Desulfitobacterium chlororespirans Co23 is capable of using 3-chloro-4-hydroxybenzoate as terminal electron acceptor for growth. Membrane preparations from cells grown fermentatively on pyruvate in the presence of 3-chloro-4-hydroxybenzoate dechlorinated this compound at a rate of 3.9 nmol min(sup-1) mg of protein(sup-1). Fivefold-greater dechlorination rates were measured with reduced methyl viologen as the artificial electron donor. Reduced benzyl viologen, NADH, NADPH, reduced flavin adenine dinucleotide, and reduced flavin mononucleotide could not substitute for reduced methyl viologen. The maximal initial rate of catalysis was achieved at pH 6.5 and 60(deg)C. The membrane-bound dechlorinating enzyme system was not oxygen sensitive and was stable at 57(deg)C for at least 2 h. Sulfite inhibited dechlorination in cell-free assays, whereas sulfate did not. Several chlorophenols were dehalogenated exclusively in the ortho position by cell extracts.

Initial Characterization of a Reductive Dehalogenase from Desulfitobacterium chlororespirans Co23.

Since the beginning of the twentieth century, short-chain C1 to C3 chlorinated aliphatic hydrocarbons (CAHs) have been manufactured in large amounts and extensively used in industrial, military, agricultural and household applications. The widespread use of CAHs is based on their desirable properties including low cost, easy availability, excellence as solvents, chemical stability, and fire safety (i.e., most chlorinated solvents are nonflammable and do not form explosive mixtures with air). The widespread use, careless handling and storage, ignorance of health effects and environmental dangers, and the lack of regulations over decades of extensive use has resulted in wide-ranging groundwater contamination. A major strategy for the bioremediation of these solvents is reductive dechlorination by microorganisms, most predominantly members of the Dehalococcoides group, the only microbes known to detoxify chlorinated ethenes to ethene. Members of this group have streamlined genomes highly adapted to using chlorinated hydrocarbons as electron acceptors, and are likely to play important roles in reductive dehalogenation of diverse organohalides in the environment.

Dehalococcoides and Reductive Dechlorination of Chlorinated Solvents

Fe2+ binding to both apo- and holo- bacterial ferritin from Azotobacter vinelandii (AVBF) was measured as a function of pH under carefully controlled anaerobic conditions. Fe2+ binding to apo-AVBF is strongly pH dependent with 25 Fe2+ ions/apo-AVBF binding tightly at pH 5.5 and over 150 Fe2+/apo-AVBF at pH 9.0. Holo-AVBF gave a similar pH-dependent binding profile with over 400 Fe2+/AVBF binding at pH of 9.0. Proton release per Fe2+ bound to either AVBF protein increases with increasing pH until a total of about two protons are released at pH 9.0. These binding results are both qualitatively and quantitatively different from corresponding measurements (Jacobs et al., 1989) on apo- and holo- mammalian ferritin (MF) where less Fe2+ binds in both cases. The high level of Fe2+ binding to holo-AVBF relative to that of mammalian ferritin is a consequence of the higher phosphate content in the core of AVBF. Reduction of AVBF by either dithionite or methyl viologen in the absence of chelating agents demonstrated that phosphate, but not Fe2+, is released from the AVBF core in amounts commensurate with the degree of iron reduction, although even at 100% reduction considerable phosphate remains associated with the reduced mineral core. Fe2+ binding to holo-AVBF made deficient in phosphate was lower than that of native AVBF, while the addition of phosphate to native holo-AVBF increased the Fe2+ binding capacity. These results clearly support the role of phosphate as the site of interaction of Fe2+ with the AVBF mineral core.(ABSTRACT TRUNCATED AT 250 WORDS)

Fe2+ and phosphate interactions in bacterial ferritin from Azotobacter vinelandii.

In order to identify the function and location of phosphate associated with the iron core of horse spleen ferritin (HoSF), the phosphate content of native HoSF was altered by two procedures. Adjustment of pH from 7.0 to 10.0 irreversibly released 53% of the phosphate and 10% of the iron, while lowering the pH to 5.0 reversibly released 43% of the phosphate and 35% of the iron. Reversible release of 85% of the initial phosphate (but little iron release) also occurs upon reduction with methyl viologen (MV) or dithionite. Most of the phosphate is released in the early stages of reduction of the iron core, suggesting that the phosphate resides primarily on the mineral core surface. Reduction followed by chelation altered both the iron and phosphate content of the HoSF mineral cores. HoSF iron cores first reconstituted in the absence of phosphate and then incubated with added phosphate did not bind phosphate. However, when HoSF was first reconstituted in the absence of phosphate and then equilibrated anaerobically with both Fe2+ and phosphate, then phosphate was incorporated in amounts similar to native HoSF. Fe2+ binding to native, phosphate altered, and reconstituted HoSF in the presence and absence of phosphate clearly showed that Fe2+ binding to the mineral core depends on the presence of core-bound phosphate. Fe2+ binding to phosphate-depleted mineral cores or to cores reconstituted with 621, 2158, and 3013 Fe/HoSF core in the absence of phosphate bound only eight Fe2+ per entire ferritin molecule, clearly showing that Fe2+ has no measurable affinity for the phosphate-free, reconstituted mineral core.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of phosphate in fe2+ binding to horse spleen holoferritin

The usefulness in structure/function studies of molybdenum-containing hydroxylases in work with rosy mutant strains of Drosophila melanogaster has been investigated. At least 23 such strains are available, each corresponding to a single known amino acid change in the xanthine dehydrogenase sequence. Sequence comparisons permit identification, with some certainty, of regions associated with the iron-sulphur centres and the pterin molybdenum cofactor of the enzyme. Procedures have been developed and rigorously tested for the assay in gel-filtered extracts of the flies, of different catalytic activities of xanthine dehydrogenase by the use of various oxidizing and reducing substrates. These methods have been applied to 11 different rosy mutant strains that map to different regions of the sequence. All the mutations studied cause characteristic activity changes in the enzyme. In general these are consistent with the accepted assignment of the cofactors to the different domains and with the known reactivities of the molybdenum, flavin and iron-sulphur centres. Most results are interpretable in terms of the mutation affecting electron transfer to or from one redox centre only. The activity data provide evidence that FAD and the NAD+/NADH binding sites are retained in mutants mapping to the flavin domain. Therefore, despite some indications from sequence comparisons, it is concluded that the structure of this domain of xanthine dehydrogenase cannot be directly related to that of other flavoproteins for which structural data are available. The data also indicate that the artificial electron acceptor phenazine methosulphate acts at the iron-sulphur centres and suggest that these centres may not be essential for electron transfer between molybdenum and flavin. The work emphasizes the importance of combined genetic and biochemical study of rosy mutant xanthine dehydrogenase variants in probing the structure and function of enzymes of this class.

Use of rosy mutant strains of Drosophila melanogaster to probe the structure and function of xanthine dehydrogenase.

A chemical preparation of pure reduced viologens for use as biomolecular reducing reagents.

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A chemical preparation of pure reduced viologens for use as biomolecular reducing reagents.