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Originally published In Press as doi:10.1074/jbc.M601750200 on March 29, 2006

J. Biol. Chem., Vol. 281, Issue 21, 15021-15028, May 26, 2006
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A Second Nitrogenase-like Enzyme for Bacteriochlorophyll Biosynthesis

RECONSTITUTION OF CHLOROPHYLLIDE a REDUCTASE WITH PURIFIED X-PROTEIN (BchX) AND YZ-PROTEIN (BchY-BchZ) FROM RHODOBACTER CAPSULATUS*{diamondsuit}

Jiro Nomata{ddagger}, Tadashi Mizoguchi§, Hitoshi Tamiaki§, and Yuichi Fujita{ddagger}1

From the {ddagger}Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan and the §Department of Bioscience and Biotechnology, Ritsumeikan University, Kusatsu 525-8577, Japan

Received for publication, February 23, 2006 , and in revised form, March 28, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 nitrogenase-like enzymes to determine the ring structure of (bacterio)chlorophyll pigments are discussed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 Mg2+ into protoporphyrin IX (bchI, bchD, bchH), (ii) the methylation of propinonate at the C-13 position (bchM), (iii) the formation of an isopentanone fifth ring (bchE), (iv) the reduction of a vinyl group at the C-8 position (bchJ), and (v) the formation of a chlorin ring by the D-ring reduction of protochlorophyllide (Pchlide; bchL, bchN, bchB). Most genes responsible for these reactions are conserved in cyanobacteria and plants (5, 6), with some exceptions, such as the isopentanone ring formation (chl27 (7) and xantha-I (8)), the C-8 vinyl reduction (dvr (9) and pcb2 (10)), and the alternative (light-dependent) Pchlide reductase (LPOR; Refs. 11 and 12). Chlorophyllide a (Chlide a), the final product of these five reactions, has spectral properties identical to Chl a; that is, it absorbs red light with enough energy to perform oxygenic photosynthesis. These enzymes involved in BChl a biosynthesis, which are common to plants, have been biochemically studied as model enzymes for Chl a biosynthesis (13-16), as has LPOR, which is a key enzyme in the light-dependent greening of angiosperms (11, 12).

Compared with the enzymes common to Chl a biosynthesis, enzymes specific for BChls, such as BChls a, b, c, d, and e, are poorly understood, except for C-20 methyl transferase BchU, which operates in BChl c biosynthesis (17, 18). In BChl a biosynthesis, two additional biosynthetic steps, the conversion of a chlorin ring to a bacteriochlorin ring by the B-ring reduction and modification of the C-3-vinyl group to an acetyl group, transform Chlide a to bacteriochlorophyllide a. These structural changes exert effects on the spectral properties of these compounds enabling them to absorb infrared light to perform anoxygenic photosynthesis. Directed mutagenesis and phenotypic analysis of the resultant mutants suggest that three genes, bchX, bchY, and bchZ, are involved in the B-ring reduction (19, 20) and that two genes, bchF and bchC, are required for the C-3 group modification (2, 21).

A similarity search of amino acid sequences of bch gene products has suggested that two nitrogenase-like enzymes are involved in BChl a biosynthesis (22). The first nitrogenase-like enzyme is the dark-operative (light-independent) Pchlide reductase (DPOR) that catalyzes the stereo-specific reduction of the D-ring of Pchlide (Fig. 1; Ref. 23). The three subunits of DPOR, BchL, BchN, and BchB, show significant similarities to the three nitrogenase subunits NifH, NifD, and NifK, respectively. The nitrogenase-like features of DPOR have recently been exemplified by the reconstitution of the DPOR reaction by purified proteins and have confirmed that DPOR has properties common to nitrogenase (24, 25). DPOR consists of two separable components, an L-protein (BchL dimmer) and an NB-protein (BchN-BchB heterotetramer), which is similar to nitrogenase (Fe-protein and MoFe-protein), and DPOR catalysis depends on ATP and dithionite or reduced ferredoxin.


Figure 1
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  		FIGURE 1.
The two sequential reactions, from Pchlide to 3VBChlide, in BChl a biosynthesis. The porphyrin cyclic tetrapyrrole structure of Pchlide ({lambda}max of Qy band: 625 nm in ether) is converted to chlorin (Chlide a) by D-ring reduction catalyzed by the nitrogenase-like enzyme DPOR. Chlide a has spectral properties ({lambda}max of Qy band: 661 nm in ether) identical to Chl a and is then converted to Chl a by the attachment of a phytol tail in oxygenic photosynthetic organisms. A second nitrogenase-like enzyme, COR, converts the chlorin ring structure to bacteriochlorin by B-ring reduction, forming 3VBChlide ({lambda}max of Qy band: 745 nm in ether). The vinyl group at the C-3 position of 3VBChlide is then converted to an acetyl group followed by the attachment of a phytol tail to produce BChl a.

 
The other nitrogenase-like enzyme involved in BChl a biosynthesis is Chlide a reductase (COR), which catalyzes the stereo-specific reduction of the B-ring of Chlide a (Fig. 1). The deduced amino acid sequences of BchX, BchY, and BchZ also show significant similarities to the amino acid sequences of the nitrogenase subunits NifH, NifD, and NifK, respectively. BchX from R. capsulatus is a protein of 333 amino acid residues and shows 31.5% identity to that of the NifH protein from Azotobacter vinelandii. The Fe-protein, a homodimer of NifH, functions as an ATP-dependent electron donor for the MoFe-protein of nitrogenase. To accomplish its functions, the Fe-protein carries a [4Fe-4S] cluster, bridged by two Cys residues from each NifH protomer, and the Walker motif A for ATP-binding, which is conserved in the amino-terminal region (26). BchX has two completely conserved Cys residues (Cys130 and Cys165) for the Fe-S cluster chelation and the Walker motif A(Gly42-Ser49; Ref. 23). This sequence conservation suggests that BchX functions as an ATP-dependent reductase that is specific for the other catalytic component as well as the Fe-protein of nitrogenase. BchY and BchZ from R. capsulatus are proteins of 497 amino acid residues and 490 amino acid residues, respectively, and show lower yet apparent similarity to NifD and NifK of nitrogenase, respectively. The MoFe-protein contains novel metalloclusters, FeMo-cofactor (FeMo-co), and P-cluster (a [8Fe-7S] cluster) that occur as pairs. FeMo-co is a [1Mo-7Fe-9S-X-homocitrate] cluster held by His and Cys residues in NifD. Six Cys residues (three from NifD and three from NifK) are involved in the chelating of P-cluster (26). His and Cys residues for FeMo-co chelation are not conserved in BchY. Instead, three Cys in BchY, and one Cys in BchZ, of the six Cys residues in NifD and NifK are conserved (23). These features of sequence conservation suggest that BchY and BchZ form a catalytic component as well as MoFe-protein with slightly different metallocluster(s). However, to our best knowledge, there is no biochemical evidence for the nitrogenase-like features of COR.

Here, we first report the reconstitution of the COR reaction by the purified proteins, BchX and BchY-BchZ, from R. capsulatus, and the identification of the COR product as 3-vinyl bacteriochlorophyllide a (3VBChlide). These results demonstrate that the sequential actions of two nitrogenase-like enzymes, DPOR and COR, convert the cyclic tetrapyrrole ring structure from porphyrin (Pchlide) to bacteriochlorin in the BChl a biosynthetic pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Culture Conditions—The photosynthetically competent R. capsulatus mutant DB176 (ORF176::KmR; 25) was used as the host strain to overexpress Strep-tagged bchX and bchY genes. The transconjugants were grown heterotrophically in PY medium (27) overnight with vigorous shaking at 200 rpm in the dark at 34 °C. To prepare crude extract for protein purification, the transconjugants were grown photosynthetically in RCV medium (28) illuminated with incandescent lamps (90 watts; National, Osaka, Japan) for 4 days at 34 °C. PY and RCV media contained 5 µg ml-1 kanamycin and 10 µg ml-1 spectinomycin.

Construction of Overexpression Plasmids—Two plasmids, pJN4X and pJN4YZ, were constructed to overexpress BchX and BchY as fusion proteins with an affinity tag (Strep-tagII) under the control of the puc promoter in R. capsulatus (Fig. 2). The Strep-tagII is a short peptide consisting of eight amino acid residues (WSHPQFEK) that binds specifically to an engineered streptavidin, Strep-Tactin. Two primers, bchXf2 (5'-AGTTCGAGAAGTCGGGGGTGACTGACGCACCCAACCTGAA-3') and bchXr1 (5'-CAGGTACCTCAGACATCGTCGTAGATCAC-3'), were used to amplify the entire coding region for bchX by PCR (the sequences that anneal to the bchX coding region are underlined; Fig. 2, PCR1-1). Another two primers, bchYf2 (5'-AGTTCGAGAAGTCGGGGGTGACCGATCTTCCGCAAGCCGA-3') and bchZr1 (5'-CAGGTACCTCAGTTCCCCCCCTTCCGATC-3'), were used to amplify the chromosomal region covering the entire coding regions of bchY and bchZ, which are located contiguously on the chromosome, by PCR (the sequences that anneals to the bchY and bchZ coding regions are underlined; Fig. 2, PCR1-2). PCR was performed with KOD polymerase (KOD-plus; Toyobo, Osaka, Japan) using genomic DNA of R. capsulatus as the template. A 2-kb fragment consisting of the spectinomycin omega cartridge and the puc promoter was amplified with two primers, pBBRT7f1 (5'-TCGGTACCGTAATACGACTCACTATAGGGC-3') and pPucStrepr2 (5'-CACCCCCGACTTCTCGAACTGCGGATGCGACCACGAGGCCATTGTCCCGAATCCTCCAA-3'; the sequence encoding the Strep-tag is double-underlined), using pJN3 as the template (Fig. 2, PCR1). pJN3 is a pBBR1MCS2 derivative containing the spectinomycin omega cartridge and the puc promoter. The second PCR reactions were followed by amplification with the primer pairs "pBBRT7f1" and "bchXr1" (Fig. 2, PCR2-1) or "pBBRT7f1" and "bchZr1" (Fig. 2, PCR2-2). The final amplified fragments consisting of the spectinomycin resistance gene, the puc promoter, the Strep-tag, and the entire coding regions for bchX or bchY-bchZ were digested with KpnI (indicated by italics in the sequences) and ligated into the KpnI site of pBBR1MCS2 (29), yielding the final overexpression plasmids, pJN4X and pJN4YZ. The amino-terminal sequence of Strep-tagged BchX and BchY is commonly MASWSHPQFEKSGV followed by the second amino acid residues of their native sequences. The nucleotide sequence encoding the Strep-tag (5'-ATGGCCTCGTGGTCGCATCCGCAGTTCGAGAAGTCGGGGGTG-3') was designed to match the codon usage of R. capsulatus.

Isolation of R. capsulatus Strains JNX and JNYZ—The plasmids pJN4X and pJN4YZ were transferred into R. capsulatus DB176 cells by triparental mating with Escherichia coli strain JM105 containing the relevant plasmids (30). Transconjugants were selected on PY plates containing rifampicin (100 µg ml-1), kanamycin (5 µg ml-1), and spectinomycin (10 µgml-1). The resulting transconjugants expressing Strep-tagged BchX and BchY proteins under the control of the puc promoter were designated JNX and JNYZ, respectively.

Purification of BchX and BchY—Crude extracts for protein purification were prepared as described previously (25). Cultures at the optical density (OD660) of about 1.5 were collected in a 1-liter bottle and placed in an anaerobic chamber (model A; Coy Laboratory Products, Grass Lake, MI). Approximately 100 mg of sodium dithionite (Sigma) was added to the culture, and the cells were harvested by centrifugation at 10,000 x g for 5 min at 4 °C (TA24BH rotor; Tomy, Tokyo, Japan). All subsequent procedures were carried out in an anaerobic chamber using solutions that had been degassed and stored in the chamber. Sodium dithionite (final concentration of 1.7 mM) was added just before use to remove residual oxygen. The collected cells were suspended in lysis buffer (24) and disrupted by sonication. The sonicate was then transferred to 30PC tubes (Hitachi, Tokyo, Japan) and centrifuged at 37,000 x g for 30 min (RP50-2; Hitachi) at 4 °C. Approximately 15 ml of the resulting supernatant was then loaded onto a Strep-Tactin-Sepharose column (2 ml of Strep-Tactin-Sepharose in a 1.5 x 1.2-cm column; IBA, Göttingen, Germany) that was equilibrated with wash buffer containing 100 mM HEPES-KOH (pH 8.0), 150 mM NaCl, 0.1% Triton X-100, and 12 µM sodium dithionite. The column was washed in 12 ml of wash buffer and Strep-tagged proteins were then eluted using wash buffer containing 2.5 mM desthiobiotin. Protein concentrations were determined using the BCA method (Protein Assay; Bio-Rad), with bovine serum albumin as the standard.

SDS-PAGE and Amino-terminal Sequence Analysis—Purified proteins were electrophoresed on a 12% acrylamide gel that was stained with CBB ("CBB Stain One"; Nakarai, Osaka, Japan). For the aminoterminal sequence analysis, 300 ng of purified YZ-protein was loaded onto a 10% acrylamide gel of 1.5-mm thickness. After electrophoresis, the proteins were electrically transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Billerica, MA) using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) according to the instructional manual. Each blot of the BchY and BchZ proteins was excised and washed in distilled water, then 50% methanol, 0.1% trifluoroacetic acid, methanol, and anhydrous acetonitrile, before being dried, and loaded onto protein sequencers PPSQ-23A (Shimadzu, Kyoto, Japan) and Procise cLC 494cLC (Applied Biosystems, Foster City, CA), respectively, to determine the amino-terminal sequences (Toray Research Center, Kamakura, Japan).

Preparation of Chlide a—The R. capsulatus mutant CB1200 ({Delta}bchF, {Delta}bchZ; Ref. 2) was grown in RCV-2/3PY medium (80 ml in a 200-ml flask) containing 10 µg ml-1 spectinomycin at 34 °C in the dark with slow shaking at 130 rpm. The culture medium was collected by centrifugation. The Chlide a in the culture medium was then extracted in one-third volume of diethyl ether. The ether was then evaporated to dryness by a stream of nitrogen. The dried Chlide a was dissolved in Me2SO to a final concentration of 150 µM. The Chlide a concentration was determined in 80% acetone using the extinction coefficient at 663 nm (77.1 mM-1 cm-1) of Chl a (31).

Chlide a Reductase Assay—COR assays were carried out in a 250-µl volume containing 100 mM HEPES-KOH (pH 8.0), 5 mM MgCl2, 5 mM dithiothreitol, 2 mM ATP, 20 mM creatine phosphate, 21 units µl-1 creatine phosphokinase, 0.7 mM sodium dithionite, 1-2 µM Chlide a, and 12.5 µl of purified protein(s). The assay mixtures were incubated in anaerobic conditions in the dark for 50 min at 34 °C. To stop the reactions, an aliquot (200 µl) of the assay mixture was mixed with acetone (final concentration 80%). After phase partitioning with 700 µl of hexane, absorption spectra of the lower acetone phase were recorded on a Jasco V550 spectrophotometer (Jasco, Hachioji, Japan). To monitor the change in absorption spectra of the COR reaction mixture, the assay mixture was prepared in a cuvette with an air-tight screw cap in the anaerobic chamber and the cuvette was set in the Jasco V550 spectrophotometer with a temperature control module (model ETC-477, set at 34 °C; Jasco). The absorption spectra were periodically recorded. The extinction coefficient of 3VBChlide, the COR reaction product, at 734 nm in 80% acetone (hexane-extracted) was experimentally determined to be 44.7 mM-1 cm-1 based on the extinction coefficient (82.5 mM-1 cm-1) of 3-vinyl bacteriochlorophyll a (3VBChl) in ether (32).

LC/MS Analysis of COR Product—Extraction of the COR products from the above assay mixtures after 0- and 50-min incubations was performed as follows. Each aliquot of the assay mixtures was mixed with acetone. After washing with hexane, the lower acetone-phase was extracted with a mixture of petroleum ether, diethyl ether, and ethyl acetate (1:1:2 (v/v/v)). The upper phase containing the COR products was evaporated to dryness by a stream of nitrogen. The extract thus obtained was dissolved in methanol for LC/MS analysis. LC/MS was performed using a Shimadzu LCMS-2010EV system (Shimadzu) comprising a liquid chromatograph (SCL-10Avp system controller, LC-10ADvp pump, and SPD-M10Avp photodiode-array detector) and a quadrupole mass spectrometer equipped with an electrospray ionization probe. HPLC was performed using reverse-phase chromatography under the following conditions: column, Inertsil ODS-EP (3.0 x 150 mm; GL Sciences, Tokyo, Japan); eluent, methanol:75 mM ammonium acetate (pH 5.25), 75:25 (v/v); flow rate, 0.35 ml min-1; and detection wavelengths, 725 and 415 nm. MS conditions were as follows: capillary temperature, 250 °C; electrospray ionization voltage, 4.5 kV; sheath gas flow, 1.5 liters min-1; and drying gas pressure, 0.16 megapascals. The absorption spectrum of 3VBChlide was also recorded using a photodiode-array detector.


Figure 2
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  		FIGURE 2.
Construction of plasmids for the overexpression of BchX and BchY as Strep-tag fusion proteins. A DNA fragment, consisting of the spectinomycin resistance cartridge (SpR), the puc promoter, and the Strep-tagII (shaded box), was amplified by PCR using the recombinant plasmid pJN3 as a template (PCR1). The coding regions of bchX and bchY-bchZ were amplified by PCR from the genomic DNA of R. capsulatus (PCR1-1 and PCR1-2, respectively). Two chimeric DNA fragments were produced by a second cycle of PCR with the two DNA fragments (PCR2-1 and PCR2-2). Then, two plasmids, pJN4X and pJN4YZ, were constructed by ligating these amplified fragments into the KpnI site of the broad host-range vector, pBBR1MCS2. These plasmids were introduced into DB176 cells by triparental mating. KmR, kanamycin resistance marker.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Purification of BchX and BchY-BchZ—To facilitate purification of proteins, we set up a system to overexpress affinity tagged (Strep-tagII) BchX and BchY in R. capsulatus DB176 (Fig. 2, 25). Because of the structural similarity to nitrogenase, we carefully performed all procedures involving the characterization of COR, such as harvesting of cells, cell lysis, protein purification, and the activity assay under anaerobic conditions using an anaerobic chamber (24, 25). The Strep-tagged BchX protein (Strep-BchX) was purified from crude JNX extract by Strep-Tactin-Sepharose. Fig. 3 shows the CBB-stained protein profiles of crude JNX extract and purified Strep-BchX protein on SDS-PAGE (lanes 1 and 2). Purified Strep-BchX protein showed an apparent molecular mass of 39 kDa, which is in good agreement with the calculated molecular mass of 37,015 Da.

Unlike Strep-BchX, purified Strep-tagged BchY (Strep-BchY) showed a doublet with apparent molecular masses of 54 and 55 kDa, which were at almost equimolar amounts as judged by CBB staining (Fig. 3, lane 4). The amino-terminal sequence of the upper band was ASWS, which matched the Strep-tag sequence without the amino-terminal Met, indicating that the upper band was Strep-BchY. The amino-terminal sequence of the lower band was MFLLD, which is identical to that of BchZ as deduced from the nucleotide sequence (Z11165 [GenBank] ), indicating that BchZ was co-purified with Strep-BchY. The mobility of the two proteins on SDS-PAGE was consistent with the calculated molecular mass of Strep-BchY (54,060 Da) and BchZ (53,217 Da). Similar yields of amino-terminal residues (3.3 pmol of Ala for Strep-BchY and 3.5 pmol of Met for BchZ) indicated that the BchY-BchZ complex forms in equimolar ratios of BchY and BchZ. This result provides the first experimental evidence that the BchY and BchZ proteins form a complex.


Figure 3
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  		FIGURE 3.
SDS-PAGE of purified X-protein and YZ-protein. Crude extracts of JNX (16 µg, lane 1) and JNYZ (23 µg, lane 3) cells grown photosynthetically were prepared as described under "Experimental Procedures." Purified Strep-tagged BchX (0.84 µg) and BchY (0.87 µg) proteins were loaded in lanes 2 and 4, respectively. A protein size marker with the indicated molecular mass is shown in lane M.

 


Figure 4
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  		FIGURE 4.
Reconstitution of Chlide a reduction with purified X-protein and YZ-protein. Absorption spectra of the lower acetone phase after phase-partitioning with hexane from COR assays containing purified Strep-BchX (trace a), only purified Strep-BchY-BchZ (trace b), or both Strep-BchX and Strep-BchY-BchZ (trace c), are shown. The assay mixtures were incubated for 50 min at 34 °C in the dark under anaerobic conditions.

 
Reconstitution of COR Activity in Purified Proteins—The purified proteins were examined for COR activity (Fig. 4). We devised an in vitro assay system, which is similar to the assay for DPOR activity (24, 25). Purified BchX (0.95 µg) and purified BchY-BchZ (1.0 µg) were added individually or together to assay mixtures, which contained ATP, an ATP-regeneration system, sodium dithionite, and Chlide a. The reactions were incubated under anaerobic conditions. As shown in Fig. 4, the absorption spectrum of the substrate, Chlide a, was unaltered in the assay mixtures, which contained only one of the purified proteins, BchX or BchY-BchZ (Fig. 4, traces a and b). However, when both purified proteins were added to the assay mixture, a new pigment with a peak at 734 nm appeared, concomitant with a decrease in the Chlide a peak at 665 nm (Fig. 4, trace c). This result suggested that Chlide a was converted to a new pigment (P734) with a Qy peak of longer wavelength that was dependent on both protein components. Assuming that P734 is the expected product of the B-ring-reduced Chlide a, 3VBChlide, the amount of P734 that was formed and the amount of Chlide a that was used during the incubation period (50 min) were estimated to be 264 pmol and 243 pmol, respectively, supporting the equimolar conversion of Chlide a to P734.

We also assessed whether ATP, as well as a reductant (sodium dithionite), were required for P734 formation. As shown in Fig. 5, P734 was not formed when ATP was omitted from the reaction (traces b and d). However, P734 was produced in the reaction without an ATP-regeneration system in amounts similar to the complete reaction system (trace c), and dithionite was necessary for the reaction (traces e and f). These results indicate that the formation of P734 is dependent on ATP and dithionite; however, it is not dependent on the ATP-regeneration system. These results are similar to those for DPOR, except that DPOR is dependent on an ATP-regeneration system (24).

The formation of P734 was monitored without acetone extraction by recording the absorption spectra of the reaction mixture (Fig. 6A). During the assay, the peak for P734 constantly increased, whereas the Chlide a peak at 670 nm decreased. Isosbestic points were obvious at 690 and 591 nm, suggesting that Chlide a was converted to P734 in equimolar amounts. The time course of P734 formation as 3VBChlide in this assay is shown in Fig. 6B. 3VBChlide was formed in a linear manner within the first 6 min followed by a slower increase until 50 min. The initial velocity of 3VBChlide formation was about 32.5 nmol3VBChlide min-1 mg(X + YZ)-1.


Figure 5
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  		FIGURE 5.
ATP and dithionite requirements of the COR reaction. Traces a and e represent complete reactions containing 1 mM ATP, the ATP regeneration system, and 0.7 mM sodium dithionite; trace b represents a reaction without ATP; trace c represents a reaction without the ATP regeneration system; trace d represents a reaction without ATP and the ATP regeneration system; and trace f represents a reaction without sodium dithionite. Assay mixtures contained the same amount of BchX and BchY-BchZ proteins, as in Fig. 4.

 
Identification of the COR Product—The pigment P734 in the COR assay was analyzed by LC/MS (Figs. 7 and 8). The HPLC profile of the 0-min assay indicated that the assay mixture contained two major pigments with absorptions at 415 nm. These two pigments were Chlide a, which eluted at 9.9 min, and pheophorbide a (Pheobide), which eluted at 44 min (Fig. 7, trace a). No pigments with absorptions at 725 nm were detected (trace b). After the reaction, the Chlide a peak (9.9 min) decreased, and three new peaks (14.5, 17.6, and 61 min) appeared in the profile at 415 nm (trace c). Because these pigments did not have any significant absorption at 725 nm, they did not correspond to P734 and appeared to be by-products. A new peak eluted at 10.6 min, which was detected as a single peak in the profile at 725 nm and appeared to correspond to P734 (trace d). The absorption spectrum of the pigment that eluted at 10.6 min is shown in Fig. 8A. This pigment in the elution solvent showed Soret, Qx, and Qy peaks at 350, 583, and 723 nm, respectively. The property of the absorption spectrum of the pigment that eluted at 10.6-min suggested it has a bacteriochlorin ring structure, and we concluded that this pigment corresponds to P734 in the COR assay. Mass spectrometry was carried out on the pigment that eluted at 10.6 min (Fig. 8B). The main molecular ion peak was observed at an m/z value of 616.1 in the region of m/z values from 500 to 700. The m/z value matched the calculated molecular mass of the B-ring reduced Chlide a, 3VBChlide (calculated for 616.25). An adduct with methanol originated from the eluent was also detected as a minor peak (648.1). These results indicated that P734 is 3VBChlide in the COR assay. Thus, we concluded that the B-ring of Chlide a is reduced by another nitrogenase-like enzyme consisting of the two components, BchX and BchY-BchZ, which are dependent on ATP and dithionite.


Figure 6
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  		FIGURE 6.
Absorption spectral change of the COR reaction mixture. A COR assay was performed in a cuvette (200 µl) with an air-tight screw cap set in a temperature-controlled unit of the spectrophotometer at 34 °C, and the absorption spectra were recorded periodically (A). Traces a, b, c, d, e, f, and g are absorption spectra at 0, 4, 8, 12, 25, 40, and 50 min, respectively. B, time course of 3VBChlide formation in the assay. The amount of 3VBChlide at each time point was calculated in a proportional manner from the final 3VBChlide amount (50 min, 1.32 µM), which was determined by the absorption spectrum of the acetone extract of the reaction.

 


Figure 7
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  		FIGURE 7.
HPLC profiles of COR reaction products. Aliquots of the assay mixtures at 0 min (traces a and b) and 50 min (traces c and d) were loaded onto an ODS column (Intersil ODS-EP), and the elution profile was monitored by absorption at 415 nm (traces a and c) and 725 nm (traces b and d).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we demonstrated that the B-ring reduction to form a bacteriochlorin ring from Chlide a is catalyzed by a second nitrogenase-like enzyme, COR.


Figure 8
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  		FIGURE 8.
Absorption (A) and mass spectra (B) obtained on-line during LC/MS analysis of COR products after 50-min incubation. The molecular ion peaks of 3VBChlide corresponding to M+· and [M + CH3OH]+· were observed at m/z 616.1 and 648.1, respectively.

 
X-protein (BchX)—The reconstitution system of COR indicated that ATP is required for the reaction, suggesting that the X-protein functions as an ATP-dependent reductase for the YZ-protein, as expected from the sequence similarity. The Fe-protein-like protein, BchL, in DPOR shows almost the same degree of similarity to the NifH protein (29.0% identity to A. vinelandii NifH) as BchX and has been demonstrated to function as a homodimer transferring electrons to the other catalytic component in an ATP-dependent manner (24, 25). Thus, we have confirmed that the X-protein is a new member of the ATP-dependent reductase component of the nitrogenase-like enzyme family. BchX also shows 29.8% identity to BchL. About 30% sequence identity may be the lowest limit required to maintain the structure and function of the ATP-dependent reductase component of these enzymes.

YZ-protein (BchY-BchZ)—Co-purification of BchZ with Strep-BchY indicates that BchY and BchZ form an equimolar complex, BchY-BchZ, namely the YZ-protein. The MoFe-protein and the NB-protein of DPOR are heterotetramers of NifD and NifK, and BchN and BchB, respectively. The equimolar complex of the YZ-protein suggested that the YZ-protein has a heterotetrameric structure as well. The YZ-protein probably serves as the catalytic component, by which the double bond of the Chlide a B-ring is stereospecifically reduced, whereas the MoFe-protein and NB-protein are sites for the reduction of the substrates, dinitrogen and Pchlide, respectively. The heterotetrameric structure could be a common structural feature of the catalytic components (MoFe-protein, NB-protein, and YZ-protein) of the nitrogenase-like enzyme family.

The His and Cys residues that are involved in the chelation of FeMo-co in NifD are not conserved in BchY. Instead, three Cys residues in BchY (Cys50, Cys70, and Cys133), and one Cys residue in BchZ (Cys35), of the six Cys residues that are involved in the P-cluster chelation in the MoFe-protein are conserved. This arrangement of Cys conservation appears in the NifE-NifN complex, which is another MoFe-protein-related complex involved in the assembly of FeMo-co as a scaffold. The NifE-NifN complex contains two [4Fe-4S] clusters, instead of FeMo-co and P-cluster (33). Thus, it is suggested that YZ-protein also carries two [4Fe-4S] clusters.

The COR Reconstitution System—The COR reconstitution system with purified components also indicated the requirement of both an electron donor (dithionite) and ATP (Fig. 5), which are common to nitrogenase and DPOR. The physiological source of electrons for COR has yet to be identified. R. capsulatus has six different ferredoxins, of which ferredoxin I functions as a specific electron donor to nitrogenase (34). A reduced ferredoxin (from maize) supports the Chlide a formation in the DPOR reaction (25). Thus, ferredoxin is the most probable candidate for being the electron donor in the COR reaction. A common ferredoxin could donate electrons to COR and DPOR, to convert Pchlide to 3VBChlide.

We have demonstrated that the COR reaction can be spectrophotometrically monitored in a continuous manner (Fig. 6). The course of the reaction of LPOR has been traced by changes in absorption and fluorescence emission spectra (12, 35), and some intermediates of the reaction have been identified leading to the hypothesized reaction schemes (35, 36). To investigate the reaction mechanism of nitrogenase, a continuous and spectrophotometric assay overcomes the limitation of fixed time point assays (37). The continuous assay could be applicable for DPOR and provides a promising system for further detailed kinetic analysis to understand the molecular mechanisms of nitrogenase-like enzymes, including DPOR.

COR Product, 3VBChlide—The COR product, P734, has been identified as 3VBChlide. The spectral properties of 3VBChl have been reported previously (32). Because the attachment of a phytol chain does not cause any effect on the spectral properties of the phytol-free tetrapyrrole pigments, 3VBChlide has identical spectral properties to 3VBChl. The pigment that eluted at 10.6 min in HPLC showed peaks at 350, 583, and 723 nm in the aqueous solvent (Fig. 8A), which appears quite different from the reported peaks of 3VBChl at 351, 560, and 745 nm (in ether). The P734 pigment extracted from the COR reaction mixture with ether showed Qx and Qy peaks at 563 and 744 nm, respectively, which are in good agreement with that reported for 3VBChl (data not shown). This suggests that the polarity of the solvent strongly affects the Qy and Qx peaks of 3VBChlide.

Substrate Specificity of COR—The Chlide a sample used in this study was prepared from the culture medium of CB1200 ({Delta}bchZ-{Delta}bchF mutant). Absorption spectra of the preparation and the HPLC profile of the 0-min sample indicated that the preparation contained a significant amount of Pheobide. After the COR reaction, the peak for Pheobide was still detected in almost the same amount as that detected before the reaction (Fig. 7), suggesting that COR did not reduce the B-ring of Pheobide and that the presence of a central metal is required for the reaction. The requirement of the central metal is a common features among many enzymes involved in (B)Chl biosynthesis (12, 18). When Pchlide was added to the COR reaction instead of Chlide a, no spectral change was detected after the reaction (data not shown). This result indicated that the reduced D-ring is recognized by COR and that Pchlide does not serve as the substrate for COR. This substrate specificity for the ring structure determines the order of the D- and B-ring reduction reactions; the first to occur is the D-ring reduction by DPOR, and the second is the B-ring reduction by COR in the BChl biosynthetic pathway. Another issue is whether COR discriminates between the vinyl group and the hydroxyethyl group at the C3 position. The two reactions, B-ring reduction by COR and the conversion of the vinyl group to the hydroxyethyl group at the C3 position by BchF, appear to occur concurrently in BChl a biosynthesis (2, 38). In accordance with this model, COR should catalyze both Chlide a and 3-hydroxyethyl Chlide a, which could be clarified by the COR reconstitution system.

Evolutionary Implications—The genes for COR and DPOR share ancestral genes with those of nitrogenase. An evolutionary scenario for (B)Chl biosynthesis in photosynthetic organisms could be depicted as follows (23). In the early evolution of photosynthesis, a primitive and undifferentiated type of DPOR/COR diverged from the common ancestral enzyme to catalyze the reduction of both D- and B-rings of Pchlide, forming 3VBChlide, in BChl biosynthesis. Then, another gene duplication event appears to have generated DPOR and COR with substrate specificity for the Pchlide D-ring and Chlide a B-ring, respectively, and the sequential actions of these two enzymes converted porphyrin to bacteriochlorin, as seen in contemporary photosynthetic bacteria. In a lineage leading to cyanobacteria, the loss of COR resulted in a shortcut of the BChl biosynthetic pathway to Chlide a, the direct precursor of Chl a, to give rise to the Chl a biosynthetic pathway. This change in the biosynthetic pathway may have led to the change in the photosynthetic pigment from BChl a to Chl a, which has provided the molecular basis for the evolution of oxygenic photosynthesis.

In addition to COR and DPOR, some other nitrogenase-like genes have been found in the genomes of methanogens and some nitrogen-fixing bacteria, including the purple nonsulfur bacteria (e.g. Methanosarcina acetivorans, Methanosarcina mazei, Desulfibacterium hafnience, R. capsulatus, Rhodopseudomonas palustris, Group IV; Ref. 39). The presence of nitrogenase-like genes in a small subset of prokaryotes implies that nitrogenase-like genes were a significant source of gene recruitment, from which existing enzymes evolved into new enzymes during the early evolution of photosynthesis and nitrogen fixation.

The reconstitution of COR with purified X-protein and YZ-protein components should allow detailed analysis for molecular mechanisms to recognize ring structures of porphyrin and chlorin, which are key in the evolution of photosynthesis. These future studies could provide new clues to tracing the evolutionary path of photosynthesis from the aspect of pigment biosynthesis.


   FOOTNOTES
 
* This work was supported by Grants-in-aid for Scientific Research Nos. 15570033, 14390051, and 13CE2005 and the 21st Century COE Program (to Y. F.); Nos. 17029065 and 15350107 (to H.T.); and No. 17750167 (to T. M.) and by an "Academic Frontier" Project for Private Universities (to H. T.), a matching fund subsidy, from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{diamondsuit} This article was selected as a Paper of the Week. Back

1 To whom correspondence should be addressed. Tel.: 81-52-789-4105; Fax: 81-52-789-4107; E-mail: fujita{at}agr.nagoya-u.ac.jp.

2 The abbreviations used are: BChl a, bacteriochlorophyll a; Chl a, chlorophyll a; Pchlide, protochlorophyllide; Chlide a, chlorophyllide a; DPOR, dark-operative protochlorophyllide reductase; LPOR, light-dependent protochlorophyllide reductase; COR, chlorophyllide a reductase; 3VBChl, 3-vinyl bacteriochlorophyll a; 3VBChlide, 3-vinyl bacteriochlorophyllide a; LC, liquid chromatography; MS, mass spectrometry; HPLC, high performance liquid chromatography; CBB, Coomassie Brilliant Blue; Pheobide, pheophorbide a. Back


   ACKNOWLEDGMENTS
 
We thank Carl E. Bauer for kindly donating the broad host-range vector pBBR1MSC2, the mutant CB1200, and the triparental mating system for R. capsulatus. We thank Yuka Moriwaki (Toray Research Center, Kamakura, Japan) for determining the amino-terminal sequences of BchY and BchZ proteins. We thank Tatsuo Omata, Hirozo Oh-oka, and Kazuki Terauchi for valuable discussions.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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