Sulfide dehydrogenase activity of the monomeric flavoprotein SoxF of Paracoccus pantotrophus.
Flavocytochrome c-sulfide dehydrogenases (FCSDs) are complexes of a flavoprotein with a c-type cytochrome performing hydrogen sulfide-dependent cytochrome c reduction in vitro. The amino acid sequence analysis revealed that the phylogenetic relationship of different flavoproteins reflected the relationship of sulfur-oxidizing bacteria. The flavoprotein SoxF of Paracoccus pantotrophus is 29-67% identical to the flavoprotein subunit of FCSD of phototrophic sulfur-oxidizing bacteria. Purification of SoxF yielded a homogeneous emerald-green monomeric protein of 42 797 Da. SoxF catalyzed sulfide-dependent horse heart cytochrome c reduction at the optimum pH of 6.0 with a k(cat) of 3.9 s(-1), a K(m) of 2.3 microM for sulfide, and a K(m) of 116 microM for cytochrome c, as determined by nonlinear regression analysis. The yield of 1.9 mol of cytochrome c reduced per mole of sulfide suggests sulfur or polysulfide as the product. Sulfide dehydrogenase activity of SoxF was inhibited by sulfur (K(i) = 1.3 microM) and inactivated by sulfite. Cyanide (1 mM) inhibited SoxF activity at pH 6.0 by 25% and at pH 8.0 by 92%. Redox titrations in the infrared spectral range from 1800 to 1200 cm(-1) and in the visible spectral range from 400 to 700 nm both yielded a midpoint potential for SoxF of -555 +/- 10 mV versus Ag/AgCl at pH 7.5 and -440 +/- 20 mV versus Ag/AgCl at pH 6.0 (-232 mV versus SHE') and a transfer of 1.9 electrons. Electrochemically induced FTIR difference spectra of SoxF as compared to those of free flavin in solution suggested a strong cofactor interaction with the apoprotein. Furthermore, an activation/variation of SoxF during the redox cycles is observed. This is the first report of a monomeric flavoprotein with sulfide dehydrogenase activity.
Quentmeier
Armin
A
Lehrstuhl für Technische Mikrobiologie, Fachbereich Bio- und Chemieingenieurwesen, Universität Dortmund, Emil-Figge-Strasse 66, D-44221 Dortmund, Germany.
Hellwig
Petra
P
Bardischewsky
Frank
F
Wichmann
Rolf
R
Friedrich
Cornelius G
CG
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002384:Catalysis; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D045304:Cytochromes c / Q000737:chemistry; D004563:Electrochemistry; D004789:Enzyme Activation; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D016680:Genome, Bacterial; D006736:Horses; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D042084:Paracoccus pantotrophus / Q000201:enzymology* / Q000235:genetics; D017550:Spectroscopy, Fourier Transform Infrared
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D005420:Flavoproteins; D009097:Multienzyme Complexes; C107539:SoxF protein, Paracoccus; D045304:Cytochromes c; D010088:Oxidoreductases; C064500:flavocytochrome c sulfide dehydrogenase