Reconstitution of Light-independent Protochlorophyllide Reductase from Purified Bchl and BchN-BchB Subunits

Protochlorophyllide reductase catalyzes the reductive formation of chlorophyllide from protochlorophyllide during biosynthesis of chlorophylls and bacteriochlorophylls. The light-independent (dark) form of protochlorophyllide reductase plays a key role in the ability of gymnosperms, algae, and photosynthetic bacteria to green (form chlorophyll) in the dark. Genetic and sequence analyses have indicated that dark protochlorophyllide reductase consists of three protein subunits that exhibit significant sequence similarity to the three subunits of nitrogenase, which catalyzes the reductive formation of ammonia from dinitrogen. However, unlike the well characterized features of nitrogenase, there has been no previous biochemical characterization of dark protochlorophyllide reductase. In this study, we report the first reproducible demonstration of dark protochlorophyllide reductase activity from purified protein subunits that were isolated from the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus. Two of the three subunits (Bchl and BchN) were expressed in R. capsulatus as S tag fusion proteins that facilitated affinity purification. The third subunit (BchB) was co-purified with the BchN protein indicating that BchN and BchB proteins form a tight complex. Dark protochlorophyllide reductase activity was shown to be dependent on the presence of all three subunits, ATP, and the reductant dithionite. The similarity of dark protochlorophyllide reductase to nitrogenase is discussed. Protochlorophyllide reductase catalyzes the reductive formation of chlorophyllide from protochlorophyllide during biosynthesis of chlorophylls and bacteriochlorophylls. The light-independent (dark) form of protochlorophyllide reductase plays a key role in the ability of gymnosperms, algae, and photosynthetic bacteria to green (form chlorophyll) in the dark. Genetic and sequence analyses have indicated that dark protochlorophyllide reductase consists of three protein subunits that exhibit significant sequence similarity to the three subunits of nitrogenase, which catalyzes the reductive formation of ammonia from dinitrogen. However, unlike the well characterized features of nitrogenase, there has been no previous biochemical characterization of dark protochlorophyllide reductase. In this study, we report the first reproducible demonstration of dark protochlorophyllide reductase activity from purified protein subunits that were isolated from the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus. Two of the three subunits (Bchl and BchN) were expressed in R. capsulatus as S tag fusion proteins that facilitated affinity purification. The third subunit (BchB) was co-purified with the BchN protein indicating that BchN and BchB proteins form a tight complex. Dark protochlorophyllide reductase activity was shown to be dependent on the presence of all three subunits, ATP, and the reductant dithionite. The similarity of dark protochlorophyllide reductase to nitrogenase is discussed. protochlorophyllide chlorophyll chlorophyllide light-dependent protochlorophyllide oxidoreductase light-independent (dark) protochlorophyllide reductase polymerase chain reaction base pairs polyacrylamide gel electrophoresis Protochlorophyllide (Pchlide)1 is a key intermediate in the biosynthesis of chlorophylls (Chl) and bacteriochlorophylls. Among photosynthetic organisms, there are two different enzymes known to catalyze stereo-specific double-bond reduction of ring-D of Pchlide to form chlorophyllide a(Chlide), which is a direct precursor of Chl a (1Fujita Y. Plant Cell Physiol. 1996; 37: 411-421Crossref PubMed Scopus (118) Google Scholar, 2Suzuki J.Y. Bollivar D.W. Bauer C.E. Annu. Rev. Genet. 1997; 31: 61-89Crossref PubMed Scopus (141) Google Scholar, 3Armstrong G.A. J. Photochem. Photobiol. B Biol. 1998; 43: 87-100Crossref Scopus (120) Google Scholar) (Fig.1). One enzyme is light-dependent Pchlide oxidoreductase (LPOR, EC 1.3.1.33). LPOR utilizes NADPH to catalyze reduction of Pchlide with the interesting requirement that the substrate (Pchlide) must also absorb light in order for the enzyme to promote double bond reduction. LPORs have been extensively studied for some time, because the requirement for light makes this enzyme a key player in light-dependent greening of flowering plants (angiosperms) (4Levedev N. Timko M.P. Photosynth. Res. 1998; 58: 5-23Crossref Scopus (112) Google Scholar). The other enzyme that reduces Pchlide is light-independent (dark) Pchlide reductase (DPOR). This enzyme catalyzes Pchlide reduction irrespective of light. Genetic and sequence studies have shown that primitive anoxygenic (nonoxygen evolving) photosynthetic bacteria contain only DPOR (5Xiong J. Inoue K. Bauer C.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14851-14856Crossref PubMed Scopus (142) Google Scholar). In contrast, cyanobacteria, algae, and gymnosperms (nonflowering plants) contain both DPOR and LPOR, whereas angiosperms only contain LPOR (1Fujita Y. Plant Cell Physiol. 1996; 37: 411-421Crossref PubMed Scopus (118) Google Scholar, 3Armstrong G.A. J. Photochem. Photobiol. B Biol. 1998; 43: 87-100Crossref Scopus (120) Google Scholar). Because of the presence of DPOR, photosynthetic bacteria, algae, and gymnosperms are capable of synthesizing bacteriochlorophylls and chlorophylls in the dark, whereas the lack of DPOR in angiosperms makes a requirement for light for Chl synthesis in these cells. Genetic studies of the purple nonsulfur bacterium Rhodobacter capsulatus indicated that three genes, bchL,bchN and bchB, are involved in light-independent Pchlide reduction during biosynthesis of bacteriochlorophyll (6Yang Z. Bauer C.E. J. Bacteriol. 1990; 172: 5001-5010Crossref PubMed Google Scholar, 7Burk D.H. Alberti M. Hearst J.E. J. Bacteriol. 1993; 175: 2414-2422Crossref PubMed Google Scholar, 8Bollivar D.W. Suzuki J.Y. Beaty J.T. Dobrowski J.M. Bauer C.E. J. Mol. Biol. 1994; 237: 622-640Crossref PubMed Scopus (164) Google Scholar). Studies with the cyanobacterium Plectonema boryanum (9Fujita Y. Takahashi Y. Chuganji M. Matsubara H. Plant Cell Physiol. 1992; 33: 81-92Google Scholar, 10Fujita Y. Matsumoto H. Takahashi Y. Matsubara H. Plant Cell Physiol. 1993; 34: 305-314PubMed Google Scholar, 11Fujita Y. Takagi H. Hase T. Plant Cell Physiol. 1996; 37: 313-323Crossref PubMed Scopus (45) Google Scholar) and the green alga Chlamydomonas reinhardtii (12Choquet Y. Rahire M. Girard-Bascou J. Erickson J. Rochaix J.-D. EMBO J. 1992; 11: 1697-1704Crossref PubMed Scopus (65) Google Scholar, 13Suzuki J.Y. Bauer E.C. Plant Cell. 1992; 4: 929-940Crossref PubMed Scopus (101) Google Scholar, 14Li J. Goldschmidt-Clermont M. Timko M.P. Plant Cell. 1993; 5: 1817-1829Crossref PubMed Google Scholar, 15Liu X.-Q. Xu H. Huang C. Plant Mol. Biol. 1993; 23: 297-308Crossref PubMed Scopus (38) Google Scholar) have also demonstrated that these organisms use similar genes for Pchlide reduction during Chl biosynthesis that are called chlL, chlN, and chlB. Analysis of the deduced amino acid sequences surprisingly showed the presence of significant sequence similarity between the putative BchL/ChlL, BchN/ChlN, and BchB/ChlB subunits of DPOR with the NifH, NifD, and NifK subunits of nitrogenase, respectively (1Fujita Y. Plant Cell Physiol. 1996; 37: 411-421Crossref PubMed Scopus (118) Google Scholar, 10Fujita Y. Matsumoto H. Takahashi Y. Matsubara H. Plant Cell Physiol. 1993; 34: 305-314PubMed Google Scholar, 16Fujita Y. Takahashi Y. Shonai F. Ogura Y. Matsubara H. Plant Cell Physiol. 1991; 32: 1093-1106Crossref Scopus (44) Google Scholar, 17Burke D.H. Hearst J.E. Sidow A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7134-7138Crossref PubMed Scopus (129) Google Scholar). Nitrogenase is a well characterized enzyme that consists of two separable components, the Fe-protein (also called dinitrogenase reductase) and the MoFe protein complex that catalyzes the reduction of dinitrogen (N2) to form ammonia (2NH3) (18Howard J.B. Rees D.C. Annu. Rev. Biochem. 1994; 63: 235-264Crossref PubMed Scopus (165) Google Scholar, 19Peters J.W. Fisher K. Dean D.R. Annu. Rev. Microbiol. 1995; 49: 335-366Crossref PubMed Scopus (140) Google Scholar, 20Dean D.R. Bolin J.T. Zheng L. J. Bacteriol. 1993; 175: 6737-6744Crossref PubMed Google Scholar). The Fe-protein complex transfers electrons from ferredoxin to the MoFe protein concomitant with Mg-ATP hydrolysis. This complex is comprised of a dimer of NifH proteins that together form a 4Fe:4S redox cluster that is bridged by two Cys from each subunit. The MoFe protein, which serves as the catalytic site for dinitrogen reduction, is comprised of the α2β2 tetramer of the NifD (α) and NifK (β) proteins. The MoFe protein complex contains two types of metallocenters, an 8Fe:7S cluster (P cluster) held at the interface between the NifD and NifK proteins, as well as a 1Mo:7Fe:9S:1homocitrate cofactor (FeMo cofactor) that is present in each NifD subunit. The P-cluster is thought to mediate electron transfer from the iron-protein complex to the FeMo cofactor that is the catalytic site for dinitrogen reduction. The structural similarity between DPOR and nitrogenase is most evident between the BchL/ChlL and NifH (dinitrogenase reductase) subunits where there is 33% overall identity and 50% similarity (13Suzuki J.Y. Bauer E.C. Plant Cell. 1992; 4: 929-940Crossref PubMed Scopus (101) Google Scholar, 16Fujita Y. Takahashi Y. Shonai F. Ogura Y. Matsubara H. Plant Cell Physiol. 1991; 32: 1093-1106Crossref Scopus (44) Google Scholar, 17Burke D.H. Hearst J.E. Sidow A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7134-7138Crossref PubMed Scopus (129) Google Scholar). Most notable is the fact that a critical feature such as the ATP-binding motif and the two Cys residues that are involved in coordinating the 4Fe:4S cluster are completely conserved among NifH and BchL/ChlL proteins (13Suzuki J.Y. Bauer E.C. Plant Cell. 1992; 4: 929-940Crossref PubMed Scopus (101) Google Scholar, 16Fujita Y. Takahashi Y. Shonai F. Ogura Y. Matsubara H. Plant Cell Physiol. 1991; 32: 1093-1106Crossref Scopus (44) Google Scholar, 17Burke D.H. Hearst J.E. Sidow A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7134-7138Crossref PubMed Scopus (129) Google Scholar). This indicates that the BchL/ChlL proteins might catalyze ATP-dependent transfer of electrons from a reductant, such as ferredoxin, to a catalytic protein complex via the Fe:S center. The amino acid sequences of the N proteins (BchN and ChlN) and B proteins (BchB and ChlB) also exhibits similarity to NifD and NifK, respectively (10Fujita Y. Matsumoto H. Takahashi Y. Matsubara H. Plant Cell Physiol. 1993; 34: 305-314PubMed Google Scholar, 11Fujita Y. Takagi H. Hase T. Plant Cell Physiol. 1996; 37: 313-323Crossref PubMed Scopus (45) Google Scholar). Interestingly, only four of the six Cys residues that are involved in forming the 8Fe:7S P cluster in nitrogenase are conserved in the N and B proteins. This implies that the N and B proteins might instead form a 4Fe:4S redox center. There is also no conservation of the residues that are involved in formation of the FeMo cofactor in nitrogenase indicating that the catalytic site, where Pchlide is reduced is highly diverged from the site in nitrogenase where dinitrogen undergoes reduction (1Fujita Y. Plant Cell Physiol. 1996; 37: 411-421Crossref PubMed Scopus (118) Google Scholar, 10Fujita Y. Matsumoto H. Takahashi Y. Matsubara H. Plant Cell Physiol. 1993; 34: 305-314PubMed Google Scholar). Despite the interesting structural similarity between DPOR and nitrogenase, biochemical analysis of DPOR has not yet been undertaken. The absence of biochemical analysis of DPOR can be traced to the fact that there has been no reliable published procedures for assaying DPOR activity in cell-free extracts of photosynthetic cells. Although there have been a few prior reports of DPOR activity in crude cell-free extracts (21Peschek G.A. Hinterstoisser B. Wastyn M. Kuntner O. Pineau B. Missbichler A. Lang J. J. Biol. Chem. 1989; 264: 11827-11832Abstract Full Text PDF PubMed Google Scholar, 22Peschek G.A. Hinterstoisser B. Pineau B. Missbichler A. Biochem. Biophys. Res. Commun. 1989; 162: 71-78Crossref PubMed Scopus (18) Google Scholar, 23Forreiter C. Apel K. Planta. 1993; 190: 536-545Crossref PubMed Scopus (76) Google Scholar), there has been no independent confirmation of these reports nor any attempts at purification of the DPOR enzyme from these systems. Heterologous expression of DPOR subunits in Escherichia coli has also not resulted in the generation of extracts that exhibit DPOR activity. Because the purple nonsulfur bacterium R. capsulatus naturally expresses and assembles DPOR in an active form, we believed that this organism would provide an ideal system to overexpress and purify DPOR. To perform this analysis, we constructed two R. capsulatus strains, one that overexpresses an S tag fusion derivative of the BchN protein and the other that overexpresses an S tag fusion derivative of BchL. The S tag BchL protein was purified as a single polypeptide by affinity purification, whereas the S tag BchN protein was affinity purified as a 1:1 complex with the BchB protein. DPOR activity was measured in an assay mixture comprised of purified protein fractions, ATP and dithionite. The observed biochemical characteristics of isolated DPOR strongly support “nitrogenase-like” features of this Chl biosynthesis enzyme. The procedure to construct nonreplicable plasmids, pYCSFXN1 and pYCSFXL3, is summarized in Fig. 2, A and C. A chimeric DNA fragment consisting of the puc promoter (24Nickens D.G. Bauer C.E. J. Bacteriol. 1998; 180: 4270-4277Crossref PubMed Google Scholar), S tag (25Kim J.-S. Raines R.T. Protein Sci. 1993; 2: 348-356Crossref PubMed Scopus (182) Google Scholar), and 5′-part of bchN was obtained by an overlap extension method using two-step PCR (26Good L. Nazar R. Nucleic Acids Res. 1992; 20: 4934Crossref PubMed Scopus (53) Google Scholar) (Fig. 2 A). The puc promoter part (corresponding to −216 to −1, Ref. 24Nickens D.G. Bauer C.E. J. Bacteriol. 1998; 180: 4270-4277Crossref PubMed Google Scholar) was amplified with a pair of primers; and and S tag are and double using (24Nickens D.G. Bauer C.E. J. Bacteriol. 1998; 180: 4270-4277Crossref PubMed Google Scholar) as the The 5′-part of bchN from the was amplified with pair of and and S tag are and double using J. Bacteriol. PubMed Google Scholar) as the the the obtained chimeric DNA fragment was with and in and and the of derivative of with the instead of the J.M. 1992; PubMed Scopus Google Scholar), (Fig. 2 A). Because the BchN protein by not have the site for was constructed as A chimeric DNA fragment consisting of the puc S and site was amplified by of and site of and S tag and double using as the The fragment DNA was with and and the of to the (Fig. 2, A and The of bchN and were amplified with pairs of and using as the The of bchN and of were with and in and and the of the pYCSFXN1 and pYCSFXL3, respectively (Fig. 2 nonreplicable in coli in the presence of R. capsulatus was as the to the S bchN The nonreplicable plasmids, pYCSFXN1 and pYCSFXL3, were first to coli which is capable of transfer of the nonreplicable to was by the method Bauer C.E. Mol. Genet. 1989; PubMed Scopus Google Scholar), with on and were for of the puc promoter in of bchN genes by of S tag fusion BchN tag BchN) and BchL tag proteins in the was by analysis using S protein The and expressed S tag BchN and S tag the of the puc promoter (Fig. 2, and R. were in Bauer C.E. Mol. Genet. 1989; PubMed Scopus Google Scholar) of in the dark at with at which that are to the puc promoter (24Nickens D.G. Bauer C.E. J. Bacteriol. 1998; 180: 4270-4277Crossref PubMed Google Scholar). at the of were and and in an and of was to the and the by at for using procedures were in the using that been and in the to which at a was use to The were in and and by for with with a The was to and at for of S was to of and for with for of S tag BchN S tag BchL protein. The S was in of the three by and in of and The S was in of and of at with for the protein was in the was from the by to the at a of was to the to activity of Protein was using a assay with crude and purified proteins were on a gel that was with the sequence analysis, a of of purified BchN and co-purified BchB proteins 2 were a gel with the proteins were a of using Cell to the of the BchN and BchB proteins was and in of each protein was with a of R. capsulatus was in in a at in the dark with at The was by by a Pchlide in the was in of in the was as the on was to by a of The Pchlide was in to of Pchlide was in using the of at M. Photosynth. Res. 4: Scopus Google Scholar). DPOR were in a of ATP, 2 and an of purified The assay were in in the dark for at of the assay mixture was with and the of each was on a of a and Pchlide in the was by the by H. Scholar). The bchB, and genes from R. capsulatus have been to for subunits of DPOR (6Yang Z. Bauer C.E. J. Bacteriol. 1990; 172: 5001-5010Crossref PubMed Google Scholar, 7Burk D.H. Alberti M. Hearst J.E. J. Bacteriol. 1993; 175: 2414-2422Crossref PubMed Google Scholar, 8Bollivar D.W. Suzuki J.Y. Beaty J.T. Dobrowski J.M. Bauer C.E. J. Mol. Biol. 1994; 237: 622-640Crossref PubMed Scopus (164) Google Scholar). of these genes is in the the of which is the of a promoter C.E. Z. Mol. Genet. 1991; PubMed Scopus Google Scholar) (Fig. 2 To purification of DPOR, we to overexpress affinity BchN and BchL genes in R. capsulatus. To this we first constructed the nonreplicable the of which are shown in Fig. 2 A. This has a DNA that contains a promoter for the proteins and the site in with amino acid residues that for an affinity purification S tag sequence and site (Fig. 2 of the site are three and that can be to construct an in fusion of a with the S tag extension To the affinity tag and overexpress the bchN and we first and DNA of the of bchN and genes, and the PCR the of the pYCSFXN1 and pYCSFXL3, respectively (Fig. 2 shown in Fig. 2 of pYCSFXN1 the via single resulted in of the that overexpresses the BchN tag BchN) protein. of resulted in of that overexpresses BchL tag protein of the proteins was observed in cell-free extracts as on an 2 and as well as with analysis using an S protein that to the S tag not that DPOR has structural features similar to nitrogenase, was to perform all procedures such as of protein purification, and assaying for activity using an that a mixture of and The S tag BchL protein was purified from the of cell-free by affinity S tag BchL to S by of protein from the The S tag BchL protein was from the by which BchL protein that contains a acid extension (Fig. 2 shown in the in Fig. the protein is as on a single of which is in with the of for BchL the four amino acid BchN protein was purified from the of using the S and affinity purification procedure as for BchL. However, unlike analysis of purified BchN demonstrated the presence of two proteins of and that are at as by (Fig. The of BchN protein the amino acid extension is whereas the of which is the third DPOR is at To the that BchB is with the affinity BchN we sequence analysis in the and proteins that were a by The sequence analysis indicated that the protein an sequence of which is to the BchB polypeptide as deduced from the sequence and that the protein the sequence which the BchN sequence that is as a of of the S tag extension from S tag BchN (Fig. 2 of BchB with S tag BchN the first for the formation of a complex between the BchN and BchB proteins. the similar of of from BchB and of from BchN during the amino acid sequence analysis indicates that these two proteins form a complex in an the purified proteins DPOR activity by an in assay system that is similar to that to assay for nitrogenase activity PubMed Scopus Google Scholar). this purified protein were of the of the BchL together to an assay mixture that ATP, an ATP system and electron and 2 The reaction was in at for which of the reaction were with to a of to shown by the in Fig. the Pchlide at was in the assay that contain only one of the purified protein (Fig. a and However, both purified protein were to the assay the Pchlide was reduced concomitant with the of a at (Fig. The activity of the DPOR was to be (Fig. also there is a requirement for ATP, as well as a reductant for DPOR activity. shown in no DPOR activity was ATP was from the also observed activity in in which the ATP system was (Fig. indicating that DPOR a of A requirement for was also because no activity was was from the reaction (Fig. that the BchL and proteins subunits of DPOR. also that DPOR consists of two separable components, the and a complex. This is not unlike that observed for nitrogenase that is of dinitrogenase reductase that is separable from the MoFe protein complex. observed for nitrogenase, there is also a requirement for ATP and a reductant for activity. In this study, we have the first of DPOR from a photosynthetic The characterization of this enzyme indicates that contains features in with the of DPOR with purified proteins that DPOR consists of two separable components, the BchL protein and the complex (Fig. was shown to be dependent on ATP, an ATP as well as the reductant dithionite. The characteristics of in DPOR assay system are similar to that of nitrogenase, which also ATP and during in reduction of PubMed Scopus Google Scholar). Although the in reductant for DPOR has not been is that ferredoxin is the most the of structural similarity of BchL to NifH, which is known to electrons from capsulatus six different with ferredoxin a electron to nitrogenase Biophys. Scopus Google Scholar, Y. N. J. C. from to The Scholar). is which ferredoxin as the reductant for DPOR. However, is that one ferredoxin can as an electron because studies have not of that are in Pchlide reduction that to a ferredoxin the of the of the electron to DPOR, there are that can be in studies with isolated DPOR. sequence similarity between DPOR and nitrogenase subunits that the BchL protein in as a dimer that transfers electrons from ferredoxin in to the protein complex in an ATP-dependent also that the protein complex serves as the catalytic site for the Pchlide reduction similar to the MoFe protein complex that is the site of reduction. The of the BchN and BchB proteins in an is with the that the protein a similar to the MoFe protein (Fig. dimer formation by the of a The of ATP and of electrons in the DPOR reaction also to be The reduction of dinitrogen to two of ammonia by nitrogenase of ATP as well as the of of and to the only six and electrons are to one dinitrogen to two of ammonia with the two and electrons for the of be interesting to the of and electron by DPOR that is to catalyze double bond reduction of Pchlide and also be interesting to can in the DPOR Nitrogenase is also capable of a of other such as that in the in nitrogenase as well as other with double such as dinitrogen and PubMed Scopus Google Scholar). is not that a enzyme has that is capable of a double bond in In this be interesting to isolated DPOR is also capable of a of different double that to be is the presence of putative other that be present in the isolated BchL and proteins. we have not yet isolated of the DPOR enzyme to perform analysis for the presence of (Fig. we have observed that both purified protein exhibit a that is with known of proteins that contain an have also observed that DPOR activity is to by not which is a of enzymes such as nitrogenase that contain that are by that to be be present in DPOR. analysis indicates that the three Cys residues in NifD and three Cys residues in NifK that together an 8Fe:7S P cluster are only conserved in the N and B proteins of the conserved Cys residues are present in the N protein and only one conserved Cys is in the B analysis that DPOR be to protein pair known as and The which is thought to a redox role in the FeMo cofactor biosynthesis J.T. Dean D.R. 1998; 37: PubMed Scopus Google Scholar), also has a similar conservation of the P cluster Cys residues in and only one in Dean D.R. J. Bacteriol. PubMed Google J. B. T. H. Mol. Genet. 1990; PubMed Scopus Google Scholar). Studies have shown that contains two instead of the two 8Fe:7S P that are present in J.T. Dean D.R. 1998; 37: PubMed Scopus Google Scholar). There is also no conservation of the Cys and residues that are involved in formation of the active site FeMo cofactor in NifD with B N protein. that the DPOR protein complex most only contains a single pair of not unlike that observed for The of a purification and assay system for DPOR of the of dark Pchlide reduction. The characterization of the structural and biochemical of DPOR to nitrogenase to electron transfer that these of proteins studies also provide as to the between biosynthesis and that during of for analysis of the BchN and BchB proteins. for the and Hase for and critical of the also of the for and