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

The periplasmic sulfite dehydrogenase of Paracoccus pantotrophus GB17 was p urified to homogeneity by a four-step procedure from cells grown lithoautot rophically with thiosulfate. The molecular mass of native sulfite dehydroge nase was 190 kDa as determined by native gradient PAGE. SDS-PAGE showed sul fite dehydrogenase to comprise two subunits with molecular masses of 47 kDa and 50 kDa, suggesting an alpha 2 beta 2 structure. The N-terminal amino a cid sequence and immunochemical analysis using SoxC-specific antibodies ide ntified the 47-kDa protein as the soxC gene product. SoxD-specific antibodi es identified the 50-kDa protein as SoxD. Based on the molecular masses ded uced from the nucleotide sequence for mature SoxC (43,442 Da) and SoxD (37, 637 Da) sulfite dehydrogenase contained 1.30 mol molybdenum/mol alpha 2 bet a 2 sulfite dehydrogenase. The iron content was 3.17 mol/mol alpha 2 beta 2 sulfite dehydrogenase, and 3.53 mol heme/mol alpha 2 beta 2 sulfite dehydr ogenase was determined by pyridine hemochrome analysis, These data are cons istent with the two heme-binding domains (CxxCH), characteristic for c-type cytochromes, deduced from the soxD nucleotide sequence, Electrospray ioniz ation 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 So xD a mass of 38,815 Da was determined; this accounted for the polypeptide a nd two covalently bound hemes. Reconstitution of the catalytic activity of sulfite dehydrogenase required additional fractions; these eluted from Q Se pharose at 0.05, 0.25, and 0.30 M NaCl. The K-m of sulfite dehydrogenase fo r sulfite was 7.0 mu M and for cytochrome c 19 mu M Sulfite dehydrogenase a ctivity was inhibited by sulfate and phosphate. The structural and catalyti c properties make sulfite dehydrogenase from P. denitrificans GB17 distinct from sulfite oxidases of other prokaryotic or eukaryotic sources.