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 α2β2 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 SoxCD1 complex consisting of SoxC and SoxD1 of 30 kDa, which contained only the heme-1 domain. The heme-2 domain was isolated as a new cytochrome SoxD2 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 SoxCD1 and SoxD2 both exhibit a midpoint potential of E m = 278 ± 10 mV. Electrochemically induced FTIR difference spectra of Sox(CD)2, SoxCD1, and SoxD2 were distinct. A carboxy group is protonated upon reduction of the SoxD1 heme but not for SoxD2. The specific activity of SoxCD1 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 SoxCD1 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 α2β2 tetrameric structure of sulfur dehydrogenase.