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The heterodimeric hemoprotein SoxXA, essential for lithotrophic sulfur oxidation of the aerobic bacterium Paracoccus pantotrophus, was examined by a combination of spectroelectrochemistry and EPR spectroscopy. The EPR spectra for SoxXA showed contributions from three paramagnetic heme iron centers. One highly anisotropic low-spin (HALS) species (gmax = 3.45) and two "standard" cytochrome-like low-spin heme species with closely spaced g-tensor values were identified, LS1 (gz = 2.54, gy = 2.30, and gx = 1.87) and LS2 (gz = 2.43, gy = 2.26, and gx = 1.90). The crystal structure of SoxXA from P. pantotrophus confirmed the presence of three heme groups, one of which (heme 3) has a His/Met axial coordination and is located on the SoxX subunit [Dambe et al. (2005) J. Struct. Biol. 152, 229-234]. This heme was assigned to the HALS species in the EPR spectra of the isolated SoxX subunit. The LS1 and LS2 species were associated with heme 1 and heme 2 located on the SoxA subunit, both of which have EPR parameters characteristic for an axial His/thiolate coordination. Using thin-layer spectroelectrochemistry the midpoint potentials of heme 3 and heme 2 were determined: Em3 = +189 +/- 15 mV and Em2 = -432 +/- 15 mV (vs NHE, pH 7.0). Heme 1 was not reducible even with 20 mM titanium(III) citrate. The Em2 midpoint potential turned out to be pH dependent. It is proposed that heme 2 participates in the catalysis and that the cysteine persulfide ligation leads to the unusually low redox potential (-436 mV). The pH dependence of its redox potential may be due to (de)protonation of the Arg247 residue located in the active site.

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.

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.

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.

The gene region coding for lithotrophic sulfur oxidation of Paracoccus pantotrophus GB17 is located on a 13-kb insert of plasmid pEG12. Upstream of the previously described six open reading frames (ORFs) soxABCDEF with a partial sequence of soxA and soxF (C. Wodara, F. Bardischewsky, and C. G. Friedrich, J. Bacteriol. 179:5014-5023, 1997), 4,350 bp were sequenced. The sequence completed soxA, and uncovered six new ORFs upstream of soxA, designated ORF1, ORF2, and ORF3, and soxXYZ. ORF1 could encode a 275-amino-acid polypeptide of 29,332 Da with a 61 to 63% similarity to LysR transcriptional regulators. ORF2 could encode a 245-amino-acid polypeptide of 26,022 Da with the potential to form six transmembrane helices and with a 48 to 51% similarity to proteins involved in redox transport in cytochrome c biogenesis. ORF3 could encode a periplasmic polypeptide of 186 amino acids of 20,638 Da with a similarity to thioredoxin-like proteins and with a putative signal peptide of 21 amino acids. Purified SoxXA, SoxYZ, and SoxB are essential for thiosulfate or sulfite-dependent cytochrome c reduction in vitro. N-terminal and internal amino acid sequences identified SoxX, SoxY, SoxZ, and SoxA to be coded by the respective genes. The molecular masses of the mature proteins determined by electrospray ionization spectroscopy (SoxX, 14,834 Da; SoxY, 11,094 Da; SoxZ, 11,717 Da; and SoxA, 30,452 Da) were identical or close to those deduced from the nucleotide sequence with differences for the covalent heme moieties. SoxXA represents a novel type of periplasmic c-type cytochromes, with SoxX as a monoheme and SoxA as a hybrid diheme cytochrome c. SoxYZ is an as-yet-unprecedented soluble protein. SoxY has a putative signal peptide with a twin arginine motif and possibly cotransports SoxZ to the periplasm. SoxYZ neither contains a metal nor a complex redox center, as proposed for proteins likely to be transported via the Tat system.