Search

The newly discovered di-haem cytochrome c4 from the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina is the first cytochrome c4 to be crystallized from an anaerobic organism. It was crystallized using the addition of metal-ion salts to the standard vapour-diffusion method. Coloured well shaped three-dimensional crystals with dimensions of approximately 0.6 x 0.05 x 0.02 mm grew within 3-4 d at pH 5 and diffracted to 1.72 angstroms without radiation damage. Cytochrome c4 crystallized in space group P4(1)2(1)2 as a primitive tetragonal system with unit-cell parameters a = b = 75.29, c = 37.12 angstroms, alpha = beta = gamma = 90 degrees.

There are at least two membrane-bound (HynSL and HupSL) and one soluble (HoxEFUYH) [NiFe] hydrogenases in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium. Genes coding for accessory proteins that participate in the biosynthesis and maturation of hydrogenases seem to be scattered along the chromosome. Transposon-based mutagenesis was used to locate the hydrogenase accessory genes. Molecular analysis of strains showing mutant phenotypes led to the identification of hupK (hoxV ), hypC1, hypC2, hypD, hypE, and hynD genes. The roles of hynD, hupK and the two hypC genes were investigated in detail. The putative HynD was found to be a hydrogenase-specific endoprotease type protein, participating in the maturation of the HynSL enzyme. HupK plays an important role in the formation of the functionally active membrane-bound [NiFe] hydrogenases, but not in the biosynthesis of the soluble enzyme. In-frame deletion mutagenesis showed that HypC proteins were not specific for the maturation of either hydrogenase enzyme. The lack of either HypC protein drastically reduced the activity of every hydrogenase. Hence both HypCs might participate in the maturation of [NiFe] hydrogenases. Homologous complementation with the appropriate genes substantiated the physiological roles of the corresponding gene products in the H2 metabolism of T. roseopersicina.

The effects of some metal ions on the activity and activation of Thiocapsa roseopersicina hydrogenase have been studied. Inhibitory effects of Ni2+ and Cd2+ on the catalytic activity of the enzyme were reversible and competitive with respect to methyl viologen (MV) in the reaction of hydrogen oxidation. The affinity of these metal ions to the enzyme increased significantly with increasing pH, suggesting that their interactions are determined by electrostatic forces. Cu2+ and Hg2+ irreversibly inhibited the hydrogenase activity. A decrease in absorption of hydrogenase at 400 nm in the presence of these metal ions is indicative of the destruction of the FeS cluster in the enzyme.

The PHA synthase structural gene of Thiocapsa pfennigii was identified and subcloned on a 2.8-kbp BamHI restriction fragment, which was cloned recently from a genomic 15.6-kbp EcoRI restriction fragment. Nucleotide sequence analysis of this fragment revealed three open reading frames (ORFs), representing coding regions. Two ORFs encoded for the PhaE (Mr 40,950) and PhaC (Mr 40,190) subunits of the PHA synthase from T. pfennigii and exhibited high homology with the corresponding proteins of the Chromatium vinosum (52.8% and 85.2% amino acid identity) and the Thiocystis violacea (52.5% and 82.4%) PHA synthases, respectively. This confirmed that the T. pfennigii PHA synthase was composed of two different subunits. Also, with respect to the molecular organization of phaE and phaC, this region of the T. pfennigii genome resembled very much the corresponding regions of C. vinosum and of Thiocystis violacea. A recombinant strain of Pseudomonas putida, which overexpressed phaE and phaC from T. pfennigii, was used to isolate the PHA synthase by a two-step procedure including chromatography on Procion Blue H-ERD and hydroxyapatite. The isolated PHA synthase consisted of two proteins exhibiting the molecular weights predicted for PhaE and PhaC. Hybrid PHA synthases composed of PhaE from T. pfennigii and PhaC from C. vinosum and vice versa were constructed and functionally expressed in a PHA-negative mutant of P. putida; and the resulting PHAs were analyzed.