Characterization of a new type of sulfite dehydrogenase from Paracoccus pantotrophus GB17

Abstract

The periplasmic sulfite dehydrogenase of Paracoccus pantotrophus GB17 was purified to homogeneity by a four-step procedure from cells grown lithoautotrophically with thiosulfate. The molecular mass of native sulfite dehydrogenase was 190 kDa as determined by native gradient PAGE. SDS-PAGE showed sulfite dehydrogenase to comprise two subunits with molecular masses of 47 kDa and 50 kDa, suggesting an alpha2beta2 structure. The N-terminal amino acid sequence and immunochemical analysis using SoxC-specific antibodies identified the 47-kDa protein as the soxC gene product. SoxD-specific antibodies identified the 50-kDa protein as SoxD. Based on the molecular masses deduced from the nucleotide sequence for mature SoxC (43,442 Da) and SoxD (37,637 Da) sulfite dehydrogenase contained 1.30 mol molybdenum/mol alpha2beta2 sulfite dehydrogenase. The iron content was 3.17 mol/mol alpha2beta2 sulfite dehydrogenase, and 3.53 mol heme/mol alpha2beta2 sulfite dehydrogenase was determined by pyridine hemochrome analysis. These data are consistent with the two heme-binding domains (CxxCH), characteristic for c-type cytochromes, deduced from the soxD nucleotide sequence. Electrospray ionization revealed two masses for SoxC of 43,503 and 43,897 Da. The difference in molecular mass was attributed to the molybdenum cofactor of SoxC. For SoxD a mass of 38,815 Da was determined; this accounted for the polypeptide and two covalently bound hemes. Reconstitution of the catalytic activity of sulfite dehydrogenase required additional fractions; these eluted from Q Sepharose at 0.05, 0.25, and 0.30 M NaCl. The K(m) of sulfite dehydrogenase for sulfite was 7.0 microM and for cytochrome c 19 microM. Sulfite dehydrogenase activity was inhibited by sulfate and phosphate. The structural and catalytic properties make sulfite dehydrogenase from P. denitrificans GB17 distinct from sulfite oxidases of other prokaryotic or eukaryotic sources.

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

“…SoxCD 1 is the construct in which the second heme-2 domain of SoxD is deleted (7). The SoxCD 1 complex was purified from P. pantotrophus strain GB17 as described previously (2,23). SoxCD 1 was enriched from the cell extract by differential centrifugation and ammonium sulfate fractionation.…”

SoxV transfers electrons to the periplasm of Paracoccus pantotrophus – an essential reaction for chemotrophic sulfur oxidation

“…The resulting cell-free extract was subjected to centrifugation at 200 000 g for 2 h to separate the soluble periplasmic and cytoplasmic proteins from the membranes. Proteins of the supernatant were precipitated at 65 % saturation ammonium sulfate as described by Quentmeier et al (2000) and designated the A65 fraction. The 200 000 g pellet was washed twice with 50 mM sodium phosphate buffer, pH 7?4, and designated the membrane fraction.…”

“…The assay (3?0 ml) contained 10 mg protein of the membrane fraction and 30 mg protein of the A65 fraction. Thiosulfate-dependent cytochrome c reducing activity of the A65 fraction and of periplasmic extracts was determined spectrophotometrically at 550 nm as described by Quentmeier et al (2000). One unit (U) of enzyme activity was defined as 1 mmol cytochrome c reduced or O 2 utilized per minute at 30 uC.…”

“…The A65 fraction and the membrane fraction were prepared from cells disrupted with a French press (Quentmeier et al, 2000). The extract was subjected to differential centrifugation at 4 uC.…”

Sulphur oxidising bacteria in mangrove ecosystem: A review

“…Sox (CD) 2 then oxidize the remaining sulphane sulphur, acting as a sulphur dehydrogenase. Sox (CD) 2 are composed of the molybdoprotein SoxC and the diheme c-type cytochrome SoxD (Quentmeier et al, 2000). SoxF gene encodes the monomeric flavoprotein SoxF that has sulphide dehydrogenase activity .…”