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.

Chlorophyllide a Oxidoreductase Works as One of the Divinyl Reductases Specifically Involved in Bacteriochlorophyll a Biosynthesis

“…COR carries out its enzymatic reduction under anaerobic conditions but shows no activity in the presence of dioxygen (7). In contrast, BciA and BciB are active even under aerobic conditions (12,19). Most purple bacteria still synthesize BChl a under semiaerobic and dark conditions.…”

“…One of those orthologs, the Ctha_1208 gene of the green sulfur bacterium Chloroherpeton thalassium, was observed (18); although a genetic manipulation of this bacterium is not available, the Ctha_1208 gene could complement the DVR ability in the bciA mutant of C. tepidum and was denoted as bciB for BChl synthetic pathways. Very recently, the recombinant BciB and cyano-type DVR were studied in vitro and showed the 8-vinyl reduction activity using ferredoxin as an electron donor (19,20).…”

Crystal Structure and Catalytic Mechanism of 7-Hydroxymethyl Chlorophyll a Reductase

“…The homologue of HCAR in green sulfur bacteria, BciB, is a DVR (19). In the cyanobacterium Synechocystis, the HCAR homologue possesses promiscuous enzymatic activities besides its primary function as an DVR and can work as NADH dehydrogenase, chlorophyll b reductase, and HCAR (17,19,44).…”

“…They perform different roles, such as the F 420 -binding subunit of F 420 -reducing [NiFe] hydrogenase (Frh) in methanogenic archaea (16) and 3,8-divinyl (proto)chlorophyllide a 8-vinyl reductase (DVR) involved in (bacterio)chlorophyll biosynthesis (17,18). The cofactors FAD and [4Fe-4S] cluster have been found to participate in electron transfer within Frh and DVR (18,19). The structure of Frh from Methanothermobacter marburgensis has been solved by cryo-microscopy and crystallography (20 -22).…”

“…Frh is a heterotrimer, with subunit FrhA carrying a [NiFe] center, subunit FrhG carrying three [4Fe-4S] clusters, and subunit FrhB carrying one [4Fe-4S] cluster and one FAD. It has been suggested that the HCAR homologues adopt structures similar to that of FrhB, the F 420 -binding subunit (19), but arrangements of the cofactors and hence the details of the electron pathway within HCAR remain unclear.…”