Characterization of the Redox Centers in Dimethyl Sulfide Dehydrogenase from Rhodovulum sulfidophilum

• Published in: Biochemistry (Easton) 2002-12, Vol.41 (51), p.15234-15244
• Main Authors: McDevitt, Christopher A, Hanson, Graeme R, Noble, Christopher J, Cheesman, Myles R, McEwan, Alastair G
• Format: Article
• Language: English
• Subjects: Alcohol Oxidoreductases - chemistry; Alcohol Oxidoreductases - genetics; Alphaproteobacteria - enzymology; Alphaproteobacteria - growth & development; Bacterial Proteins - chemistry; Circular Dichroism; Cytochrome b Group - chemistry; Electron Spin Resonance Spectroscopy; Ferric Compounds - chemistry; Heme - chemistry; Iron-Sulfur Proteins - chemistry; Ligands; Molybdenum - chemistry; Oxidation-Reduction; Oxidoreductases - chemistry; Potentiometry; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi026221u

Dimethyl sulfide dehydrogenase from the purple phototrophic bacterium Rhodovulum sulfidophilum catalyzes the oxidation of dimethyl sulfide to dimethyl sulfoxide. Recent DNA sequence analysis of the ddh operon, encoding dimethyl sulfide dehydrogenase (ddhABC), and biochemical analysis ( ) have revealed that it is a member of the DMSO reductase family of molybdenum enzymes and is closely related to respiratory nitrate reductase (NarGHI). Variable temperature X-band EPR spectra (120−122 K) of purified heterotrimeric dimethyl sulfide dehydrogenase showed resonances arising from multiple redox centers, Mo(V), [3Fe-4S]+, [4Fe-4S]+, and a b-type heme. A pH-dependent EPR study of the Mo(V) center in 1H2O and 2H2O revealed the presence of three Mo(V) species in equilibrium, Mo(V)−OH2, Mo(V)−anion, and Mo(V)−OH. Above pH 8.2 the dominant species was Mo(V)−OH. The maximum specific activity occurred at pH 9.27. Comparison of the rhombicity and anisotropy parameters for the Mo(V) species in DMS dehydrogenase with other molybdenum enzymes of the DMSO reductase family showed that it was most similar to the low-pH nitrite spectrum of Escherichia coli nitrate reductase (NarGHI), consistent with previous sequence analysis of DdhA and NarG. A sequence comparison of DdhB and NarH has predicted the presence of four [Fe-S] clusters in DdhB. A [3Fe-4S]+ cluster was identified in dimethyl sulfide dehydrogenase whose properties resembled those of center 2 of NarH. A [4Fe-4S]+ cluster was also identified with unusual spin Hamiltonian parameters, suggesting that one of the iron atoms may have a fifth non-sulfur ligand. The g matrix for this cluster is very similar to that found for the minor conformation of center 1 in NarH [Guigliarelli, B., Asso, M., More, C., Augher, V., Blasco, F., Pommier, J., Giodano, G., and Bertrand, P. (1992) Eur. J. Biochem. 307, 63−68]. Analysis of a ddhC mutant showed that this gene encodes the b-type cytochrome in dimethyl sulfide dehydrogenase. Magnetic circular dichroism studies revealed that the axial ligands to the iron in this cytochrome are a histidine and methionine, consistent with predictions from protein sequence analysis. Redox potentiometry showed that the b-type cytochrome has a high midpoint redox potential (E° = +315 mV, pH 8).