted for the heme-2 domain, which produced SoxCD(1) and transferred electrons from thiosulfate to oxygen. These data demonstrated the crucial role of the heme-1 domain of SoxD for catalytic activity, electron yield, and transfer of the electrons to the cytoplasmic membrane, while the heme-2 domain mediated the alpha(2)beta(2) tetrameric structure of sulfur dehydrogenase.

Amino Acid Sequence↗

PubMed2005

Prokaryotic sulfur oxidation.

Recent biochemical and genomic data differentiate the sulfur oxidation pathway of Archaea from those of Bacteria. From these data it is evident that members of the Alphaproteobacteria harbor the complete sulfur-oxidizing Sox enzyme system, whereas members of the beta and gamma subclass and the Chlorobiaceae contain sox gene clusters that lack the genes encoding sulfur dehydrogenase. This indicates a different pathway for oxidation of sulfur to sulfate. Acidophilic bacteria oxidize sulfur by a system different from the Sox enzyme system, as do chemotrophic endosymbiotic bacteria.

Acidianus↗

PubMed2005

SoxRS-mediated regulation of chemotrophic sulfur oxidation in Paracoccus pantotrophus.

Paracoccus pantotrophus GB17 requires thiosulfate for induction of the sulfur-oxidizing (Sox) enzyme system. The soxRS genes are divergently oriented to the soxVWXYZA-H genes. soxR predicts a transcriptional regulator of the ArsR family and soxS a periplasmic thioredoxin. The homogenate mutant GBOmegaS carrying a disruption of soxS by the Omega-kanamycin-resistance-encoding interposon expressed a low thiosulfate-oxidizing activity under heterotrophic and mixotrophic growth conditions. This activity was repressed by complementation with soxR, suggesting that SoxR acts as a repressor and SoxS is essential for full expression. Sequence analysis uncovered operator characteristics in the intergenic regions soxS-soxV and soxW-soxX. In each region a transcription start site was identified by primer extension analysis. Both regions were cloned into the vector pRI1 and transferred to P. pantotrophus. Strains harbouring pRI1 with soxS-soxV or soxW-soxX expressed the sox genes under heterotrophic conditions at a low rate, indicating repressor titration. Sequence analysis of SoxR suggested a helix-turn-helix (HTH) motif at position 87-108 and uncovered an invariant Cys-80 and a cysteine residue at the C-terminus. SoxR was overproduced in Escherichia coli with an N-terminal His6-tag and purified to near homogeneity. Electrophoretic gel mobility shift assays with SoxR retarded the soxS-soxV region as a single band while the soxW-soxX region revealed at least two protein-DNA complexes. These data demonstrated binding of SoxR to the relevant DNA. This is believed to be the first report of regulation of chemotrophic sulfur oxidation at the molecular level.

Amino Acid Sequence↗

PubMed2002

The cytochrome complex SoxXA of Paracoccus pantotrophus is produced in Escherichia coli and functional in the reconstituted sulfur-oxidizing enzyme system.

The heterodimeric c-type cytochrome complex SoxXA of Paracoccus pantotrophus was produced in Escherichia coli. The soxX and soxA genes, separated by two genes in the sox gene cluster of P. pantotrophus, were fused with ribosome binding sites optimal for E. coli and combined to give soxXA in pRD133.27. The cytochrome complex SoxXA was produced in E. coli M15 containing pRD133.27, pREP4 encoding the Lac repressor and plasmid pEC86, carrying essential cytochrome c maturation genes. SoxX and SoxA were formed in a ratio of about 2.5:1. SoxA appeared to be unstable when not complexed with SoxX. The cytochrome complex SoxXA, purified to homogeneity from periplasmic extracts of E. coli M15 (pRD133.27, pREP4, pEC86), exhibited identical biochemical and biophysical properties as compared to SoxXA of P. pantotrophus. Moreover, this cytochrome complex was shown to be equally catalytically active with respect to rates and reactivity with different sulfur substrates in the reconstituted sulfur-oxidizing enzyme system using homogeneous Sox-proteins of P. pantotrophus. Homogeneous SoxX was catalytically inactive.

Bacterial Proteins↗