Competitive Inhibitions of the Chlorophyll Synthase of Synechocystis sp. Strain PCC 6803 by Bacteriochlorophyllide a and the Bacteriochlorophyll Synthase of Rhodobacter sphaeroides by Chlorophyllide a

Bacterial strains and growth conditions. Synechocystis sp. PCC 6803 was cultured at 30°C in 300-ml Erlenmeyer flasks containing 50 ml of a BG11 medium (4) supplemented with 10 mM d-glucose, which was agitated on a rotary shaker at 100 rpm under 40 microeinsteins m⁻² s⁻¹ white light. Rhodobacter sphaeroides 2.4.1 was grown at 28°C in Sistrom's succinate-based minimal (Sis) medium as described previously (31). Escherichia coli was grown at 37°C in Luria-Bertani (LB) medium. Kanamycin (Km), tetracycline (Tc), streptomycin (Sm), and spectinomycin (Sp) for R. sphaeroides and E. coli cultures were added as indicated previously (14).

Addition of Bchlide a and Chlide a to the bacterial cultures.The concentrations of Bchlide a and Chlide a in acetone were determined as described previously (22, 32; also see below). The pigments were dried and dissolved in dimethyl sulfoxide (DMSO). Aliquots of stock solutions (10 mM) were taken out and diluted in up to 250 μl of DMSO prior to their addition to the culture (50 ml) of Synechocystis sp. PCC 6803. Accordingly, all the cultures were given the same volume (250 μl) of DMSO in which Bchlide a with or without Chlide a was dissolved. The final concentration of pigments varied from 0 to 50 μM. The growth of Synechocystis sp. PCC 6803 is not affected by 0.5% DMSO.

Likewise, Bchlide a and Chlide a were dissolved in ethanol, and aliquots of stock solutions (14 mM) were taken out and diluted in up to 500 μl of ethanol prior to their addition to the culture (50 ml) of R. sphaeroides. All the cultures were given the same volume (500 μl) of ethanol in which Bchlide a with or without Chlide a was dissolved. The final concentration of pigments varied from 0 to 70 μM. The growth of R. sphaeroides is not affected by 1% ethanol.

Growth measurement by total protein determination.The growth of bacterial cells which had been treated with light-absorbing pigments (Chlide a, Bchlide a, or both) was measured by total protein determination. Cell aliquots (1 ml) were harvested, and protein synthesis was arrested by chloramphenicol (50 μg/ml). Cells were centrifuged and washed three times with 1 ml of phosphate-buffered saline (PBS; pH 7.4); vigorous vortexing for 5 min followed each wash to facilitate pigment diffusion out of the cells. Cells were broken by sonication and kept frozen at −70°C. Cells were further subjected to three freeze-thaw cycles for complete lysis. Total proteins were determined by the modified Lowry method, as described previously (19). Bovine serum albumin (BSA) was used as a standard. Total proteins have been shown as a suitable parameter to express cyanobacterial biomass (11). The relative levels of total protein of R. sphaeroides were compared only with cells grown under the same growth conditions and were found to be in direct proportion to numbers of CFU/ml culture.

Construction of mutants. (i) Construction of a bchFNB-bchZ mutant.A 523-bp DNA fragment upstream from the 24th residue of BchF was PCR amplified with the forward primer (5′-G T C GA C GAA GTG AGA GAC GG-3′, where the SalI site is in bold and the mutated sequence is underlined, unless noted otherwise) and reverse primer (5′-GCG CGT GAT ATC GCG TCG-3′, where the EcoRV site is in bold) and cloned into T-vector. Likewise, a 401-bp DNA fragment downstream from the 482nd residue of BchB was amplified with the forward primer (5′-GA T ATC GTC TGG CTG ACG-3′, where the EcoRV site is in bold) and reverse primer (5′-GT C GA C ATG CTC CTC GAT G-3′, where the SalI site is in bold) and cloned into T-vector. The inserted DNA fragments were isolated after digestion of the recombinant plasmids with EcoRV and SalI. The SalI-EcoRV upstream DNA and EcoRV-SalI downstream DNA were cloned into the SalI site of suicide plasmid pLO1 (Km^r) (18) to yield pLO-ΔbchFNB, in which a 3,213-bp DNA between the 24th residue of BchF and the 482nd residue of BchB was deleted. The plasmid was mobilized into wild-type R. sphaeroides to obtain the bchFNB mutant. The single-crossover exconjugant (Km^r) was isolated and subjected to segregation for double crossover (bchFNB mutant, Km^s) on Sis medium containing sucrose (15%). In order to disrupt bchZ, the recombinant suicide plasmid pSUPZ100 (16), in which the BamHI site of bchZ was interrupted with a 2.2-kb Ω (Km^r) DNA, was mobilized into the bchFNB mutant to obtain the bchFNB-bchZ mutant (Km^r and Tc^s) on Sis medium containing Km.

(ii) Construction of the bchG mutant.A 544-bp XhoI-XhoI DNA fragment, which corresponds to the region between the 19th and 200th residues of BchG, was excised from a 1.9-kb EcoRI-NsiI DNA and replaced by a 2.2-kb Ω (Sm^r/Sp^r) DNA. The resulting recombinant DNA was cloned into the EcoRI-PstI sites of pSUP202 (30) to generate pSUPBG. It was mobilized into R. sphaeroides to obtain the bchG mutant (Sm^r/Sp^r and Tc^s) on Sis medium containing Sm/Sp.

The chromosomal structures of the resulting mutants were examined by Southern hybridization analysis (26).

Purification and determination of Bchl a.Bchl a was purified from R. sphaeroides as described previously (8, 12). The level of Bchl a, which had been collected in n-hexane, was calculated with an extinction coefficient of 83.9 mM⁻¹ cm⁻¹ at 771 nm (32).

Purification of Chlide a and Bchlide a.Chlide a was purified from a culture supernatant of the bchZ-bchF mutant (16), while Bchlide a was purified in a similar way with the bchG mutant of R. sphaeroides. The mutants were cultured in Sis medium supplemented with Tween 80 (0.03%) under dark anaerobic conditions (with dimethyl sulfoxide [DMSO]) until a turbidity of 100 Klett units (KU) was reached. Chlide a and Bchlide a were extracted from the culture supernatant with diethyl ether and dried as described previously (22). They were dissolved in aqueous acetone, and their levels were determined with an extinction coefficient of 77.1 mM⁻¹ cm⁻¹ for Chlide a (22) and a coefficient of 42.1 mM⁻¹ cm⁻¹ for Bchlide a (32).

Alternatively, the C₂₀ moieties of Chl a and Bchl a were removed by chlorophyllase to yield Chlide a and Bchlide a, respectively. Chl a was purchased from Sigma-Aldrich, and Bchl a was purified from R. sphaeroides as described above. Chlorophyllase was purified from leaves of Ailanthus altissima as described previously (21). Chlide a and Bchlide a were separated from the reaction mixture as previously described (23) and dissolved in aqueous acetone to determine their levels with the extinction coefficients mentioned above.

PPP synthesis.PPP was synthesized after the pyrophosphorylation of phytol (Sigma-Aldrich) with di-(triethylammonium) phosphate (9), as described previously (13).

Construction of ChlG and BchG expression plasmids. (i) ChlG expression plasmid.A 975-bp DNA fragment encompassing the chlG gene of Synechocystis sp. PCC 6803 was PCR amplified with forward primer Fc1 (5′-CTG CAG AGT CTC GGT TAG-3′) and reverse primer Rc1 (5′-CCA AGC CCG GTC TAC TC-3′), and the PCR product was cloned into T-vector to yield pTchlG. Another primer set of Fc2 and T3 was used to amplify chlG from pTchlG; the forward primer Fc2 (5′-CAA CCT TTG G AT C C T CTA TGT C-3′), in which the BamHI site is in bold and the mutated sequence is underlined) contains the BamHI site, which was mutated from the immediate upstream DNA of the initiation codon of ChlG, whereas the reverse primer T3 (5′-ATT AAC CCT CAC TAA AGG GA-3′) corresponds to the immediate downstream DNA of the multiple cloning sites in T-vector. The PCR fragment was digested with BamHI and EcoRI (at multiple cloning sites) and cloned into the BamHI-EcoRI sites of pRSET-A (Invitrogen) to yield pRchG.

(ii) BchG expression plasmid.A 1,058-bp DNA fragment containing the bchG gene of R. sphaeroides was PCR amplified with forward primer Fb1 (5′-CGC TGA TGT GCA ATC TG-3′) and reverse primer Rb1 (5′-GTC AGC ACC ACC ATG C-3′); the PCR product was then cloned into T-vector to yield pTbchG. Another primer set of Fb2 and T7 was used to amplify bchG from pTbchG; the forward primer Fb2 (5′-GCT TAG G G G A TC CAC ATG-3′), in which the BamHI site is in bold and the mutated sequence is underlined) contains the BamHI site that was mutated from the immediate upstream DNA of the initiation codon of BchG, whereas the reverse primer T7 (5′-GTA ATA CGA CTC ACT ATA GGG C-3′) corresponds to the immediate downstream DNA of the multiple cloning sites in T-vector. The PCR fragment was digested with BamHI and HindIII (at multiple cloning sites) and cloned into the BamHI-HindIII sites of pRSET-C (Invitrogen) to yield pRbchG.

Expression of ChlG and BchG.The E. coli BL21(DE3)/pLysS strain was transformed with the recombinant plasmids pRchG and pRbchG, and each strain was grown in a 1-liter flask containing 500 ml LB medium with vigorous shaking at 30°C. When the culture reached a density (A ₆₀₀) of 0.5, isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM. After 6 h, cells were harvested by centrifugation at 10,000 × g for 10 min at 4°C, followed by washing with 10 mM Tris-HCl (pH 8.0). Cells were kept frozen at −70°C until used.

Assays of chlorophyll synthase and bacteriochlorophyll synthase.Chlorophyll synthase and bacteriochlorophyll synthase reactions were performed as described previously (23), with minor modifications. Frozen cells (1 ml) were subjected to three freeze-thaw cycles and incubated with DNase (10 mg/ml in 5 mM MgCl₂) for 1 h at room temperature. Cell lysate (8 mg protein) as an enzyme source was mixed into 290 μl of reaction buffer (120 mM potassium acetate, 10 mM magnesium acetate, 50 mM HEPES-KOH, pH 7.6, 14 mM β-mercaptoethanol, 10% glycerol, and 0.5 mM ATP) containing GGPP (200 μM) or PPP at various concentrations. The reaction was initiated by the addition of 10 μl of Chlide a or Bchlide a at various concentrations. After incubation at 30°C, the reaction was stopped by the addition of 750 μl acetone. HCl (20 μl) was added for high-performance liquid chromatography (HPLC) analysis (23), and precipitates were removed by centrifugation. The relative levels of Chl a and Bchl a were determined by HPLC.

HPLC analysis.Reverse-phase HPLC analyses were performed on a CLASS-VP LC (Shimadzu, Japan) equipped with a Gemini C₁₈ column (Phenomenex, Torrance, CA; particle size, 5 μm; column length by diameter, 250 mm by 4.6 mm), a UV-visible-light source, and a fluorescence detector. The sample was eluted with acetone-water linear gradients for 30 min from acetone-water (70:30) to acetone only. The column was then flushed with 100% acetone for 10 min. The fluorescence detector was set at 405 nm for excitation and at 675 nm for emission. Chl a and Bchl a were used as standards.

Spectrophotometric assay.Cell-free lysates of R. sphaeroides grown under photoheterotrophic and dark anaerobic (DMSO) conditions were prepared as described previously (17). Absorption spectra of R. sphaeroides extracts were analyzed with a model UV 2550-PC spectrophotometer (Shimadzu, Japan). The same levels of protein were examined when the spectral profiles of cells grown under different conditions were compared. Protein was determined by a modified Lowry method with BSA as a standard.

The photoheterotrophic growth of Synechocystis sp. PCC 6803 is inhibited by exogenously added Bchlide a, but the growth inhibition is reversed by exogenously added Chlide a.Although chlorophyll synthase and bacteriochlorophyll synthase share significant similarity in their predicted amino acid sequences, both enzymes have very strict specificities for their Mg-tetrapyrrole substrates. Nonetheless, one enzyme reaction may be affected by the other enzyme's Mg-tetrapyrrole substrate, since their chemical structures are similar to each other (Fig. 1). It was examined whether the photoheterotrophic growth of Synechocystis sp. PCC 6803 is affected by exogenously added Bchlide a. Remarkably, the bacterial growth was hampered by Bchlide a in a dose-dependent way to reveal no growth at 50 μM (Fig. 2). Other intermediates, which are specific to the metabolic branch for Bchl a biosynthesis, were extracted from the dark anaerobic (DMSO) culture of the bchF, bchZ, and bchC mutants. They were added to the culture of Synechocystis sp. PCC 6803 but did not show any effects on bacterial growth (data not shown, but the same growth without the addition of Bchlide a is shown in Fig. 2). Thus, Bchlide a specifically inhibits the growth of Synechocystis sp. PCC 6803. Since the level of Bchlide a detected from Synechocystis sp. PCC 6803 varied in direct proportion to the concentration added exogenously (data not shown), it is thought to readily diffuse into cells.

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FIG. 2.

Effect of exogenously added Bchlide a on the growth of Synechocystis sp. PCC 6803. The bacterium was grown photoheterotrophically in glucose-containing liquid BG11 supplemented with various concentrations of Bchlide a (0 to 50 μM).

Bchlide a is regarded as a structural analog of Chlide a, so we examined whether the growth inhibition caused by Bchlide a was reversed by exogenously added Chlide a. Indeed, the Bchlide a-exerted inhibition was relieved by Chlide a, illustrating a near-complete reversion in the presence of equimolar Chlide a (Fig. 3). The results strongly suggest that the chlorophyll synthase of Synechocystis sp. PCC 6803 is a target of Bchlide a.

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FIG. 3.

Effect of exogenously added Chlide a on the Bchlide a-exerted growth inhibition of Synechocystis sp. PCC 6803. The bacterium was grown photoheterotrophically in glucose-containing liquid BG11 supplemented with Bchlide a (25 μM) with or without addition of Chlide a (0 to 25 μM).

Chlorophyll synthase of Synechocystis sp. PCC 6803, which catalyzes the esterification reaction by a ping-pong pathway, is competitively inhibited by Bchlide a.The chlG DNA coding for the chlorophyll synthase of Synechocystis sp. PCC 6803 was cloned, overexpressed in E. coli, and used as an enzyme source. Since the enzyme was examined in terms of whether its esterification is affected by the presence of Bchlide a, the reaction was performed in the presence of a sufficient level (200 μM) of geranylgeranyl pyrophosphate (GGPP). However, GGPP, once mixed with E. coli lysate, is readily metabolized to lower its effective concentration (27), so the velocity (V ₀) was measured during the initial 15 min, in which the reaction rate falls within a linear range (data not shown). The K_m value (0.1 mM) of chlorophyll synthase for Chlide a (Table 1) apparently increased in the presence of Bchlide a (140 μM) (Fig. 4). However, the inhibitory effect of Bchlide a on V _max was absent at the elevated level of Chlide a (Fig. 4A). The results are diagnostic of competitive inhibition with an inhibition constant (K _I) for Bchlide a binding of 0.30 mM (Table 1). Phytyl pyrophosphate (PPP), which is also a natural substrate for chlorophyll synthase, is not metabolized rapidly in E. coli lysate (27). Accordingly, PPP was employed as a substrate instead of GGPP. The same inhibition mode was observed (Fig. 4B), and the K _I (1.14 mM) for Bchlide a in the presence of PPP is higher than that (0.30 mM) observed with GGPP (Table 1). This suggests that Bchlide a binding to the enzyme is not favored when the phytyl moiety is present in the active site.

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FIG. 4.

Inhibition kinetics of chlorophyll synthase by Bchlide a. Lineweaver-Burk diagrams of Chlide a esterification by chlorophyll synthase in the absence or presence of Bchlide a (140 μM). GGPP (A) and PPP (B) were used as a source of C₂₀.

The K_m of chlorophyll synthase for Chlide a is 0.32 mM in the presence of PPP, which is larger than the 0.1 mM observed with GGPP (Table 1). The results suggest that Chlide a binding to the geranylgeranylated enzyme is preferred to its binding to the phytylated enzyme. Thus, GGPP is a preferred C₂₀ source for chlorophyll synthase to esterify Chlide a.

Whether chlorophyll synthase of Synechocystis sp. PCC 6803 follows a ping-pong reaction as observed with chlorophyll synthase from the oat plant Avena sativa (27) was investigated. The double-reciprocal (Lineweaver-Burk) plots of the chlorophyll synthase reaction were determined either with various concentrations of Chlide a and four fixed concentrations of PPP (Fig. 5A1) or with various concentrations of PPP and four fixed concentrations of Chlide a (Fig. 5A2). Both plots exhibited straight parallel lines, which are characteristic of a ping-pong pathway involving the formation of a covalently altered enzyme after reaction with the first substrate and its subsequent reaction with the second substrate, yielding the final product.

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FIG. 5.

Kinetics of bi-substrate reactions of chlorophyll synthase and bacteriochlorophyll synthase. Lineweaver-Burk diagrams of Chlide a esterification by chlorophyll synthase (A1 and A2) and Bchlide a esterification by bacteriochlorophyll synthase (B1 and B2). PPP was used as a source of C₂₀. The concentration of Chlide a varied in the presence of constant PPP (A1); this experiment was repeated for several concentrations of PPP (1 to 200 μM). On the other hand, the concentration of PPP was varied in the presence of constant Chlide a (A2); this experiment was repeated for several concentrations of Chlide a (5 to 300 μM). Likewise, the Bchlide a concentration varied in the presence of constant PPP (B1); this experiment was repeated for several concentrations of PPP (1 to 200 μM). On the other hand, the concentration of PPP varied in the presence of constant Bchlide a (B2); this experiment was repeated for several concentrations of Bchlide a (5 to 300 μM).

The rate of Chl a formation was examined during the first 5 min of the reaction with chlorophyll synthase that had been preincubated for 15 min with either PPP or Chlide a (Fig. 6). The reaction rate for the first 1 min with an enzyme preincubated with PPP was reproducibly higher than that observed with an enzyme preincubated with Chlide a (Fig. 6A). No differences in rates between the two were observed thereafter. Thus, enzyme phytylation is a rate-limiting step, as observed with chlorophyll synthase from A. sativa (27). The result also reflects that PPP may be the first substrate, since the accelerated rate would be expected only if the first reaction were rate limiting and completed during preincubation. Accordingly, PPP and possibly GGPP appear to be the first substrate in a double-displacement reaction.

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FIG. 6.

Initial reaction rates of chlorophyll synthase and bacteriochlorophyll synthase. (A) The rate of Chl a formation was measured with the chlorophyll synthase that had been preincubated for 15 min with either PPP or Chlide a. (B) Likewise, the rate of Bchl a formation was measured with the bacteriochlorophyll synthase that had been preincubated for 15 min with either PPP or Bchlide a.

Bacteriochlorophyll synthase of Rhodobacter sphaeroides, which also catalyzes the esterification reaction by the ping-pong pathway, is competitively inhibited by Chlide a.Since chlorophyll synthase is competitively inhibited by Bchlide a, we examined whether bacteriochlorophyll synthase is also affected by Chlide a. The bchG DNA coding for the bacteriochlorophyll synthase of R. sphaeroides was cloned, overexpressed in E. coli, and used as an enzyme source. As illustrated with chlorophyll synthase, the bacteriochlorophyll synthase reaction was examined in the presence of sufficient GGPP (200 μM). Surprisingly, the K_m (0.14 mM) of bacteriochlorophyll synthase for Bchlide a apparently increased in the presence of Chlide a (280 μM), but the inhibitory effect of Chlide a on V _max disappeared at the elevated concentration of Bchlide a (Fig. 7A). Thus, the bacteriochlorophyll synthase is indeed inhibited by Chlide a in a competitive mode, with a K _I of 0.54 mM (Table 1). The same inhibition mode was observed with PPP (Fig. 7B). The K _I (0.77 mM) for Chlide a in the presence of PPP is not much different from that (0.54 mM) observed with GGPP (Table 1), which suggests that Chlide a binding to bacteriochlorophyll synthase is not affected by the C₂₀ types at its active site.

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FIG. 7.

Inhibition kinetics of bacteriochlorophyll synthase by Chlide a. Lineweaver-Burk diagrams of Bchlide a esterification by bacteriochlorophyll synthase in the absence or presence of Chlide a (280 μM). GGPP (A) and PPP (B) were used as a source of C₂₀.

The K_m of bacteriochlorophyll synthase for Bchlide a is 0.26 mM in the presence of PPP, which is larger than the 0.14 mM observed with GGPP. The results suggest that Bchlide a binding to the geranylgeranylated enzyme is also preferred to its binding to the phytylated enzyme.

Whether the bacteriochlorophyll synthase of R. sphaeroides also follows a ping-pong mechanism was also investigated. PPP was employed as a C₂₀ source, and the Lineweaver-Burk plots of the bacteriochlorophyll synthase reaction were determined as described above for chlorophyll synthase. Both plots also exhibited straight parallel lines, indicative of a ping-pong pathway (Fig. 5B1 and B2). The preincubation of bacteriochlorophyll synthase with PPP for 15 min also resulted in a higher rate of Bchl a formation at the initial stage of reaction (Fig. 6B), suggestive of PPP (and possibly GGPP) as the first substrate in a double-displacement reaction.

The metabolic conversion of exogenously added Bchlide a into Bchl a by the R. sphaeroides bchFNB-bchZ mutant is inhibited by exogenously added Chlide a, but the metabolic inhibition is reversed by an increased level of exogenously added Bchlide a.Whether exogenously added Chlide a affects the bacteriochlorophyll synthase of R. sphaeroides to result in growth arrest was examined. Given the metabolic conversion of Chlide a into Bchlide a, it is not possible to examine the effect of exogenously added Chlide a on the photoheterotrophic growth of wild-type R. sphaeroides. Accordingly, the Chlide a-exerted effect was examined with its bchFNB-bchZ mutant, which neither synthesizes nor metabolizes Chlide a (Fig. 8A). The mutant is not able to grow photoheterotrophically due to the lack of Bchl a. In the presence of exogenously added Bchlide a (35 μM), however, the mutant exhibits photoheterotrophic growth up to approximately 40 μg of cell protein per ml culture, which is almost equivalent to a culture density (A ₆₀₀) of 1.0 (Fig. 8B). The photoheterotrophic growth of the bchFNB-bchZ mutant in the presence of Bchlide a (35 μM) was significantly inhibited by the addition of 70 μM Chlide a (Fig. 8B). However, the inhibition was nearly reversed by doubling the Bchlide a level to 70 μM (Fig. 8B), corroborating the in vitro results for the competitive inhibition of bacteriochlorophyll synthase by Chlide a.

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FIG. 8.

Effect of exogenously added Chlide a on the photoheterotrophic growth of the R. sphaeroides bchFNB-bchZ mutant in the presence of exogenously added Bchlide a. (A) The R. sphaeroides bchFNB-bchZ mutant neither synthesizes nor metabolizes Chlide a. However, the mutant is expected to form Bchl a if Bchlide a is added exogenously. The mutated metabolic steps are denoted with dotted lines. The bchLNB genes code for three subunits comprising the dark-operative protochlorophyllide oxidoreductase. (B) The photoheterotrophic growth of the bchFNB-bchZ mutant was examined in the presence of exogenously added Bchlide a (0 to 70 μM) with or without exogenously added Chlide a (70 μM).

The spectral complexes of the R. sphaeroides bchFNB-bchZ mutant were examined with cells harvested at 110 h after photoheterotrophic growth (Fig. 8B) in the presence of exogenously added Bchlide a (35 μM) (Fig. 9A). The mutant exhibits both B800-B850 and B875 complexes, as well as carotenoids, under the given conditions (Fig. 9A, culture 1). The more Chlide a was added exogenously, the fewer spectral complexes were formed (Fig. 9A, culture 3), resulting in retardation of photoheterotrophic growth (Fig. 8B). However, an increase of Bchlide a to 70 μM reversed growth retardation (Fig. 8B), with an increase of B800-B850 and B875 complex levels (Fig. 9A, culture 2). The absorbance of the residual Bchlide a in the cell interfered with the measurement of the B875 complex, so total Bchl a was examined after extraction from the cell. The cellular Bchl a content, which amounted to 4.72 nmol/mg protein at 35 μM Bchlide a, was drastically decreased by the addition of 70 μM Chlide a, but its inhibition was significantly reversed by doubling the Bchlide a level to 70 μM (Fig. 9C).

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FIG. 9.

Effect of exogenously added Chlide a on the metabolic conversion of exogenously added Bchlide a into Bchl a. (A) The spectral complexes of the R. sphaeroides bchFNB-bchZ mutant were examined with cells harvested at 110 h after photoheterotrophic growth in the presence of exogenously added Bchlide a (0 to 70 μM) with or without exogenously added Chlide a (70 μM) (Fig. 8B). (B) The spectral complexes of the R. sphaeroides bchFNB-bchZ mutant were examined with cells grown anaerobically in the dark with DMSO in the presence of Bchlide a (0 to 70 μM) with or without exogenously added Chlide a (70 μM). (C) Total Bchl a, which was examined after extraction from the R. sphaeroides bchFNB-bchZ mutant grown photoheterotrophically (A) and anaerobically in the dark with DMSO (B), is illustrated as a mean ± standard deviation.

The R. sphaeroides bchFNB-bchZ mutant was grown anaerobically in the dark with DMSO in the presence of Bchlide a with or without Chlide a, as described above. Neither Bchlide a nor Chlide a affected the growth of R. sphaeroides under the given conditions (data not shown). The spectral complexes (Fig. 9B) and Bchl a contents (Fig. 9C) were almost the same as those from the R. sphaeroides bchFNB-bchZ mutant grown photoheterotrophically. Thus, the inhibition of Bchl a formation by Chlide a is not due to any phototoxicity of exogenously added intermediates but is rather due to the specific inhibition of bacteriochlorophyll synthase by Chlide a, as illustrated by enzyme kinetics (Fig. 7).

Taken together, the photoheterotrophic growth of Synechocystis sp. PCC 6803 is inhibited by exogenously added Bchlide a, while the metabolic conversion of exogenously added Bchlide a to Bchl a by the R. sphaeroides bchFNB-bchZ mutant is inhibited by exogenously added Chlide a. The growth inhibition of Synechocystis sp. PCC 6803 is reversed by exogenously added Chlide a, while the metabolic inhibition of the R. sphaeroides bchFNB-bchZ mutant is reversed by the increased level of exogenously added Bchlide a. The results are explained by the competitive inhibition of chlorophyll synthase by Bchlide a and the same type of inhibition of bacteriochlorophyll synthase by Chlide a. Kinetic analyses further revealed the same ping-pong reactions. Thus, functional and structural similarities are suggested to exist between their active sites.