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J Biol Chem, Vol. 274, Issue 47, 33594-33600, November 19, 1999

Magnesium Insertion by Magnesium Chelatase in the Biosynthesis of Zinc Bacteriochlorophyll a in an Aerobic Acidophilic Bacterium Acidiphilium rubrum^*

Tatsuru Masuda, Kazuhito Inoue^§, Munehisa Masuda, Miho Nagayama, Atsuko Tamaki^§, Hiroyuki Ohta, Hiroshi Shimada, and Ken-ichiro Takamiya

From the Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan and the ^§ Department of Biological Sciences, Faculty of Science, Kanagawa University, Hiratsuka 259-1293, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To elucidate the mechanism for formation of zinc-containing bacteriochlorophyll a in the photosynthetic bacterium Acidiphilium rubrum, we isolated homologs of magnesium chelatase subunits (bchI, -D, and -H). A. rubrum bchI and -H were encoded by single genes located on the clusters bchP-orf168-bchI-bchD-orf320-crtI and bchF-N-B-H-L as in Rhodobacter capsulatus, respectively. The deduced sequences of A. rubrum bchI, -D, and -H had overall identities of 59.8, 40.5, and 50.7% to those from Rba. capsulatus, respectively. When these genes were introduced into bchI, bchD, and bchH mutants of Rba. capsulatus for functional complementation, all mutants were complemented with concomitant synthesis of bacteriochlorophyll a. Analyses of bacteriochlorophyll intermediates showed that A. rubrum cells accumulate magnesium protoporphyrin IX monomethyl ester without detectable accumulation of zinc protoporphyrin IX or its monomethyl ester. These results indicate that a single set of magnesium chelatase homologs in A. rubrum catalyzes the insertion of only Mg²⁺ into protoporphyrin IX to yield magnesium protoporphyrin IX monomethyl ester. Consequently, it is most likely that zinc-containing bacteriochlorophyll a is formed by a substitution of Zn²⁺ for Mg²⁺ at a step in the bacteriochlorophyll biosynthesis after formation of magnesium protoporphyrin IX monomethyl ester.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Naturally occurring cyclic tetrapyrroles such as chlorophylls and hemes usually contain a metal atom chelated to the nitrogen atoms at the center of the macrocyclic ring. Chlorophylls and bacteriochlorophylls (Bchls)¹ have a chelated magnesium atom and function in light harvesting and energy-generating charge separation. Very recently, however, an exception was reported that a purple bacterium, Acidiphilium rubrum, possesses zinc-containing Bchl a (zinc-Bchl a) as its major chlorophyll (1). This finding modifies our common understanding that naturally occurring chlorophylls and Bchls ubiquitously have a chelated magnesium atom at the center of macrocyclic ring.

In the biosynthetic pathway of chlorophylls and Bchls, magnesium chelation into protoporphyrin IX (Proto) is catalyzed by magnesium chelatase to successively form magnesium-Proto monomethyl ester, and the magnesium atom is retained in the subsequent synthesis of chlorophylls. Magnesium chelatase genes have been cloned from higher plants, cyanobacteria and photosynthetic bacteria, and they are composed of three cognate genes, chlI (bchI), chlD (bchD), and chlH (bchH) (2-5). Homologous or heterologous combination of these three cognate gene products overexpressed in Escherichia coli reconstituted the magnesium chelatase activity (3-5).

Considering many investigations on metal chelation of tetrapyrroles in vivo and in vitro, the following three possible mechanisms can be proposed for the formation of zinc-Bchl a in A. rubrum: 1) The zinc atom is directly inserted into Proto by the catalysis of a novel "zinc chelatase." 2) The zinc atom is directly inserted into Proto by a presently existing metal chelatase such as magnesium chelatase or ferrochelatase. 3) After magnesium is inserted into Proto by magnesium chelatase, a zinc atom is substituted for the magnesium atom enzymatically or non-enzymatically. In all cases, the zinc atom is thought to be retained throughout the rest of the biosynthesis, since it is known that enzymes involved in the steps after magnesium insertion in the chlorophyll biosynthetic pathway, such as NADPH-protochlorophyllide oxidoreductase (6) and chlorophyll synthetase (7), can use both magnesium and zinc chlorophyll derivatives as substrates. Besides these possibilities, formation of zinc-Proto has been reported in several biological materials. Ferrochelatase is known to catalyze in vitro chelation of divalent metals including zinc, and may form zinc-Proto in vivo in erythrocytes of patients affected with lead poisoning and iron deficiency anemia (8). In photosynthetic organisms, formation of zinc-Proto has been demonstrated with cell-free systems of such as Rhodobacter sphaeroides (9) and greening cucumber cotyledons (10). However, it may be formed nonenzymatically. Furthermore, it is well known that among chlorophyll derivatives containing metals other than magnesium, only zinc-containing chlorophylls have chemical features comparable to those of magnesium-chlorophylls (11), and have been widely used in studies of artificial photosynthesis by virtue of good stability of zinc-porphyrin derivatives (12). Thus, we cannot exclude the possibility that in A. rubrum zinc-Bchl is synthesized from such nonspecifically formed zinc-Proto, although no in vivo formation of zinc-complexed chlorophyll has been reported in these organisms,

All known species of the genus Acidiphilium are chemoheterotrophic aerobes and extremely acidophilic bacteria showing optimum pH for growth at around 3.0. They belong to the -subclass of Proteobacteria that includes many typical Bchl-utilizing photosynthetic bacteria (13). It is, therefore, likely that the Acidiphilium species utilize zinc-Bchl instead of acid-labile Bchl in order to adapt themselves to the acidic environment. In fact, our previous study (14) showed that the ratio of zinc-Bchl/Bchl in A. rubrum cells increases as the pH of the growth medium decreases to pH 3.5, suggesting that zinc-Bchl accumulation may be correlated with the acidic environment of A. rubrum.

In this study, we demonstrate that in A. rubrum magnesium is initially inserted into Proto by a magnesium chelatase homolog to form magnesium-Proto monomethyl ester and it is most likely that in a later step the magnesium atom is substituted by zinc atom.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Strains, Medium, and Growth Conditions-- A. rubrum (ATCC35905) was grown aerobically in darkness by shaking at 60 strokes per min at 30 °C in 5 × BYG medium (pH 3.5) containing mineral salts, 0.05% yeast extract, and 0.5% fructose in L-shaped tubes (1). The photosynthetic bacterium Rba. sphaeroides strain 2.4.1 was cultured photosynthetically as described previously (15). The insertional mutants of bchI (strain DB350), bchD (strain DB561), and bchH (strain ZY6) of Rhodobacter capsulatus (16, 17) were grown chemoheterotrophically or phototrophically in PYS or RCV 2/3 PCV medium as described (18).

Cloning of the A. rubrum bchI, -D, and -H Genes-- Two sets of degenerate primers were designed from conserved regions of the BchI/ChlI and the BchH/ChlH protein sequences. For bchI, an upstream primer (DG-UI) 5'-GG(C/G)GA(A/G)AAGGC(C/G)TTCGA(A/G)CC(C/G)GG(C/G)CT(C/G)CT(C/G)GC-3' and a downstream primer (DG-DI) 5'-TGCGG(C/G)CG(C/G)AG(A/G)TC(C/G)CC(C/T)TC(C/T)TC(C/G)GGGTT(C/G)CC-3' were made from the invariant amino acid motifs of GEKAFEPGLLA and GNPEEG(D/E)LRPQ, respectively. Likewise, for BchH, an upstream primer (DG-UH) 5'-GC(C/G)TTCGG(C/G)TA(C/T)GA(A/G)GG(C/G)GA(C/T)CC(C/G)ATGCG-3' and a downstream primer (DG-UH) 5'-GT(C/G)GC(C/T)TC(C/G)GA(C/G)GGGTTGTT(C/G)GC(C/G)GC(A/G)TA-3' were designed from the amino acid motifs (T/V) FGYEGDPMR and YAANNPSEAT, respectively. These primers were applied by PCR to amplify DNA from the A. rubrum genome. LA Taq polymerase (Takara Shuzo) was used according to the manufacturer's instruction. Temperature regime used was as follows: 95 °C for 1 min, followed by 30 cycles of 98 °C for 0.5 min and 68 °C for 2 min. Fragments of 221 and 257 base pairs were PCR amplified and cloned by standard procedures (19).

For isolation of the A. rubrum bchI gene, approximately 10 µg of A. rubrum genomic DNA was digested with BamHI and StuI, and separated by agarose gel electrophoresis. Since the radiolabeled bchI PCR product hybridized to 5 kilobases of BamHI- and 4 kilobases of StuI-digested genomic DNA, DNA fragments around these sizes were recovered from gels and cloned into pZErO-2.1 vector (Invitrogen). The two obtained mini-genomic plasmid libraries were screened with the bchI probe. For isolation of the A. rubrum bchH gene, a genomic clone of A. rubrum, designated pAr2, which functionally complemented bchN-disrupted mutant of Rba. capsulatus, appeared to contain A. rubrum bchH gene (see "Results"). The cosmid pAr2 was digested with BamHI and StuI, and the digested fragments were cloned into pZErO-2.1 vector. Obtained plasmid clones were screened with the radiolabeled bchH PCR fragment. Library screening, DNA manipulations, and hybridizations were performed according to standard procedures (19). Double-stranded plasmids containing cloned gene fragments were sequenced with LI-COR Model 4000L sequencer and the Thermo Sequenase cycle sequencing kit (Amersham Pharmacia Biotech). Results were analyzed by the GENETYX program (Software Development).

Genomic Southern Hybridization-- Genomic DNA was prepared from A. rubrum cells according to standard procedures (19). After digestion by restriction enzymes, 10 µg of each digested DNA was separated on a 0.7% agarose gel and then transferred to a nylon membrane. This blot was hybridized with the respective ³²P-labeled PCR product for 16 h at 65 °C. After hybridization, the blot was washed twice with 0.2 × SSC, 0.1% SDS for 15 min at 65 °C.

Overexpression of A. rubrum bchI, -D, and -H Genes in E. coli-- To construct pET24a (Novagen) derivatives containing the bchI, -D, and -H genes, specific oligonucleotides including the stop and start codons from the respective gene sequences were designed with preceding NdeI site containing a start codon of each gene. These oligonucleotides were used to amplify DNA by PCR and to introduce NdeI and relevant restriction sites for cloning the PCR fragments into pET24a vector. After sequence confirmation, these constructs were transformed into E. coli BL21(DE3) (Novogen) strain. Expression of recombinant proteins were induced by addition of isopropyl--D-thiogalactopyranoside to the cultures at a final concentration of 1 mM at 37 °C. After 3 h, the cells were harvested and disrupted by sonication. The disrupted cells were centrifuged at 15,000 × g for 15 min and the resultant supernatants were subjected for SDS-polyacrylamide gel electrophoresis (20) and used for magnesium chelatase activity assay as described by Kannangara et al. (21). For assay of zinc chelatase activity, zinc chloride was added to the reaction mixture instead of magnesium chloride. For in vitro transcription and translation of recombinant proteins, DNA templates of the pET24a derivatives were reacted with [³⁵S]methionine (ICN) and PROTEINscript kit (Ambion) according to the manufacturer's instruction.

Complementation of Rba. capsulatus Mutants-- Each gene fragment of the pET24a derivatives was introduced into NdeI and blunt ended PstI sites of pZJ102 vector (gifted from Z.-Y. Jiang and C. E. Bauer), yielding pZJ-bchI, pZJ-bchD, and pZJ-bchH. Plasmid pZJ102 is a derivative of pBBR1MCS5 (accession number U25061), which contains Rba. capsulatus puc promoter for high level of expression in Rba. capsulatus, and the ATG in NdeI site of this vector is positioned as the start codon of pucB gene. The plasmids of pET-bch constructs were also digested with XbaI and relevant restriction enzymes, and resulting fragments containing each gene with a ribosome-binding site originated from pET24a were introduced just downstream of bch genes of the relevant pZJ-bch plasmids. All 6 combinations of genes, which were doubly ligated at the same transcriptional direction under puc promoter of pZJ102, were constructed, and designated pZJ-bchI-D, -I-H, -D-I, -D-H, -H-I, and -H-D. Likewise, all 6 combinations of triple ligated pZJ-bch plasmids were constructed by preparing 6 combinations of the double ligated fragments containing a ribosome-binding site and each gene in pBluescript II SK-, followed by introduction of these cassettes into relevant pZJ-bch plasmids, yielding pZJ-bchI-D-H, -I-H-D, -D-I-H, -D-H-I, -H-I-D, and -H-D-I. The above 15 pZJ-bch constructs and pZJ102 vector were transformed into E. coli strain S17-1 (22), permitting conjugative transfer of each of the plasmids into the insertional mutants of Rba. capsulatus (16, 17). Complementation of mutants of Rba. capsulatus strains was performed by conjugation, followed by selection for photosynthetic growth in RCV medium.

Pigment Analysis and High Performance Liquid Chromatography-- A 4-day grown 50-ml culture of cells were pelleted by centrifugation at 10,000 × g for 10 min. Cell pellets were washed in 0.1 M Tris-HCl (pH 7.0), and pigments were extracted as described by Belanger and Rebeiz (23). For analysis of Bchls, the resulting hexane fractions which contain esterified Bchls and carotenoids were subjected for high-performance liquid chromatography (HPLC) analysis. Separation of various esterified forms of Bchls was achieved using the method of Shioi et al. (24). Sample was loaded onto a Asahipak ODP-50 column (250 × 4.6 mm inner diameter, Showa Denko, Tokyo) and eluted using a linear mobile-phase gradient from methanol, 1 M ammonium acetate (8:2, v/v) to acetonitrile/acetone (7:3, v/v) with a flow rate of 0.8 ml/min at 27 °C. The duration of the gradient was 28 min and the final composition was maintained isocratic for 10 min. The elution of pigments was monitored fluorimetrically using wavelength of excitation at 370 nm and emission at 780 nm.

For analysis of polar porphyrins, the resulting diethyl ether fraction, which contains pigments free of carotenoids and esterified Bchls, was subjected to spectral analysis and HPLC. Fluorescence spectra were obtained with an excitation wavelength at 415 nm with a fluorescence spectrophotometer (Hitachi Model 850). Porphyrins were separated by HPLC on a Zorbax ODS (Du Pont) column (250 × 4.6 mm inner diameter) at a flow rate of 0.8 ml/min. Pigments were eluted with linear gradient of solvent B (90% methanol, 0.1 M ammonium acetate) in solvent A (0.1 M ammonium acetate) as follows: 20-100% over 10 min and 100% solvent B for 20 min (25). The elution of pigments was monitored fluorimetrically using excitation wavelength at 418 nm and emission at 595 nm. For identification of magnesium-porphyrins, the extract from 2,2'-dipyridyl-treated cucumber cotyledons was used as an origin containing the standards of magnesium-Proto and its monomethyl ester (26). For identification of zinc-porphyrins, commercially available zinc-Proto (Sigma) and chemically zinc-substituted porphyrins from magnesium-porphyrins were used as standard of zinc-Proto and its monomethyl ester, respectively. Zinc substitution was carried out directly into porphyrins in the extract of Rba. sphaeroides by the metal acetate method (27).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning of bchI, -D, and -H from A. rubrum-- For PCR amplification, we used two sets of degenerate primer pairs, designed from conserved regions of the BchI/ChlI and the BchH/ChlH protein sequences and subsequently cloned two fragments of A. rubrum genomic DNA. Sequence analysis revealed that the two fragments were highly identical to the Rba. capsulatus bchI (90.5%) and bchH (87%) genes, respectively. These fragments were used to screen a genomic library of A. rubrum. For isolation of the bchI gene, we obtained two overlapping plasmid clones designated pIGBB4z and pIGSS3z. Restriction mapping and sequence analysis revealed that these two clones comprised 4,965 bp of the A. rubrum genome which includes the genes bchP-orf168-bchI-bchD-orf320-crtI, showing that genes for Bchl biosynthesis in A. rubrum were clustered like Rba. capsulatus (Fig. 1). For isolation of the bchH gene, by PCR amplification and Southern hybridization analyses, we found that a genomic clone of A. rubrum in cosmid pJRD215, designated pAr2, which functionally complemented bchN-disrupted mutant of Rba. capsulatus,² contained the A. rubrum bchH gene. From pAr2 cosmid DNA, we cloned two overlapping plasmid subclones, designated pHCEN9z and pHCSS7z. These two clones comprised 7,482 base pairs of the A. rubrum genome which includes the genes bchF-N-B-H-L, the gene organization of which was identical to that of Rba. capsulatus (Fig. 1). The involvement of the bchN gene in this region coincides with the fact that pAr2 complemented the bchN mutant of Rba. capsulatus.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Organization of the gene cluster for Bchl biosynthesis in the genome of A. rubrum. For sequence determination of the bchH and bchI-D gene clusters, two sets of overlapping plasmids designated pHCEN9z and pHCSS7z, and pIGBB4z and pIGSS3z, respectively, were isolated. Sequenced subclones are shown as solid lines. Putative genes and their transcriptional directions are indicated by arrows. The corresponding regions of Rba. capsulatus genes (16) are also depicted below.

Analysis of the Deduced Amino Acid Sequences of A. rubrum bchI, -D, and -H-- The A. rubrum bchI gene encoded a protein of 345 amino acids with a calculated mass of 37.4 kDa and 59.8 and 49.4% amino acid identity to BchI of Rba. capsulatus and ChlI of Synechocystis PCC6803, respectively. A phosphate-binding motif (GX₄GKS) which is commonly conserved in other BchI homologs was also conserved in the A. rubrum BchI sequence, located about 35 amino acids from the NH₂-terminal end of the protein. The A. rubrum bchD gene encoded a protein of 558 amino acids with a calculated mass of 57.9 kDa and 40.5 and 28.5% amino acid identity to BchD of Rba. capsulatus and Synechocystis PCC6803, respectively. The primary structure of A. rubrum BchD conserved some common features to those of other BchD homologs. The NH₂-terminal half (260 residues) of the A. rubrum BchD significantly resembled the entire BchI sequences (30.4 and 28.8% amino acid identity to BchI of A. rubrum and Rba. sphaeroides, respectively). Like Rba. capsulatus, the NH₂ terminus phosphate-binding motif conserved in BchI was lost in A. rubrum BchD. Immediately after the BchI homologous region, a proline-rich sequence at the center, followed by highly charged stretch of amino acids was observed in the A. rubrum BchD like other BchD homologs. The A. rubrum bchH gene encoded a protein of 1,200 amino acids with a calculated mass of 129.3 kDa and 50.7% amino acid identity to BchH of Rba. capsulatus. Three completely conserved His residues in other BchH homologs, which are considered to be associated with binding of substrates and catalysis of magnesium chelatase (28), were also conserved in the A. rubrum BchH (positions at 589, 593, and 734). Comparison of hydropathy profiles of A. rubrum BchI, -D, and -H with those of Rba. capsulatus showed no significant difference between the two species (data not shown). Genomic Southern hybridizations with the radiolabeled fragments described in the previous section showed a single band, indicating that each gene is encoded by a respective single gene in A. rubrum (Fig. 2).

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Genomic Southern hybridizations of the A. rubrum (A) bchI and (B) bchH genes. Genomic DNA from A. rubrum was cut with (1) EcoRI, (2) BamHI, and (3) SmaI, respectively. Each genomic Southern blot was hybridized with respective PCR product as a probe. Two bands in the lane 1 in B were caused by an internal EcoRI site of the bchH probe.

Overexpression and Reconstitution of the bchI, -D, and -H Gene Products-- DNA fragments containing the A. rubrum bchI, -D, and -H genes were separately cloned into pET24a, and the E. coli cultures containing these constructs were induced with isopropyl--D-thiogalactopyranoside. In vivo expression in E. coli induced recombinant BchI and BchH proteins in the predicted molecular weight sizes on SDS-PAGE gel, whereas the BchD protein was not detected as in the case of Rba. sphaeroides (4), probably because of very low abundance (Fig. 3A). However, an in vitro transcription and translation experiment showed that all three proteins were expressed at the predicted molecular weight sizes (Fig. 3B). We tried to reconstitute metal chelatase activity by combination of all these in vivo and in vitro expressed proteins. However, unlike the case of Rba. sphaeroides (4), magnesium chelatase activity was hardly detected in various buffer systems and at the pH value tested (data not shown). In addition, when zinc was added to the reaction mixture instead of magnesium, a substantial quantity of zinc-Proto was always formed even without recombinant protein(s), indicating that zinc-Proto can be easily formed non-enzymatically in vitro. Therefore, we concluded that in the in vitro reconstitution experiment it was difficult to distinguish whether the A. rubrum chelatase catalyzes only the insertion of magnesium or both magnesium and zinc.

View larger version (51K): [in this window] [in a new window]   Fig. 3.   In vivo and in vitro expression of the A. rubrum bchI, -D, and -H genes. A, Coomassie Brilliant Blue-stained SDS-polyacrylamide gels of the cell-free extracts prepared from E. coli strains containing the bchI, -D, and -H genes. Each gel shows the proteins produced by the E. coli strains before () and after (+) the addition of isopropyl--D-thiogalactopyranoside. Molecular weight markers are indicated on the left (Mr × 103). Arrowheads indicate the expressed proteins. B, autoradiography of 35S-labeled proteins produced by in vitro transcription and translation. Arrowheads indicate the expressed proteins.

Complementation Analysis of bchI, -D, and -H Mutants of Rba. capsulatus-- In addition to the reconstitution experiments, we examined the metal chelating activity of the A. rubrum bchI, -D, and -H gene products by complementation of Rba. capsulatus bchI, -D, and -H mutants (16, 17). The A. rubrum bchI, -D, and -H genes were inserted under puc promoter of pZJ102, and introduced into the Rba. capsulatus mutants by conjugation. To examine the interaction among A. rubrum and Rba. capsulatus subunits in the complementation, we also introduced double and triple ligated A. rubrum bch genes into Rba. capsulatus mutant strains. In this case, we constructed all possible combinations of genes, in case the order of genes may affect the expression or the complex formation of the enzyme (Table I). Initially, we evaluated the ability to complement by appearance of colonies during 7 days of photosynthetic growth. In this test, pZJ-bchI-D, pZJ-bchI-H-D, pZJ-bchH-I-D, and pZJ-bchH-D-I were effective for complementation of bchI and -D mutants of Rba. capsulatus (evaluated as ++ in Table I), and no complementation of bchH mutant was observed. However, when the incubation period was prolonged, tiny colonies frequently or always appeared from all bchI, -D, and -H mutants strains. Although the frequency and the time required for colony formation varied, the Rba. capsulatus bchI mutant was essentially complemented by pZJ derivatives that contained the A. rubrum bchI gene in constructs (evaluated + or ± in Table I). With the bchD mutant, occasional or frequent complementaion by pZJ-bchD-I and pZJ-bchI-D-H was observed. The complementation of the bchH mutant was observed in conjugants with pZJ-bchH, pZJ-bchH-D, pZJ-bchI-H-D, pZJ-bchH-I-D, and pZJ-bchH-D-I. We should note that even in low frequency and weak complementations such as observed with pZJ-bchI-H for the bchI mutant, and with pZJ-bchH and pZJ-bchH-D-I for the bchH mutant, such complementations were clearly distinguishable from reversion of the mutants, which recovered the ability of photosynthetic growth, because complemented colonies were formed from most of the conjugation spots and these were able to grow in the presence of selection antibiotics for pZJ102, while the revertants occasionally formed a few colonies from conjugation spots, but these were sensitive to antibiotic.

Among 15 pZJ derivatives, pZJ-bchI-H-D was most effective for complementation of all bchI, -D, and -H mutants. Fig. 4 shows HPLC analysis of Bchls accumulated in the Rba. capsulatus bchI, -D, and -H mutants complemented by pZJ-bchI-H-D. Each complemented mutant accumulated only Bchl a (Fig. 4, traces 2, 3, and 4), and when compared with pigment extract from A. rubrum cells (trace 5), no accumulation of zinc-Bchl a was observed. HPLC analysis of other complemented mutants also showed accumulation of only Bchl a in the cells (data not shown), indicating that the A. rubrum BchI, -D, and -H proteins form an active magnesium chelatase complex in the Rba. capsulatus.

View larger version (10K): [in this window] [in a new window]   Fig. 4.   HPLC analysis of Bchls in mutants of Rba. capsulatus complemented by pZJ-bchI-H-D. Trace 1, elution profile of pigment extract from wild type Rba. capsulatus cells. Traces 2-4, elution profiles of extracts from complemented bchI, -D, and -H mutants by pZJ-bchI-H-D, respectively. Trace 5, elution profile of pigment extract from A. rubrum cells. Peaks are as follows; M, Bchl a; Z, zinc-Bchl a; P, bacteriopheophytin a. Assignment of other peaks is described by Masuda et al. (14).

Pigment Analysis of A. rubrum Cells-- To confirm whether a set of the three subunits of A. rubrum catalyzes insertion of only magnesium into Proto, we examined the formation of porphyrin intermediates in Bchl biosynthesis in A. rubrum cells. It is known that a small quantity of magnesium-Proto monomethyl ester accumulates in wild type cells of Rba. sphaeroides (29), and magnesium-Proto and its monomethyl ester accumulate abnormally in dark-grown etiolated cucumber cotyledons treated with 2,2'-dipyridyl (26). By using these extracts and commercially available zinc-Proto as standards, we examined the fluorescent properties of the extract from A. rubrum. magnesium-Proto and its monomethyl ester in diethyl ether extracts of 2,2'-dipyridyl-treated cucumber and magnesium-Proto monomethyl ester in Rba. sphaeroides extract have characteristic fluorescent properties with excitation and emission maxima at 418 and 594 nm, respectively (Fig. 5, traces 1 and 3). The peaks of emission and excitation in the spectra of zinc-Proto standard were slightly blue-shifted (excitation and emission maxima at 417 and 589 nm, respectively) with respect to those of magnesium-Proto (Fig. 5, trace 2). The excitation and emission maxima of A. rubrum extract were 417 and 593-594 nm, respectively (Fig. 5, trace 4), which are essentially same as those of magnesium-Proto and its monomethyl ester.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Fluorescence emission spectra of: 1) ethereal extract from 2,2'-dipyridyl-treated cucumber cotyledons; 2) ether solution of authentic zinc-Proto; 3) Rba. sphaeroides ethereal extract; and 4) A. rubrum ethereal extract. Excitation wavelength, 418 nm.

To confirm the identification of magnesium-Proto and its monomethyl ester in A. rubrum extract, we compared retention times of these pigments with HPLC analysis. Retention times of magnesium-Proto and its monomethyl ester in the extracts of 2,2'-dipyridyl-treated cucumber and Rba. sphaeroides were 19.57 and 21.98 min, respectively (Fig. 6, traces 1 and 3). In contrast, authentic zinc-Proto (trace 2) was eluted between magnesium-Proto and its monomethyl ester at 20.85 min. To estimate the retention time of zinc-Proto monomethyl ester, magnesium-Proto monomethyl ester in the extract of Rba. sphaeroides was chemically replaced with zinc (27) and the resulting zinc-Proto monomethyl ester was eluted at 24.63 min (trace 4). The elution profile of tetrapyrroles extracted from A. rubrum was almost identical to that of Rba. sphaeroides, indicating that A. rubrum mainly accumulated the porphyrin intermediate magnesium-Proto monomethyl ester. Neither zinc-Proto nor zinc-Proto monomethyl ester was detected. It is, therefore, concluded that magnesium is inserted into Proto at the metal chelation step in zinc-Bchl biosynthetic pathway of A. rubrum.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   HPLC elution profiles of magnesium- and zinc-Proto and their monomethyl esters and ethereal extract from A. rubrum. Trace 1, ethereal extract from 2,2'-dipyridyl-treated cucumber cotyledons (magnesium-Proto and its monomethyl ester). Trace 2, authentic zinc-Proto. Trace 3, ethereal extract from Rba. sphaeroides (magnesium-Proto monomethyl ester). Trace 4, chemically zinc-replaced magnesium-Proto monomethyl ester in extract from Rba. sphaeroides (zinc-Proto monomethyl ester). Trace 5, ethereal extract from A. rubrum. Column eluent was monitored by fluorescence (excitation, 418 nm; emission, 595 nm).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The complementation experiment and pigment analysis described in this study demonstrated that A. rubrum synthesizes a set of cognate subunits of magnesium chelatase that catalyze the insertion of magnesium but not of zinc into Proto. Furthermore, since no zinc-Proto nor its monomethyl ester was detected in A. rubrum cells, the possibilities of nonspecific ferrochelatase-mediated or non-enzymatic zinc chelation into Proto are unlikely. It is, therefore, most likely that zinc-Bchl a is formed in A. rubrum by substitution of zinc for magnesium after the biosynthesis of magnesium-Proto monomethyl ester, supporting the third possibility described in the Introduction.

Genes encoding magnesium chelatase in A. rubrum are similar to those of the phylogenetically distant bacteria Rba. sphaeroides and Rba. capsulatus with respect to the number and size, as well as the organization of the gene cluster. In Rba. capsulatus and Rba. sphaeroides, bchI and bchD reside on the same transcriptional unit (4). Although the location and the transcriptional direction of other flanking genes differ from those of Rba. capsulatus, the A. rubrum bchI and bchD genes are likely to be co-transcribed as in Rhodobacter, since the stop codon of the A. rubrum bchI gene overlaps the putative start codon of the bchD gene. In A. rubrum, the bchH gene resides in a separate transcriptional unit from other bch genes, the organization of which is identical to that of Rba. capsulatus.

It has been reported that the reaction of magnesium chelatase is a two-step (activation and chelation) reaction; activation is achieved by the association of the BchI and -D subunits with ATP, while BchH, a carrier of Proto, is required for the chelation step with the ternary complex of BchI, -D, and ATP (28). It has been shown that the I and D subunits derived from one bacterial species are reconstitutable for magnesium chelatase activity in vitro with the H subunit from another species, probably because of the necessity of formation of the active ternary BchI, -D, and ATP preactivation complex (5). The strong physical interaction of the I and D subunits was also demonstrated in tobacco by analysis with the yeast two-hybrid system (2). Our complementation results also showed interesting tendencies concerning the formation of an active heterologous magnesium chelatase complex. Since all of the constructs containing A. rubrum bchI gene were able to rescue the bchI mutant, the A. rubrum BchI can form an active complex with the Rba. capsulatus BchD subunit. On the other hand, to complement the bchD mutant, the concomitant existence of A. rubrum bchI and -D genes in the constructs was always necessary except for pZJ-bchD-I-H and pZJ-bchD-H-I, suggesting that A. rubrum BchD can only associate with A. rubrum BchI and not with Rba. capsulatus BchI. It is interesting to note that the transcriptional order of the bchD gene in double- or triple-ligated constructs seems to be critical for formation of the active complex. Except for low frequency complementation by pZJ-bchD-I, some bch gene(s) preceded the bchD in all successful cases, indicating that such precedence may be crucial for expression of the bchD gene. It is likely that such heterologous or homologous pre-activated BchI-BchD complexes can form an active magnesium-chelatase complex with Rba. capsulatus BchH. Since the bchH mutant was rescued by pZJ-bchH and pZJ-bchH-D with relatively low frequency, we presume that A. rubrum BchH can form an active but weak complex with Rba. capsulatus BchI and -D. We do not know why the transcriptional order of the bchH gene severely affected the degree of complementation. In any case, it was clearly demonstrated by HPLC analysis of Bchls that the A. rubrum bchI, -D, and -H gene products catalyze insertion of only magnesium into Proto in Rba. capsulatus in vivo.

Interesting questions are where and how the zinc substitution takes place during the biosynthesis of zinc-Bchl a in A. rubrum, and whether a biological catalyst is involved in this reaction. The chemistry of metal-porphyrins will provide insight into these questions. Metalation of porphyrins with divalent metal ions depends on deprotonation of the pyrrole nitrogen atoms, together with the removal of metal-coordinated ligands such as water molecules (30). Zinc is the second easiest metal after copper for insertion into porphyrins (31), and it is easily introduced into porphyrin without any catalyst (9, 10). The difficulty of zinc insertion is increased as the macrocyclic ring is reduced from chlorin to bacteriochlorin (32). Although in an acidic environment zinc can be introduced by direct metal exchange into magnesium complexes of porphyrins, chlorins, and bacteriochlorins, these reactions require substantial heat energy for completion. Consequently, if zinc substitution takes place non-enzymatically, one of the most probable ways is spontaneous zinc insertion into hypothetical pheophytinized intermediates after magnesium-Proto monomethyl ester formation. Concerning the possibility of the involvement of biological catalyst, there is nothing known about the presence of distinct zinc chelatase or "metal-exchange enzyme" so far. The substrate specificity of ferrochelatase seems to be limited to porphyrin derivatives, and ferrochelatase has no metal exchange activity with magnesium-porphyrins; rather than it is significantly inhibited by them (33). However, if we assume that zinc insertion takes place non-enzymatically, there should be zinc incorporation and concentration mechanism in A. rubrum cells, because there is only a trace amount of zinc as a component of the yeast extract (0.5 g/liter) in the growth medium of A. rubrum. It is estimated that zinc in the medium is quantitatively incorporated in zinc-Bchl a of A. rubrum,³ indicating that A. rubrum may vigorously incorporate zinc into the cells for synthesis of zinc-Bchl a, and without this mechanism, a reaction of non-enzymatic zinc insertion into Proto may not proceed in the medium containing such a low concentration of zinc.

From our previous results, it seems likely that the acidic environment is correlated with an increased ratio of zinc-Bchl/Bchl in A. rubrum (14). Since it is known that zinc is more soluble at acidic pH than neutral, and the magnesium-porphyrins are easily pheophytinized by acid, these circumstances may be correlated with the increased ratio of zinc-Bchl/Bchl in the cell. However, the intracellular pH of acidophilic bacteria was indirectly estimated to be neutral by the optimum pH of a cytoplasmic enzyme and by the distribution of a radioactive 5,5'-dimethyl-2,4-oxazolidinedione across the cell membrane (34-36). The relationship between the acidic environment and zinc replacement after magnesium-Proto monomethyl ester remains to be elucidated.

	ACKNOWLEDGEMENTS

We thank Prof. C. E. Bauer for critically reading of the manuscript. We are grateful to Prof. C. E. Bauer and Dr. Z.-Y. Jiang for the generous gifts of magnesium chelatase mutants of Rba. capsulatus and complementation vector pZJ102. We thank Prof. H. Tamiaki for valuable discussions. and Prof. Y. Shioi for technical advice of HPLC analysis.

	FOOTNOTES

^* This study was supported by Ministry of Education, Science and Culture of Japan Grants 09309008, 10740367, and 11640657.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AB017350 and AB017351.

To whom correspondence should be addressed: Dept. of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. Tel.: 81-45-924-5737; Fax: 81-45-924-5805; E-mail: tmasuda@bio.titech. ac.jp.

² K. Inoue, unpublished result.

³ A. Hiraishi and S. Takaichi, personal communication.

	ABBREVIATIONS

The abbreviations used are: Bchl, bacteriochlorophyll; zinc-Bchl, zinc-containing bacteriochlorophyll; Proto, protoporphyrin IX; HPLC, high-performance liquid chromatography; PCR, polymerase chain reaction.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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