Characterization of redox centers in dimethyl sulfide dehydrogenase from Rhodovulum sulfidophilum

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 (1) 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 (120122 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 (H2O)-H-1 and (H2O)-H-2 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-o = +315 mV, pH 8).

Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617

J Biol Inorg Chem (2008) 13:1321–1333 DOI 10.1007/s00775-008-0416-1

Structural and Functional Insights from the Metagenome of an Acidic Hot Spring Microbial Planktonic Community in the Colombian Andes

A taxonomic and annotated functional description of microbial life was deduced from 53 Mb of metagenomic sequence retrieved from a planktonic fraction of the Neotropical high Andean (3,973 meters above sea level) acidic hot spring El Coquito (EC). A classification of unassembled metagenomic reads using different databases showed a high proportion of Gammaproteobacteria and Alphaproteobacteria (in total read affiliation), and through taxonomic affiliation of 16S rRNA gene fragments we observed the presence of Proteobacteria, micro-algae chloroplast and Firmicutes. Reads mapped against the genomes Acidiphilium cryptum JF-5, Legionella pneumophila str. Corby and Acidithiobacillus caldus revealed the presence of transposase-like sequences, potentially involved in horizontal gene transfer. Functional annotation and hierarchical comparison with different datasets obtained by pyrosequencing in different ecosystems showed that the microbial community also contained extensive DNA repair systems, possibly to cope with ultraviolet radiation at such high altitudes. Analysis of genes involved in the nitrogen cycle indicated the presence of dissimilatory nitrate reduction to N2 (narGHI, nirS, norBCDQ and nosZ), associated with Proteobacteria-like sequences. Genes involved in the sulfur cycle (cysDN, cysNC and aprA) indicated adenylsulfate and sulfite production that were affiliated to several bacterial species. In summary, metagenomic sequence data provided insight regarding the structure and possible functions of this hot spring microbial community, describing some groups potentially involved in the nitrogen and sulfur cycling in this environment. Citation: Jimenez DJ, Andreote FD, Chaves D, Montana JS, Osorio-Forero C, et al. (2012) Structural and Functional Insights from the Metagenome of an Acidic Hot Spring Microbial Planktonic Community in the Colombian Andes. PLoS ONE 7(12): e52069. doi:10.1371/journal.pone.0052069

GENETIC ORGANIZATION OF THE STRUCTURAL GENES FOR NITRATE REDUCTASE (TN5)

Nitrate reductase in Escherichia coli is a membrane-bound anaerobic enzyme that is repressed by oxygen and induced by nitrate. The genetic organization of the structural genes for the two larger subunits of nitrate reductase ((alpha) and (beta)) was determined by immunoprecipitation analysis of the formation of these proteins in nitrate reductase-deficient mutants resulting from transposon Tn5 mutagenesis. The results suggested that the genes encoding the (alpha) and (beta) subunits (narG and H) were arranged in an operon with transcription in the direction promoter(--->)(alpha)(--->)(beta). Segments of the chromosome containing the Tn5 inserts from several of the mutants were cloned into plasmid pBR322 and the positions of the transposons determined by restriction mapping. The Tn5 insertion sites were localized on two contiguous EcoRI fragments spanning about 6.6 kilobases of DNA. The narI gene (proposed to encode the (gamma) subunit) was positioned immediately downstream from the (beta)-gene (narH) by Southern analysis of Tn10 insertions into the narI locus. A Tn10 insertion into the narK locus, proposed to encode a nitrate-sensitive repressor of other anaerobic enzymes, was located about 1.5 kilobases upstream from the narGHI operon promoter. The narL locus, proposed to encode a nitrate-sensitive positive regulator of the narGHI operon and known to be genetically linked to the other nar genes, was demonstrated to lie outside a 19.3-kilobase region of the chromosome which encompasses the other nar genes. The physical limit of the narGHI promoter was defined by studying the effect of Tn5 insertions into a hybrid plasmid containing the functional operon. The points of origin of the coding regions for the (alpha) and (beta) genes were deduced by alignment of the chromosomal map of Tn5 insertion sites with the sizes of (alpha) and (beta) subunit fragments produced by plasmids carrying these Tn5 inserts in the nar operon. The coding region for the (alpha) subunit (143,000 daltons) begins about 250 nucleotides downstream from the deduced limit of the promoter region and includes about 4.0 kilobases of DNA; the region encoding (beta) (60,000 daltons) lies immediately downstream from the (alpha)-gene and is approximately 1.6 kilobases in length. The adjacent region encoding the (gamma) subunit (19,000 daltons) is approximately 0.5 kilobase in length. ^

Dimethylsulfide dehydrogenase from rhodovulum sulfidophilum: EPR spectroscopy and biochemical analysis reveal its place in the DMSO reductase family of molybdenum enzymes

Dimethylsulfide (DMS) dehydrogenase catalyses the oxidation of DMS to dimethylsulfoxide. The purified enzyme has three subunits of Mr = 94, 38 and 32 kDa and has an optical spectrum dominated by a b-type cytochrome. The metal ion and nucleotide analysis revealed 0.5 g-atom Mo, 9.8 g-atom Fe and 1.96 mol GMP per tool of enzyme. Taken together, these data indicate that DMS dehydrogenase contains a bis(MGD)Mo cofactor. A comparison of the Nterminal amino acid sequence of DMS dehydrogenase revealed that the Mo-containing ct-subunit was most closely related to the c~-subunits of nitrate reductase (NarG) and selenate reductase (SerA). Similarly, the [~-subunit of DMS dehydrogenase was most closely related to the [3-subunits of nitrate reductase (NarH) and selenate reductase (SerB). Variable temperature X-band EPR spectra (120-2K) of 'as isolated' DMS dehydrogenase showed resonances arising from multiple redox centres, Mo(V), [3Fe-4S] +, [4Fe-4S] ÷. A pH dependent EPR study of the Mo(V) centre in lH20 and 2H20 reveals the presence of three Mo(V) species in equilibrium, Mo(V)-OH2, Mo(V)-X and Mo(V)-OH. Between pH6 and 8.2 the dominant species is Mo(V)-OH2 and Mo(V)-X is a minor component. X is probably the anion, chloride. Comparison of the rhombicity and anisotropy parameters for the Mo(V) species in DMS dehydrogenase with other Mo(V) centres in metalloproteins showed that it was most similar to the low pH nitrite spectrum of E. coli nitrate reductase (NarGHI). The spin Hamiltonian parameters (2.0158, 1.8870, 1.8620) for the [4Fe-4S] + cluster suggests the presence of histidine (N) coordination to iron in this cluster. It is suggested that this unusual [Fe-S] cluster may be associated with a histidine-cysteine rich sequence at the N-terminus of the ct-subunit of DMS dehydrogenase.