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J Biol Chem, Vol. 273, Issue 51, 34206-34213, December 18, 1998

Heterologous Expression of the Rhodobacter capsulatus BchI, -D, and -H Genes That Encode Magnesium Chelatase Subunits and Characterization of the Reconstituted Enzyme^*

Robert D. Willows and Samuel I. Beale^§

From the Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912

	ABSTRACT

Top Abstract Introduction Procedures Results Discussion References

Magnesium chelatase inserts Mg²⁺ into protoporphyrin IX in the chlorophyll and bacteriochlorophyll biosynthetic pathways. In photosynthetic bacteria, the products of three genes, bchI, bchD, and bchH, are required for magnesium chelatase activity. These genes from Rhodobacter capsulatus were cloned separately into expression plasmids pET3a and pET15b. The pET15b constructs produced NH₂-terminally His₆-tagged proteins. All proteins were highly expressed and were purified to near homogeneity. The BchI and BchH proteins were soluble. BchD proteins were insoluble, inactive inclusion bodies that were renatured by rapid dilution from 6 M urea. The presence of BchI in the solution into which the urea solution of BchD was diluted increased the yield of active BchD. A molar ratio of 1 BchI:1 BchD was sufficient for maximum renaturation of BchD. All of the proteins were active in the magnesium chelatase assay except His-tagged BchI, which was inactive and inhibited in incubations containing non-His-tagged BchI. Expressed BchH proteins contained tightly bound protoporphyrin IX, and they were susceptible to inactivation by light. Maximum magnesium chelatase activity per mol of BchD occurred at a stoichiometry of 4 BchI:1 BchD. The optimum reaction pH was 8.0. The reaction exhibited Michaelis-Menten kinetics with respect to protoporphyrin IX and BchH.

	INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

Magnesium chelatase inserts Mg²⁺ into protoporphyrin IX in the chlorophyll and bacteriochlorophyll biosynthetic pathways. This enzyme is at the branch point of the (bacterio)chlorophyll and heme biosynthetic pathways and is believed to have a regulatory role in directing biosynthetic intermediates into the (bacterio)chlorophyll pathway (1). Transposon mutagenesis studies in Rhodobacter capsulatus (2) and Rhodobacter sphaeroides (3) have shown that the products of three genes, bchI, bchD, and bchH, are required for this insertion. Genes that are homologous to bchI, bchD, and bchH have been reported in plants and photosynthetic bacteria. It has been suggested that the genes be named bchI, bchD, and bchH when referring to organisms that accumulate bacteriochlorophyll and chlI, chlD, and chlH for the respective homologous genes in organisms that accumulate chlorophyll (1). This nomenclature will be used here.

Expression of the three R. sphaeroides genes in Escherichia coli showed that all three gene products, plus ATP, Mg²⁺, and protoporphyrin IX, were required for magnesium chelatase activity (4, 5). An enzyme activation step involving the BchI and BchD subunits was observed (5) that was similar to that reported for the pea magnesium chelatase (6). However, very low expression of the BchD protein limited further characterization of magnesium chelatase (5). Heterologous expression of individual subunits and reconstitution of magnesium chelatase activity has recently been described for tobacco (7), the cyanobacterium Synechocystis sp. PCC 6803 (8), and the green bacterium Chlorobium vibrioforme (9) However, characterization of the reaction using recombinant proteins has been limited due to low levels of expression and poor recovery of activity from these systems.

We now report the cloning and high level expression of the magnesium chelatase genes from R. capsulatus, the purification of the gene products, and characterization of the reconstituted enzyme. Portions of this work were previously published in abstract form (10).

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Construction of Plasmids for Expression of bchI, bchD, and bchH-- The plasmid pRPS404 (11), which contains a 44-kilobase pair region of the R. capsulatus photosynthetic gene cluster, was used as a PCR¹ template throughout. A modified T-vector was constructed between the EcoRI and BamHI sites of pBluescript (KS) (Stratagene, La Jolla, CA) essentially as described in Ref. 12, and this vector was used to subclone the bchID and bchD genes as described below. The oligonucleotides 5'-GGATCATCTTGCGGAAACTGT-3' and 5'-ATGACTACCGCCGTCGCTCGACTTCAACCCTCTGCT-3' were used to amplify the bchID region of the photosynthetic gene cluster by PCR using Taq DNA polymerase (Stratagene). The 3-kilobase pair PCR product was then cloned directly into the T-vector, yielding plasmid pKSBchID. This cloning created an NdeI restriction site at the bchI translation start site. A region containing the bchI gene and the 5' portion of bchD was then subcloned between the NdeI and BamHI sites of either pET3a (Novagen, Madison, WI) to create pBchI or pET15b (Novagen) to create pHisBchI. The oligonucleotides 5'-ATGGACCACGAACGCCTGAAGTCGGCCCTTG-3' and 5'-ACCCTGCGTCGCGCCGCCGCCGACCGATAGG-3' were used to amplify the bchD gene by PCR using Pfu DNA polymerase (Stratagene). The 1.8-kilobase pair PCR product was cloned into the modified T-vector, yielding plasmid pKSBchD. This cloning created an NdeI restriction site at the bchD translation start site. This plasmid was digested with NdeI and XhoI, and the bchD fragment was subcloned between either the NdeI and BamHI sites of pET3a to create pBchD, or the NdeI and XhoI sites of pET15b to create pHisBchD. The oligonucleotides 5'-AGGCCCCATATGCACGATGAGTCGATGAGC-3' and 5'-CCCTCCTTTTCGTAGTCGTAGATCTCATTC-3' were used to amplify bchH by PCR using Pfu DNA polymerase. These oligonucleotides introduced NdeI and BglII restriction sites. The PCR product was then either digested and cloned directly into pET3a to create pBchH or into the pCRBlunt (Invitrogen, Carlsbad, CA) vector to create pCRBchH and then subcloned into pET15b to create pHisBchH.

The magnesium chelatase genes in these plasmids were sequenced to check for errors using a Dye-Deoxy terminator kit (PE Applied Biosystems, Foster City, CA) with the Applied Biosystems model 377 sequencer. The first subclone of bchI, which was used to make the expression clones, was sequenced completely, and the final bchI expression clones were sequenced in from the 5'-end to verify that they were in frame. The first bchD and bchH subclones were sequenced 600 base pairs in from each end, and the final bchD and bchH expression clones were sequenced in from the 5'-end to verify that they were in frame. No errors were detected.

Expression of BchI, BchD, and BchH in E. coli-- E. coli BL21 (DE3) pLysS (Novagen) strains containing the expression plasmids were grown at 25 or 37 °C in 1 liter of LB medium (13) containing 100 µg/ml ampicillin and 34 µg/ml chloramphenicol until the A₆₀₀ of the cultures was 0.8. Protein expression was induced by the addition of isopropyl -D-thiogalactopyranoside to a final concentration of 1 mM. After 4 h, the cells were harvested by centrifugation. For the expressed pET3a constructs, the cells from 1 liter of culture were resuspended in 20 ml of 50 mM Tricine-NaOH, pH 8.0, 15 mM MgCl₂, and 4 mM DTT. The suspension was then frozen at -20 °C, thawed once, and then lysed completely by passage through a French pressure cell (SLM, Urbana, IL) at 20,000 p.s.i. For expression from pET15b constructs, the cells from 1 liter of culture were resuspended in 20 ml of 20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 15 mM MgCl₂, and 5 mM imidazole. This suspension was frozen at -20 °C, thawed once, and then lysed by passage through a French pressure cell at 20,000 p.s.i.

Purification of the Expressed Proteins-- The BchH protein was purified essentially as described previously for the protein from R. sphaeroides (5).

The BchD protein was expressed as inclusion bodies, and these were purified as described in the pET system manual (Novagen). The inclusion bodies were then solubilized in 6 M urea, 50 mM Tricine-NaOH, pH 8.0, 15 mM MgCl₂, and 4 mM DTT. The solubilized protein was loaded onto a RESOURCE Q (Amersham Pharmacia Biotech) cation exchange chromatography column that was pre-equilibrated in the same buffer, and then the protein was eluted with this buffer containing a linear gradient of 0-1 M NaCl, in 10 column volumes. The BchD protein eluted at 0.4 M NaCl.

The BchI expression lysate was centrifuged at 30,000 × g for 30 min, and then polyethylene glycol-8000 (molecular weight, 8000) was added to the soluble supernatant to a concentration of 10% (w/v). The mixture was placed on ice for 30 min and then centrifuged for 30 min at 10,000 × g, the pellet was discarded, and polyethylene glycol-8000 was added to a final concentration of 25% (w/v). After standing for 2 h on ice, the solution was centrifuged at 30,000 × g for 30 min. The supernatant was discarded, and the precipitate was washed once with 30% (w/v) polyethylene glycol-8000 and then redissolved in 50 mM Tricine-NaOH, pH 8.0, 15 mM MgCl₂, and 4 mM DTT. The BchI protein was then purified by cation exchange chromatography as described above for BchD but without urea in the buffers. BchI eluted at 0.3 M NaCl.

The HisBchH and HisBchI proteins were purified on a Ni²⁺-chelating column as described in the pET system manual, except that the eluted proteins were immediately desalted by Sephadex G-25 (Amersham Pharmacia Biotech) chromatography into 6% glycerol, 50 mM Tricine-NaOH, pH 8.0, 15 mM MgCl₂, and 4 mM DTT.

The HisBchD protein was expressed as inclusion bodies, and these were purified as described in the pET system manual (Novagen). The solubilized protein was then further purified on a Ni²⁺-chelating column under denaturing conditions in 6 M urea as described in the pET system manual. After the protein was eluted, the buffer was changed to 6 M urea, 50 mM Tricine-NaOH, pH 8.0, 15 mM MgCl₂, and 4 mM DTT.

Assay for Magnesium Chelatase-- The assay mixture contained, in 50-1000 µl, the following: 50 mM Tricine-NaOH, pH 8.0, 15 mM MgCl₂, 4 mM DTT, 4 mM ATP, 20 mM phosphocreatine, 20 units/ml of rabbit muscle creatine phosphokinase, 4 µM protoporphyrin IX, and various amounts of recombinant proteins as described in the figure and table legends. The assay mixtures were incubated for 30 or 60 min at 30 °C and stopped by the addition of 9 ml of acetone:H₂O:32% (w/v) NH₃ (80:20:1, v/v/v) per ml of incubation mixture and analyzed by fluorescence spectroscopy with the excitation wavelength set at 418 nm and the emission wavelength set at 596 nm. The intensity of the emission at 596 nm of standard magnesium-protoporphyrin IX in the same solution was proportional to the concentration within the range of 1 nM to 1 µM. A standard curve was used to determine the amount of magnesium-protoporphyrin IX formed in the assay.

Antiserum to BchD and Immunoblotting-- Antiserum to HisBchD was raised in a New Zealand White rabbit using standard procedures (14). The antiserum was cross-absorbed onto an E. coli lysate column (Pierce) according to the manufacturer's instructions. Immunoblots were made using standard methods (14) with a 1/5000 dilution of antiserum and detection by a horseradish peroxidase-conjugated secondary antibody and a chemiluminescent substrate.

Other Methods-- SDS-polyacrylamide gel electrophoresis was done according to the procedure of Fling and Gregerson (15) and gels were stained with colloidal Coomassie Brilliant Blue (16). R. capsulatus strains ZY6 and DB350 (2, 17) were a gift from C. E. Bauer and D. Bollivar. They were grown in RCV+ medium in the dark at 25 °C as described by Bollivar et al. (2).

Chemicals-- Protoporphyrin IX and magnesium-protoporphyrin IX were purchased from Porphyrin Products, Inc. (Logan, UT). Except where indicated otherwise, all other chemicals were from Fisher, Sigma, or Research Organics (Cleveland, OH).

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

Purification of Expressed Subunits-- The individual magnesium chelatase recombinant subunits were purified as described under "Experimental Procedures." With the exception of non-His-tagged BchH, they were homogeneous as judged by SDS-polyacrylamide gel electrophoresis (Fig. 1).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   SDS-polyacrylamide gel electrophoresis of the purified proteins. Lane 1, 4 µg of BchI; lane 2, 0.5 µg of HisBchI; lane 3, 2 µg of BchD; lane 4, 4 µg of HisBchD; lane 5, 4 µg of BchH; lane 6, 4 µg of HisBchH. Migration positions of standard proteins with the indicated molecular masses (in kDa) are shown at the left of the image.

BchH and HisBchH were expressed in soluble form with protoporphyrin IX noncovalently bound, as was found for the R. sphaeroides BchH protein expressed in E. coli (5). The absorption peaks attributed to protoporphyrin IX are visible in the absorption spectrum of purified HisBchH (Fig. 2A). The fluorescence excitation spectrum (Fig. 2B) shows a pronounced peak at 280 nm that is not present in the spectrum of free protoporphyrin IX, indicating that one or more protein tryptophan residues are in close proximity to the protoporphyrin IX and can transfer excitation energy to the pigment. The bound protoporphyrin IX caused BchH to become very susceptible to inactivation by light. Under normal laboratory lighting, it was inactivated in 10 min at 0 °C (data not shown). To prevent this light inactivation, all of the BchH expression and purification steps were performed in the dark or under a dim green safelight. In addition, all magnesium chelatase assays were done in the dark.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Spectra of HisBchH protein. A, absorption spectrum of 166 µg of HisBchH in 1 ml of 50 mM Tricine-NaOH, pH 8.0, 15 mM MgCl2, and 1 mM DTT. B, dashed line is the fully corrected fluorescence excitation spectrum of 66 µg of HisBchH in 1 ml (1 µM) of 50 mM Tricine-NaOH, pH 8.0, 15 mM MgCl2, and 1 mM DTT; solid line is the fluorescence excitation spectrum of 0.5 µM standard protoporphyrin IX. Emission was monitored at 635 nm.

BchD and HisBchD were both expressed as inclusion bodies. As described below, these proteins, after solubilization with urea, reconstituted magnesium chelatase when added to assay buffer containing BchH and BchI.

Reconstitution of Magnesium Chelatase Activity-- In vitro magnesium chelatase activity was reconstituted only upon the addition of all three expressed proteins, BchI, BchH (or HisBchH), and BchD (or HisBchD) to the incubation mixture (Fig. 3). Some product was formed in incubations without added protoporphyrin IX, because the protoporphyrin IX that is bound to purified BchH can be used as substrate.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Emission spectra of reaction product. Acetone extracts were obtained from 100 µl of magnesium chelatase assays incubated for 30 min at 30 °C as described under "Experimental Procedures." Amounts of protein in the assays as indicated were 10.4 µg of BchD, 22 µg of BchI, and 66 µg of HisBchH, with the exception of the first assay, which contained a mixture of renatured BchD (8.7 µg) and BchI (19.6 µg).

Although both BchI and HisBchI were highly expressed in soluble form, only non-His-tagged BchI was active in the magnesium chelatase assay. HisBchI was inactive, and it inhibited the reaction in incubations containing BchI (Fig. 4).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Inhibition of magnesium chelatase by HisBchI. Assays (100 µl) contained 5 µg of BchI, 20 µg of BchH, approximately 4 µg of BchD, and the indicated amounts of HisBchI. Incubation was for 60 min at 30 °C. The line is the best fit linear extrapolation for the data points.

For reconstitution of activity, BchD (or HisBchD), solubilized in 6 M urea, was added directly to the assay mixture containing the other two protein components. BchD, therefore, refolded to an active form in the reaction mixture during the incubation, as was recently reported for C. vibrioforme BchD (9). Although this method of assaying magnesium chelatase was useful for reconstitution experiments, it was unsatisfactory for enzyme kinetic studies because the proportion of BchD that is in the active form in the assay mixture could not be controlled. The factors that mediate renaturation of BchD were therefore examined as described below. Renatured BchD (or HisBchD) provided a consistent preparation for further study of the magnesium chelatase reaction.

Efficient in vitro renaturation of BchD required DTT and ATP (Table I). Moreover, the degree of renaturation was increased by the presence of BchI. A time course of the renaturation of BchD at 0 °C with BchI, ATP, and MgCl₂ indicated that BchD was maximally renatured at 90 min (data not shown). The concentration of BchD in the medium did not appear to have an effect on the renaturation efficiency, because approximately equal specific activity was obtained with the three concentrations tested (Table I). The slightly lower activity at higher BchD concentrations was caused by the higher concentrations of urea in the assay medium, which ranged from 30 to 120 mM in this experiment. A BchI:BchD molar ratio of 1:1 was sufficient for maximal recovery of active BchD (Table II). Neither further stimulation or inhibition of the renaturation was observed at higher BchI:BchD ratios. In the R. capsulatus chromosome, bchI is 5' to bchD and is part of the same transcription unit. This implies that BchI is translated before BchD, and it is proposed that its presence may aid in the folding of BchD to its active form in vivo.

Because BchD was solubilized in 6 M urea and added to assay medium by dilution from the urea solution, it was important to determine the effect of urea on the magnesium chelatase reaction. The reaction was progressively inhibited at increasing urea concentrations, with approximately 20% inhibition at 100 mM, 50% inhibition at 250 mM, and 90% inhibition at 800 mM (Fig. 5). Because the final urea concentration in the magnesium chelatase assays was usually 30-60 mM, its presence in the assay medium had only a minor effect on activity.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Effect of urea concentration on magnesium chelatase activity. Assays (50 µl) were done in triplicate and contained 1.7 µg of renatured BchD, 3.9 µg of BchI, and 33 µg of HisBchH in assay buffer with urea added. Incubation was for 30 min at 30 °C. Error bars indicate the S.D. about the mean. The line is the best fit exponential curve for the data points.

Reconstitution of R. capsulatus Magnesium Chelatase Mutants-- R. capsulatus mutants ZY6 and DB350 contain insertional disruptions of the bchH and bchI genes, respectively (2, 17). Magnesium chelatase activity was absent in extract of either mutant alone, but was present in a mixture of the extracts from the two mutants (Table III). Activity was reconstituted in extract of ZY6 cells supplemented with recombinant BchH. Neither BchD, BchI, nor a combination of BchD and BchI reconstituted activity of ZY6 extract. Fractionated ZY6 extract from which BchD was removed by high speed centrifugation and ion exchange chromatography required addition of both BchH and BchD to restore activity. Similarly, ZY6 extract from which BchI was partially removed by high speed centrifugation required addition of both BchH and BchI to restore full activity.

View this table: [in this window] [in a new window]   Table III In vitro complementation of R. capsulatus magnesium chelatase mutants Strains ZY6 (bchH) and DB350 (bchI) were grown and extracted as described under "Experimental Procedures." Extract of strain ZY6 (3 ml) was centrifuged for 1 h at 190,000 × g, and the glassy pellet was washed once with extraction buffer and resuspended in 0.5 ml of the same buffer to yield the BchD-containing fraction ZY6 (D) (31). The supernatant was purified by cation exchange chromatography as described under "Experimental Procedures" to yield the BchI-containing fraction ZY6 (I). Recombinant BchD was dissolved in 6 M urea, and 2 µl was added to the reactions where indicated. Incubations were 60 min at 30 °C in 100 µl of complete reaction medium containing the indicated amounts of proteins from cell extracts and purified recombinant proteins. Because the various proteins added to the incubation mixture were of different degrees of purification, the tabulated relative product formation values have not been normalized to protein concentration.

Magnesium chelatase activity was not restored to extract of strain DB350 by the addition of any single recombinant protein, but activity was partially restored by the addition of both BchD and BchI. The requirement for both proteins is consistent with the absence of the BchD in extracts of strain DB350 (Fig. 6). These results suggest that BchI is needed to stabilize BchD in vivo and are consistent with the ability of BchI to enhance the renaturation of urea-denatured BchD in vitro.

View larger version (40K): [in this window] [in a new window]   Fig. 6.   Immunoblot. The probe was antibody to purified R. capsulatus BchD protein. Lane 1 contains purified HisBchD protein; lane 2 contains extract of R. capsulatus strain ZY6, which has a disrupted bchH gene; and lane 3 contains extract of R. capsulatus strain DB350, which has a disrupted bchI gene. Migration positions of standard proteins with the indicated molecular masses (in kDa) are shown at the left of the image.

Kinetic Properties of Magnesium Chelatase-- The pH optimum for magnesium chelatase was approximately 8.0 (Fig. 7). Activity dropped to near zero below pH 6 and above pH 10.5. The time course for product accumulation exhibited a small lag phase, followed by approximately constant activity for about 100 min, and lower activity at later times (Fig. 8). Preincubation of BchD and BchI in assay mixture for 10 min at 30 °C before starting the reaction by the addition of BchH increased the initial reaction rate but did not eliminate the lag phase.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   pH optimum for magnesium chelatase. Assays (100 µl) were done in triplicate and contained 1.4 µg of renatured BchD, 4.8 µg of BchI, and 66 µg of HisBchH in assay buffer adjusted to the indicated pH and then incubated for 30 min at 30 °C. Error bars indicate the S.D. about the mean.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Reaction time course. Assays (1.0 ml) contained 110 µg/ml BchD, 140 µg/ml BchI, and 660 µg/ml HisBchH. Closed circles are for an experiment in which all ingredients were added in quick succession at the start of the reaction. Open circles are for an experiment in which all ingredients except BchH were preincubated for 10 min at 30 °C before BchH was added to start the reaction.

A double reciprocal plot of reaction rate versus protoporphyrin IX concentration indicates Michaelis-Menten kinetics with respect to protoporphyrin IX (Fig. 9A). The apparent K_m for protoporphyrin IX was calculated to be 1.23 µM. A double reciprocal plot of reaction rate versus HisBchH concentration indicates Michaelis-Menten kinetics with respect to HisBchH (Fig. 9B). The apparent K_m was calculated to be 17.6 µg/100-µl reaction, a value that is equal to 1.34 µM for HisBchH, which has a molecular mass of 131 kDa.

View larger version (15K): [in this window] [in a new window]   Fig. 9.   Concentration dependence on protoporphyrin IX and BchH. All assays (100 µl) contained 5.2 µg of BchI and 8.4 µg of BchD (2 µl of a 6 M urea solution added to start the reaction). A, for protoporphyrin IX, the incubation contained 6 µg of HisBchH, and the free protoporphyrin IX concentration was varied from 0.8 to 4 µM. B, for BchH, the free protoporphyrin IX concentration was 4 µM, and HisBchH varied from 0.4 to 30 µg. Incubation was for 60 min at 30 °C. Results are plotted in double-reciprocal form. The lines are the best fit linear extrapolations for the data points.

BchI:BchD Optimal Stoichiometry-- Reactions were done in incubation mixtures containing constant amounts of BchH and BchI and varying amounts of BchD, and the activity per unit of BchD was calculated. The results indicate that the optimal ratio of BchI:BchD is 4 (Fig. 10). This ratio was also obtained when HisBchD was used (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 10.   Optimal BchI:BchD stoichiometry. Assays (100 µl) in triplicate contained 10.8 µg of BchI, 33 µg of HisBchH, and an amount of renatured BchD sufficient to provide the indicated BchI:BchD molar ratio. Incubation was 30 min at 30 °C. Error bars indicate the S.D. about the mean.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

Three R. capsulatus genes, bchI, bchD, and bchH, have been cloned and overexpressed in E. coli, and the expressed proteins have been purified and reconstituted in vitro to yield magnesium chelatase activity. The characterization of this activity and of the individual subunits that we report here suggests that Mg²⁺ chelation is a multistep reaction and that the individual subunits may have multiple roles in the assembly of active enzyme and in the reaction.

The bchI, bchD, and bchH genes were first shown to be involved in Mg²⁺ chelation in R. capsulatus by insertional mutagenesis and analysis of the mutant phenotypes (2, 17). Genes homologous to bchI, bchD, and bchH have been described in several photosynthetic bacteria and plants. Reconstitution of active magnesium chelatase from the expressed genes was first reported for R. sphaeroides (18). In that report, bchI and bchD were co-expressed, but it was not possible to express active bchD alone. Reconstituted in vitro activity was limited by the amount of BchD present in the reaction mixture. The bchI, bchD, and bchH genes from the green bacterium C. vibrioforme were cloned, separately expressed, and recombined in vitro to yield magnesium chelatase activity (9). To reconstitute activity, it was necessary to treat the BchD protein, which was expressed as inclusion bodies, with 6 M urea to solubilize it and then add the solubilized BchD to assay mixtures containing BchI and BchH. In contrast, three genes from the cyanobacterium Synechocystis sp. PCC 6803, designated chlI, chlD, and chlH, respectively, were cloned and separately expressed to produce soluble proteins, and the proteins could be recombined in vitro to yield magnesium chelatase activity (8). Although the predicted Synechocystis sp. PCC 6803 ChlI and ChlH proteins are quite similar to the predicted BchI and BchH proteins (51 and 41% identity to the R. capsulatus proteins, respectively), ChlD is less similar to BchD (32% identity), and there are large gaps in the aligned sequences. All of these reports demonstrated that all three subunits are required for magnesium chelatase activity. However, further characterization of the activity and characterization of the individual subunits have not been reported.

As an aid to rapid enzyme subunit purification by nickel-chelating column chromatography, the three R. capsulatus magnesium chelatase proteins were expressed as NH₂-terminally His-tagged fusion constructs. For two of the subunits, BchD and BchH, the His-tagged proteins were effective in reconstituting magnesium chelatase activity. However, His-tagged BchI did not reconstitute activity and, moreover, it acted as an inhibitor in incubations containing non-His-tagged BchI. The most likely explanation for the inhibition is that the His tag prevents catalysis in some way but still allows HisBchI to interact with BchH and/or BchD and blocks BchI from interacting constructively with these subunits.

The BchI and BchH proteins were expressed as soluble active proteins in E. coli, but BchD was expressed as insoluble inclusion bodies. The insoluble BchD was solubilized in 6 M urea and was refolded by dilution of the urea to yield active protein. Optimal refolding required ATP, DTT, and BchI. It has been shown that the presence of a reducing agent, such as DTT, is required for stabilizing magnesium chelatase (19-22) and that magnesium chelatase is sensitive to sulfhydryl reagents (21-24). The requirement of DTT for maintaining and reconstituting the activity of BchD, both in the refolding process and in the subsequent enzyme assay, suggests that it is this subunit that is most sensitive to oxidizing conditions. The ATP requirement is consistent with the hypothesis that ATP may help to refold and stabilize BchD by binding to its active site. It has been reported that pea magnesium chelatase undergoes irreversible loss of activity at room temperature in the absence of ATP (6, 25).

On the R. capsulatus chromosome, the bchD open reading frame is immediately downstream of the bchI open reading frame and is translated from the same transcript. The same organization of bchI and bchD occurs in C. vibrioforme (26). This conserved gene organization, together with the fact that BchI is required for optimal refolding of BchD in vitro, suggests that BchI may be essential for the efficient refolding and/or stabilization of BchD in vivo. The difference in the optimal stoichiometry of 1 BchI:1 BchD for refolding compared with 4 BchI:1 BchD for assay of magnesium chelatase activity suggests that the function of BchI in BchD refolding and magnesium chelatase activity may not be directly linked to one another. A lag phase in the kinetics of the reaction, as was observed previously for other systems (5, 6, 25), is partially overcome by preincubation of BchI with BchD and ATP, suggesting that the catalytic function of BchI is in the ATP-dependent activation of BchD. This role is consistent with the reported ATPase and ATP-ADP phosphate exchange activities of BchI (27).

The R. capsulatus magnesium chelatase proteins expressed in E. coli were effective in the in vitro reconstitution of magnesium chelatase activity when added to extracts of the R. capsulatus magnesium chelatase mutants ZY6 and DB350. Strain ZY6 has a transposon inserted into the bchH gene, and recombinant BchH protein restored activity to extracts of ZY6 cells. Strain DB350 has a transposon inserted into the bchI gene. However, recombinant BchI alone did not restore activity in extracts of DB350 cells. Restoration of activity required the addition of both BchI and BchD proteins. The Western blot of the DB350 mutant showed no detectable BchD protein. The absence of BchD in DB350 cells is consistent with the hypothesis that BchI is needed to stabilize BchD in vivo and this hypothesis is also supported by the ability of BchI to enhance the renaturation of urea-denatured BchD in vitro.

The ability of magnesium chelatase to function in relatively high concentrations of urea suggests that the predominant interactions among the subunits are ionic and/or hydrophobic and that hydrogen bonding plays a limited role (28). The fact that the BchI and BchD subunits are separable by ion exchange chromatography (5), which involves high salt concentrations, indicates that the predominant interactions between these subunits are ionic. Examination of the kinetic constants, such as K_m values, under different urea concentrations and different ionic strengths will provide further information on the types of interactions between the subunits. The K_m values calculated for BchH and protoporphyrin IX were derived from results using assays containing 120 mM urea. The K_m for BchH indicates a relatively weak interaction between this protein and the BchI and/or BchD subunits. However, it is possible that these results were influenced by the presence of urea. The K_m for protoporphyrin IX is higher than that reported for the magnesium chelatases of R. sphaeroides (5) and pea (29), and at least part of the difference may be due to the presence of urea in the assay.

The fluorescence excitation spectrum of BchH with bound protoporphyrin IX indicates that there is at least one tryptophan residue in sufficiently close proximity to the porphyrin binding site to permit efficient energy transfer from the tryptophan to the porphyrin (30). The high photosensitivity of the BchH subunit is also consistent with the close proximity of the bound protoporphyrin IX to a tryptophan residue on the protein. Excitation transfer of light absorbed by the tryptophan would provide a pathway for energetic photon energy to reach the porphyrin.

On the basis of these results, we propose the following reaction scheme for magnesium chelatase. The BchH subunit forms a stable complex with the protoporphyrin IX substrate, and the persistence of this complex throughout the purification suggests that the equilibrium shown in Reaction 1 is in favor of the complex. The rate of formation of this complex will influence the kinetics of the reaction. The optimal stoichiometry of 4:1 for the BchI and BchD proteins suggests that they may form a complex in that ratio during an ATP-dependent activation step as shown in Reaction 2. For the purposes of this model, it is postulated that the complex has the composition BchI₄:BchD, although further work will be necessary to determine whether the 4:1 optimal component concentration ratio for enzyme activity actually indicates the existence of a complex with this ratio. It is not known whether this activation is an ATP-dependent complex formation or some other type of activation, such as activation of a Mg²⁺ ion. It is not known whether this step requires ATP hydrolysis. The product of this activation is then able to use the protoporphyrin IX bound to the BchH protein as a substrate for ATP-dependent Mg²⁺ insertion as shown in Reaction 3. This reaction apparently requires ATP hydrolysis (6). This reaction probably occurs in more than one step. The constancy of the reaction rate after the initial lag phase is completed suggests that the BchI*BchD complex, once it is formed, is stable during continued Mg²⁺ chelation cycles and does not dissociate in Reaction 3.

	ACKNOWLEDGEMENTS

We thank C. E. Bauer and D. Bollivar for supplying R. capsulatus strains.

	FOOTNOTES

^* This work was supported by United States National Science Foundation Grant MCB-9506901 and United States Department of Energy Grant DE-FG02-88ER13918.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.

Present address: Macquarie University, School of Biological Science, North Ryde 2109, Australia.

^§ To whom correspondence should be addressed. Tel.: 401-863-3129; Fax: 401-863-1182; E-mail: sib{at}brown.edu.

The abbreviations used are: PCR, polymerase chain reaction; DTT, dithiothreitol; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

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