The discovery of new chlorophyllous pigments would provide greater understanding of the mechanisms and evolution of photosynthesis. Bacteriochlorophyll f has never been observed in nature, although this name was proposed ~40 years ago based on structurally related compounds. We constructed a bacteriochlorophyll f–accumulating mutant of the green sulfur bacterium Chlorobaculum limnaeum, which originally produced bacteriochlorophyll e, by knocking out the bchU gene encoding C-20 methyltransferase based on natural transformation. This novel pigment self-aggregates in an in vivo light-harvesting antenna, the chlorosome, and exhibits a Q_y peak of 705 nm, more blue-shifted than any other chlorosome reported so far; the peak overlaps the maximum (~700 nm) of the solar photon flux spectrum. Bacteriochlorophyll f chlorosomes can transfer light energy from core aggregated pigments to another bacteriochlorophyll in the chlorosomal envelope across an energy gap of ~100 nm, and is thus a promising material for development of new bioenergy applications.

The gene named bciD, which encodes the enzyme involved in C7-formylation in bacteriochlorophyll e biosynthesis, was found and investigated by insertional inactivation in the brown-colored green sulfur bacterium Chlorobaculum limnaeum (previously called Chlorobium phaeobacteroides). The bciD mutant cells were green in color, and accumulated bacteriochlorophyll c homologs bearing the 7-methyl group, compared to C7-formylated BChl e homologs in the wild type. BChl-c homolog compositions in the mutant were further different from those in Chlorobaculum tepidum which originally produced BChl c: (3¹S)-8-isobutyl-12-ethyl-BChl c was unusually predominant.

Bacteriochlorophyll a is widely distributed among anoxygenic photosynthetic bacteria. In bacteriochlorophyll a biosynthesis, the reduction of the C8 vinyl group in 8-vinyl-chlorophyllide a is catalyzed to produce chlorophyllide a by an 8-vinyl reductase called divinyl reductase (DVR), which has been classified into two types, BciA and BciB. However, previous studies demonstrated that mutants lacking the DVR still synthesize normal bacteriochlorophyll a with the C8 ethyl group and suggested the existence of an unknown “third” DVR. Meanwhile, we recently observed that chlorophyllide a oxidoreductase (COR) of a purple bacterium happened to show the 8-vinyl reduction of 8-vinyl-chlorophyllide a in vitro. In this study, we made a double mutant lacking BciA and COR of the purple bacterium Rhodobacter sphaeroides in order to investigate whether the mutant still produces pigments with the C8 ethyl group or if COR actually works as the third DVR. The single mutant deleting BciA or COR showed production of the C8 ethyl group pigments, whereas the double mutant accumulated 8-vinyl-chlorophyllide, indicating that there was no enzyme other than BciA and COR functioning as the unknown third DVR in Rhodobacter sphaeroides (note that this bacterium has no bciB gene). Moreover, some COR genes derived from other groups of anoxygenic photosynthetic bacteria were introduced into the double mutant, and all of the complementary strains produced normal bacteriochlorophyll a. This observation indicated that COR of these bacteria performs two functions, reductions of the C8 vinyl group and the C7=C8 double bond, and that such an activity is probably conserved in the widely ranging groups.

Summary: Photosynthetic green sulfur bacteria inhabit anaerobic environments with very low‐light conditions. To adapt to such environments, these bacteria have evolved efficient light‐harvesting antenna complexes called as chlorosomes, which comprise self‐aggregated bacteriochlorophyll c in the model green sulfur, bacterium Chlorobaculum tepidum. The pigment possess a hydroxy group at the C3¹ position that produces a chiral center with R‐ or S‐stereochemistry and the C3¹‐hydroxy group serves as a connecting moiety for the self‐aggregation. Chlorobaculum tepidum carries the two possible homologous genes for C3‐vinyl hydratase, bchF and bchV. In the present study, we constructed deletion mutants of each of these genes. Pigment analyses of the bchF‐inactivated mutant, which still has BchV as a sole hydratase, showed higher ratios of S‐epimeric bacteriochlorophyll c than the wild‐type strain. The heightened prevalence of S‐stereoisomers in the mutant was more remarkable at lower light intensities and caused a red shift of the chlorosomal Q_y absorption band leading to advantages for light‐energy transfer. In contrast, the bchV‐mutant possessing only BchF showed a significant decrease of the S‐epimers and accumulations of C3‐vinyl BChl c species. As trans‐ criptional level of bchV was upregulated at lower light intensity, the Chlorobaculum tepidum adapted to low‐light environments by control of the bchV transcription.

A BciC enzyme is related to the removal of the C13²-methoxycarbonyl group in biosynthesis of bacteriochlorophylls (BChls) c, d and e functioning in green sulfur bacteria, filamentous anoxygenic phototrophs and phototrophic acidobacteria. These photosynthetic bacteria have the largest and the most efficient light-harvesting antenna systems, called chlorosomes, containing unique self-aggregates of BChl c, d or e pigments, that lack the C13²-methoxycarbonyl group which disturbs chlorosomal self-aggregation. In this study, we characterized the BciC derived from the green sulfur bacterium Chlorobaculum tepidum, and examined the in vitro enzymatic activities of its recombinant protein. The BciC-catalyzing reactions of various substrates showed that the enzyme recognized chlorophyllide (Chlide) a and 3,8-divinyl(DV)-Chlide a as chlorin substrates to give 3-vinyl-bacteriochlorophyllide (3V-BChlide) d and DV-BChlide d, respectively. Since the BciC afforded a higher activity with Chlide a than that with DV-Chlide a and no activity with (DV-)protoChlides a (porphyrin substrates) and 3V-BChlide a (a bacteriochlorin substrate), this enzyme was effective for diverting the chlorosomal pigment biosynthetic pathway at the stage of Chlide a away from syntheses of other pigments such as BChl a and Chl a. The addition of methanol to the reaction mixture did not prevent the BciC activity, and we identified this enzyme as Chlide a demethoxycarbonylase, not methylesterase.