title: Characterization of BciB: a ferredoxin-dependent 8-vinyl-protochlorophyllide reductase from the green sulfur bacterium Chloroherpeton thalassium.
abstract: Two enzymes, BciA and BciB, are known to reduce the C-8 vinyl group of 8-vinyl protochlorophyllide, producing protochlorophyllide a, during the synthesis of chlorophylls and bacteriochlorophylls in chlorophototrophic bacteria. BciA from the green sulfur bacterium Chlorobaculum tepidum reduces the C-8 vinyl group using NADPH as the reductant. Cyanobacteria and some other chlorophototrophs have a second, nonhomologous type of 8-vinyl reductase, BciB, but the biochemical properties of this enzyme have not yet been described. In this study, the bciB gene of the green sulfur bacterium Chloroherpeton thalassium was expressed in Escherichia coli , and the recombinant protein was purified and characterized. Recombinant BciB binds a flavin adenine dinucleotide cofactor, and EPR spectroscopy as well as quantitative analyses of bound iron and sulfide suggest that BciB binds two [4Fe-4S] clusters, one of which may not be essential for the activity of the enzyme. Using electrons provided by reduced ferredoxin or dithionite, recombinant BciB was active and reduced the 8-vinyl moiety of the substrate, 8-vinyl protochlorophyllide, producing protochlorophyllide a. A structural model for BciB based on a recent structure for the FrhB subunit of F420-reducing [NiFe]-hydrogenase of Methanothermobacter marburgensis is proposed. Possible reasons for the occurrence and distribution of BciA and BciB among various chlorophototrophs are discussed.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.
authors.forename: Allison H
authors.initials: AH
authors.lastname: Saunders
authors.forename: John H
authors.initials: JH
authors.lastname: Golbeck
authors.forename: Donald A
authors.initials: DA
authors.lastname: Bryant
mesh_terms: D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019414:Chlorobi / Q000201:enzymology* / Q000254:growth & development; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008665:Metalloporphyrins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011521:Protochlorophyllide / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D001051:Apoenzymes; D001426:Bacterial Proteins; D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D007527:Isoenzymes; D008665:Metalloporphyrins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D011521:Protochlorophyllide; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase