Menaquinone reduction by an HMT2-like sulfide dehydrogenase from Bacillus stearothermophilus.

The gene-encoding HMT2-like sulfide dehydrogenase from Bacillus stearothermophilus JCM2501 was amplified and expressed in Escherichia coli and the enzymatic features were examined. The enzyme was detected mainly in the membrane fraction. It catalyzed the sulfide-dependent menaquinone (MK) reduction showing special enzymatic features distinct from other sulfide-quinone oxidoreductases (SQRs) from autotrophic bacteria. The purified protein from E. coli brought about the sulfide-dependent 2,3-dimethyl-1,4-naphthoquinone (DMN) reduction in vitro. The reduction was accelerated in the presence of either cyanide or 2-mercaptoethanol and phospholipids. The high reduction was followed by a change in Km values for sulfide and DMN. The purified enzyme utilized MK as an electron acceptor in the membrane fraction from E. coli. Under anaerobic conditions, sulfide was oxidized with reduction of fumarate or nitrate via the MK pool. The dehydrogenase was different from SQR in autotrophic bacteria in terms of the low affinity for sulfide and the activity enhancement in the presence of cyanide or 2-mercaptoethanol. The sulfide oxidation via MK in the cellular membrane of Gram-positive bacteria was certified.

Characterization of a HMT2-like enzyme for sulfide oxidation from Pseudomonas putida.

The open reading frame pp0053, which has a high homology with the sequence of mitochondrial sulfide dehydrogenase (HMT2) conferring cadmium tolerance in fission yeast, was amplified from Pseudomonas putida KT2440 and expressed in Escherichia coli JM109(DE3). The isolated and purified PP0053-His showed absorption spectra typical of a flavin adenine dinucleotide (FAD)--binding protein. The PP0053-His catalyzed a transfer of sulfide-sulfur to the thiophilic acceptor, cyanide, which decreased the Km value of the enzyme for sulfide oxidation and elevated the sulfide-dependent quinone reduction. Reaction of the enzyme with cyanide elicited a dose-dependent formation of a charge transfer band, and the FAD-cyanide adduct was supposed to work for a sulfur transfer. The pp0053 deletion from P. putida KT2440 led to activity declines of the intracellular catalase and ubiquinone-H2 oxidase. The sulfide-quinone oxidoreductase activity in P. putida KT2440 was attributable to the presence of pp0053, and the activity showed a close relevance to enzymatic activities related to sulfur assimilation.