In most photosynthetic organisms, the chlorin ring structure of chlorophyll a is formed by the reduction of the porphyrin D-ring by the dark-operative nitrogenase-like enzyme, protochlorophyllide reductase (DPOR). Subsequently, the chlorin B-ring is reduced in bacteriochlorophyll biosynthesis to form a bacteriochlorin ring structure. Phenotypic analysis of mutants lacking one of three genes, bchX, bchY, or bchZ, which show significant sequence similarity to the structural genes of nitrogenase, suggests that a second nitrogenase-like enzyme is involved in the chlorin B-ring reduction. However, there is no biochemical evidence for this. Here, we report the reconstitution of chlorophyllide a reductase (COR) with purified proteins. Two Rhodobacter capsulatus strains that overexpressed Strep-tagged BchX and BchY were isolated. Strep-tagged BchX was purified as a single polypeptide, and BchZ was co-purified with Strep-tagged BchY. When BchX and BchY-BchZ components were incubated with chlorophyllide a, ATP, and dithionite under anaerobic conditions, chlorophyllide a was converted to a new pigment with a Qy band of longer wavelength at 734 nm (P734) in 80% acetone. The formation of P734 was dependent on ATP and dithionite. High performance liquid chromatography and mass spectroscopic analysis indicated that P734 is 3-vinyl bacteriochlorophyllide a, which is formed by the B-ring reduction of chlorophyllide a. These results demonstrate that the B-ring of chlorin is reduced by a second nitrogenase-like enzyme and that the sequential actions of two nitrogenase-like enzymes, DPOR and COR, convert porphyrin to bacteriochlorin. The evolutionary implications of nitrogenaselike enzymes to determine the ring structure of (bacterio)chlorophyll pigments are discussed.
Bacteriochlorophyll a (BChl a)2 is an important pigment for anoxygenic photosynthesis in photosynthetic bacteria. BChl a functions not only as light-harvesting pigments in antenna complexes, it also acts as "special pair" to drive the photosynthetic electron transfer in the reaction center (1). Biosynthesis of BChl a is a complex multienzymatic process involving at least 15 steps from 5-aminolevulinic acid (2, 3). The first half of the biosynthetic pathway from 5-aminolevulinic acid to protoporphyrin IX is shared with heme biosynthesis, and the latter half, the so-called Mg-branch that starts with the insertion of a magnesium ion into the porphyrin ring, is specific to BChl a. In the purple nonsulfur bacteria, Rhodobacter capsulatus and Rhodobacter sphaeroides, all genes of the Mg-branch (bch genes) are clustered in several tightly linked operons called a photosynthesis gene cluster that spans a region (46 and 41 kb in length, respectively) of the chromosome together with other genes involved in photosynthesis (puc, puf, and crt genes; Refs. 2 and 4). Most of the steps in the Mg-branch of BChl a biosynthesis are similar if not identical to those of chlorophyll a (Chl a) biosynthesis in oxygenic photosynthetic organisms. These steps include: (i) the insertion of Mg 2ϩ...