Redox and chemical activities of the hemes in the sulfur oxidation pathway enzyme SoxAX.

BACKGROUND: SoxAX enzymes initiate microbial oxidation of reduced inorganic sulfur compounds. Their catalytic mechanism is unknown. RESULTS: Cyanide displaces the CysS(-) ligand to the active site heme following reduction by S(2)O(4)(2-) but not Eu(II). CONCLUSION: An active site heme ligand becomes labile on exposure to substrate analogs. SIGNIFICANCE: Elucidation of SoxAX mechanism is necessary to understand a widespread pathway for sulfur compound oxidation. SoxAX enzymes couple disulfide bond formation to the reduction of cytochrome c in the first step of the phylogenetically widespread Sox microbial sulfur oxidation pathway. Rhodovulum sulfidophilum SoxAX contains three hemes. An electrochemical cell compatible with magnetic circular dichroism at near infrared wavelengths has been developed to resolve redox and chemical properties of the SoxAX hemes. In combination with potentiometric titrations monitored by electronic absorbance and EPR, this method defines midpoint potentials (E(m)) at pH 7.0 of approximately +210, -340, and -400 mV for the His/Met, His/Cys(-), and active site His/CysS(-)-ligated heme, respectively. Exposing SoxAX to S(2)O(4)(2-), a substrate analog with E(m) ~-450 mV, but not Eu(II) complexed with diethylene triamine pentaacetic acid (E(m) ~-1140 mV), allows cyanide to displace the cysteine persulfide (CysS(-)) ligand to the active site heme. This provides the first evidence for the dissociation of CysS(-) that has been proposed as a key event in SoxAX catalysis.

Rhodovulum kholense sp. nov.

A yellowish brown bacterium was isolated from photoheterotrophic enrichment cultures obtained from a mud sample collected from a mangrove forest located in Khola, India. Enrichment and isolation in medium containing 2 % NaCl (w/v) yielded strain JA297(T), cells of which were ovoid and motile. Cells of strain JA297(T) contained vesicular internal membranes and bacteriochlorophyll a and carotenoids of the spheroidene series. Strain JA297(T) grew optimally at 30 degrees C and at pH 6.0-7.0. Photo-organoheterotrophy was the preferred mode of growth. Strain JA297(T) was also able to grow photolithoautotrophically and chemo-organotrophically but not by chemolithoautotrophy or by fermentation. Strain JA297(T) was able to utilize sulfide, sulfite, sulfate, thiosulfate, thioglycolate and cysteine as sulfur sources. Biotin, niacin and thiamine were required as growth factors. On the basis of 16S rRNA gene sequence analysis, strain JA297(T) was shown to belong to the class Alphaproteobacteria and was closely related to the type strains of Rhodovulum marinum (96.72 % similarity), Rhodovulum visakhapatnamense (96.42 %) and Rhodovulum sulfidophilum (96.32 %). The DNA G+C composition was 63 mol%. Based on 16S rRNA gene sequence analysis and morphological and physiological characteristics, strain JA297(T) could be distinguished from all recognized species of the genus Rhodovulum. This strain is therefore suggested to represent a novel species of the genus Rhodovulum, for which the name Rhodovulum kholense sp. nov. is proposed. The type strain is JA297(T) (=ATCC BAA-1544(T)=JCM 14888(T)=CCUG 55397(T)).

Characterization of a mutant strain of Rhodovulum sulfidophilum lacking the pufA and pufB genes encoding the polypeptides for the light-harvesting complex 1 (B 870).

Contradictory results on the effectiveness of energy transfer from the light harvesting complex 2 (LH2) directly to the reaction center (RC) in mutant strains lacking the core light-harvesting complex 1 (LH1) have been obtained with cells of Rhodobacter capsulatus and Rhodobacter sphaeroides. A LH1(-) mutant of Rhodovulum sulfidophilum, named rsLRI, was constructed by deletion of the pufBA genes, resulting in a kanamycin resistant photosynthetically positive clone. To restore the wild type phenotype, a complemented strain C2 was constructed by inserting in trans a DNA segment containing the pufBA genes. Light-induced FTIR difference spectra indicate that the RC in the rsLRI mutant and in the C2 complemented strains are functionally and structurally identical with those in the wild type strain, demonstrating that the assembly and the function of the RC is not impaired by the LH1 deletion. The photosynthetic growth rate of the rsLRI strain increased with decreasing light intensity. At 50 W m(-2 )no photosynthetic growth was observed. These results indicate that the light energy harvested by the LH2 complex was not or inefficiently transferred to the RC; thus most of the energy necessary for photosynthetic growth is in the LH1(-) strain directly absorbed by the RC. It is supposed that in the mutant strain, RC and LH2 cannot interact in an efficient way.