Armin Quentmeier

Oxidation of Reduced Inorganic Sulfur Compounds by Bacteria: Emergence of a Common Mechanism?

Biological oxidation of hydrogen sulfide to sulfate is one of the major reactions of the global sulfur cycle. Reduced inorganic sulfur compounds (referred to below as sulfur) are exclusively oxidized by prokaryotes, and sulfate is the major oxidation product. Sulfur oxidation in members of the

Novel Genes of the sox Gene Cluster, Mutagenesis of the Flavoprotein SoxF, and Evidence for a General Sulfur-Oxidizing System in Paracoccus pantotrophus GB17

The novel genes soxFGH were identified, completing the sox gene cluster of Paracoccus pantotrophus coding for enzymes involved in lithotrophic sulfur oxidation. The periplasmic SoxF, SoxG, and SoxH proteins were induced by thiosulfate and purified to homogeneity from the soluble fraction. soxF coded for a protein of 420 amino acids with a signal peptide containing a twin-arginine motif. SoxF was 37% identical to the flavoprotein FccB of flavocytochrome c sulfide dehydrogenase of Allochromatium vinosum. The mature SoxF (42,832 Da) contained 0.74 mol of flavin adenine dinucleotide per mol. soxG coded for a novel protein of 303 amino acids with a signal peptide containing a twin-arginine motif. The mature SoxG (29,657 Da) contained two zinc binding motifs and 0.90 atom of zinc per subunit of the homodimer. soxH coded for a periplasmic protein of 317 amino acids with a double-arginine signal peptide. The mature SoxH (32,317 Da) contained two metal binding motifs and 0.29 atom of zinc and 0.20 atom of copper per subunit of the homodimer. SoxXA, SoxYZ, SoxB, and SoxCD (C. G. Friedrich, A. Quentmeier, F. Bardischewsky, D. Rother, R. Kraft, S. Kostka, and H. Prinz, J. Bacteriol. 182:4476-4487, 2000) reconstitute a system able to perform thiosulfate-, sulfite-, sulfur-, and hydrogen sulfide-dependent cytochrome c reduction, and this system is the first described for oxidizing different inorganic sulfur compounds. SoxF slightly inhibited the rate of hydrogen sulfide oxidation but not the rate of sulfite or thiosulfate oxidation. From use of a homogenote mutant with an in-frame deletion in soxF and complementation analysis, it was evident that the soxFGH gene products were not required for lithotrophic growth with thiosulfate.

The cysteine residue of the SoxY protein as the active site of protein‐bound sulfur oxidation of Paracoccus pantotrophus GB17

Four proteins of Paracoccus pantotrophus are required for hydrogen sulfide‐, sulfur‐, thiosulfate‐ and sulfite‐dependent horse heart cytochrome c reduction. The lack of free intermediates suggested a protein‐bound sulfur oxidation mechanism. The SoxY protein has a novel motif containing a cysteine residue. Electrospray ionization and matrix‐assisted laser desorption ionization mass spectrometry of the SoxYZ protein revealed one mass for SoxZ and different masses for SoxY, indicating native SoxY (10 977 Da) and SoxY with additional masses of +32, +80, +112 and +144 Da, suggesting addition of sulfur, sulfite, thiosulfate and thioperoxomonosulfate. Reduction of SoxY removed the additional masses, indicating a thioether or thioester bond. N‐Ethylmaleimide inhibited thiosulfate‐oxidation and the kinetics suggested a turn‐over‐dependent mode of action. These data were evidence that the sulfur atom to be oxidized was covalently linked to the thiol moiety of the cysteine residue of SoxY and the active site of sulfur oxidation.

Purification and characterization of the hydrogenase from Thiobacillus ferrooxidans

Abstract Hydrogenase of Thiobacillus ferrooxidans ATCC 19859 was purified from cells grown lithoautotrophically with 80% hydrogen, 8.6% carbon dioxide, and 11.4% air. Hydrogenase was located in the 140,000 ×g supernatant in cell-free extracts. The enzyme was purified 7.3-fold after chromatography on Procion Red and Q-Sepharose with a yield of 19%, resulting in an 85% pure preparation with a specific activity of 6.0 U (mg protein)–1. With native PAGE, a mol. mass of 100 and 200 kDa was determined. With SDS-PAGE, two subunits of 64 (HoxG) and of 34 kDa (HoxK) were observed. Hydrogenase reacted with methylene blue and other artificial electron acceptors, but not with NAD. The optimum of enzyme activity was at pH 9 and at 49° C. Hydrogenase contained 0.72 mol nickel and 6.02 mol iron per mol enzyme. The relationship of the T. ferrooxidans hydrogenase to other proteins was examined. A 9.5-kb EcoRI fragment of T. ferrooxidans ATCC 19859 hybridized with a 2.2-kb XhoI fragment from Alcaligenes eutrophus encoding the membrane-bound hydrogenase. Antibodies against this enzyme did not react with the T. ferrooxidans hydrogenase in Western blot analysis. The N-terminal amino acid sequence (40 amino acids) of HoxK was 46% identical to that of the hydrogen sensor HupU of Bradyrhizobium japonicum and 39% identical to that of the HupS subunit of the Desulfovibrio baculatus hydrogenase. The N-terminal sequence of 20 amino acids of HoxG of T. ferrooxidans was 83.3% identical to that of the 60-kDa subunit. HupL, of the hydrogenase of Anabaena sp. Sequences of ten internal peptides of HoxG were 50–100% identical to the respective sequences of HupL of the Anabaena sp. hydrogenase.

Interaction between Sox proteins of two physiologically distinct bacteria and a new protein involved in thiosulfate oxidation.

Organisms using the thiosulfate‐oxidizing Sox enzyme system fall into two groups: group 1 forms sulfur globules as intermediates (Allochromatium vinosum), group 2 does not (Paracoccus pantotrophus). While several components of their Sox systems are quite similar, i.e. the proteins SoxXA, SoxYZ and SoxB, they differ by Sox(CD)2 which is absent in sulfur globule‐forming organisms. Still, the respective enzymes are partly exchangeable in vitro: P. pantotrophus Sox enzymes work productively with A. vinosum SoxYZ whereas A. vinosum SoxB does not cooperate with the P. pantotrophus enzymes. Furthermore, A. vinosum SoxL, a rhodanese‐like protein encoded immediately downstream of soxXAK, appears to play an important role in recycling SoxYZ as it increases thiosulfate depletion velocity in vitro without increasing the electron yield.

Multifrequency EPR analysis of the dimanganese cluster of the putative sulfate thiohydrolase SoxB of Paracoccus pantotrophus

A detailed analysis of the EPR signatures at X-band and Q-band of an enzyme (SoxB) involved in sulfur oxidation from Paracoccus pantotrophus is presented. EPR spectra are attributed to an exchange-coupled dimanganese Mn2(II,II) complex. An antiferromagnetic exchange interaction of J=−7.0 (±1) cm−1 (H=−2JS1S2) is evidenced by a careful examination of the temperature dependence of the EPR spectra. The spin Hamiltonian parameters for a total spin of ST =1, 2 and 3 are obtained and an inter-manganese distance of 3.4 (±0.1) Å is estimated. The comparison with exchange coupling and inter-manganese distance data of other dimanganese proteins and model compounds leads to a tentative assignment of the Mn bridging ligands to bis(μ-hydroxo) (μ-carboxylato).

Sulfide-quinone reductase activity in membranes of the chemotrophic bacterium Paracoccus denitrificans GB17

Abstract Reduction of exogenous ubiquinone and of cytochromes by sulfide in membranes of the chemotrophic bacterium Paracoccus denitrificans GB17 was studied. For sulfide-ubiquinone reductase activity, Km values of 26 ± 4 and 3.1 ± 0.6 μM were determined from titrations with sulfide and decyl-ubiquinone, respectively. A maximal rate of up to 0.3 μmol decyl-ubiquinone reduced (mg protein)–1 min–1 was estimated. The reaction was sensitive to quinone-analogous inhibitors, but insensitive to cyanide. Reduction of cytochromes by sulfide was monitored with an LED-array spectrophotometer. Under oxic conditions, reduction rates and extents of reduction were lower than those under anoxic conditions. Reoxidation of cytochromes was oxygen-dependent and cyanide-sensitive. The multiphasic behavior of transient reduction of cytochrome b with limiting amounts of sulfide reflects that sulfide, in addition to acting as an electron donor, is a slowly binding inhibitor of cytochrome c oxidase. The initial peak of cytochrome b reduction is dependent on electron flow to an oxidant, either oxygen or ferricyanide, and is stimulated by antimycin A. This oxidant-induced reduction of cytochrome b suggests that electron transport from sulfide in P. denitrificans GB17 employs the cytochrome bc1 complex via the quinone pool.

Structural Basis for the Oxidation of Protein-bound Sulfur by the Sulfur Cycle Molybdohemo-Enzyme Sulfane Dehydrogenase SoxCD

The sulfur cycle enzyme sulfane dehydrogenase SoxCD is an essential component of the sulfur oxidation (Sox) enzyme system of Paracoccus pantotrophus. SoxCD catalyzes a six-electron oxidation reaction within the Sox cycle. SoxCD is an α2β2 heterotetrameric complex of the molybdenum cofactor-containing SoxC protein and the diheme c-type cytochrome SoxD with the heme domains D1 and D2. SoxCD1 misses the heme-2 domain D2 and is catalytically as active as SoxCD. The crystal structure of SoxCD1 was solved at 1.33 Å. The substrate of SoxCD is the outer (sulfane) sulfur of Cys-110-persulfide located at the C-terminal peptide swinging arm of SoxY of the SoxYZ carrier complex. The SoxCD1 substrate funnel toward the molybdopterin is narrow and partially shielded by side-chain residues of SoxD1. For access of the sulfane-sulfur of SoxY-Cys-110 persulfide we propose that (i) the blockage by SoxD-Arg-98 is opened via interaction with the C terminus of SoxY and (ii) the C-terminal peptide VTIGGCGG of SoxY provides interactions with the entrance path such that the cysteine-bound persulfide is optimally positioned near the molybdenum atom. The subsequent oxidation reactions of the sulfane-sulfur are initiated by the nucleophilic attack of the persulfide anion on the molybdenum atom that is, in turn, reduced. The close proximity of heme-1 to the molybdopterin allows easy acceptance of the electrons. Because SoxYZ, SoxXA, and SoxB are already structurally characterized, with SoxCD1 the structures of all key enzymes of the Sox cycle are known with atomic resolution.

Identification of two inactive forms of the central sulfur cycle protein SoxYZ of Paracoccus pantotrophus

The central protein of the sulfur‐oxidizing enzyme system of Paracoccus pantotrophus, SoxYZ, reacts with three different Sox proteins. Its active site Cys110Y is on the carboxy‐terminus of the SoxY subunit. SoxYZ “as isolated” consisted mainly of the catalytically inactive SoxY‐Y(Z)2 heterotetramer linked by a Cys110Y‐Cys110Y interprotein disulfide. Sulfide activated SoxYZ “as isolated” 456‐fold, reduced the disulfide, and yielded an active SoxYZ heterodimer. The reductant tris(2‐carboxyethyl)phosphine (TCEP) inactivated SoxYZ. This form was not re‐activated by sulfide, which identified it as a different inactive form. In analytical gel filtration, the elution of “TCEP‐treated” SoxYZ was retarded compared to active SoxYZ, indicating a conformational change. The possible enzymes involved in the re‐activation of each inactive form of SoxYZ are discussed.

Spectroscopic Characterization of the Molybdenum Cofactor of the Sulfane Dehydrogenase SoxCD from Paracoccus pantotrophus

The bacterial sulfane dehydrogenase SoxCD is a distantly related member of the sulfite oxidase (SO) enzyme family that is proposed to oxidize protein-bound sulfide (sulfane) of SoxY as part of a multienzyme mechanism of thiosulfate metabolism. This study characterized the molybdenum cofactor of SoxCD1, comprising the catalytic molybdopterin subunit SoxC and the truncated c-type cytochrome subunit SoxD1. Electron paramagnetic resonance spectroscopy of the Mo(V) intermediate generated by dithionite reduction revealed low- and high-pH species with g and A((95,97)Mo) matrices nearly identical to those of SO, indicating a similar pentacoordinate active site in SoxCD1. However, no sulfite-induced reduction to Mo(V) was detected, nor could a strongly coupled (1)H signal or a phosphate-inhibited species be generated. This indicates that the outer coordination sphere controls substrate binding in SoxCD, permitting access only to protein-bound sulfur via the C-terminal tail of SoxY.