Protochlorophyllide Reductase Research Articles

Discovery of Evolutionary Divergence of Biological Nitrogen Fixation and Photosynthesis: Fine Tuning of Biogenesis of the NifH and the ChlL by a Peptidyl-Prolyl <I>Cis/Trans</I> Isomerase

Problem statement: Despite the structural and functional similarities between the nitrogenase that performs biological nitrogen fixation reaction and the Dark Protochlorphyllide Oxidoreductase (DPOR) that performs chlorophyll-biosynthesis, attempts to substitute nitrogenase-components with DPOR-components have hitherto failed. This investigation was undertaken to test if Chlamydomonas reinhardtii protochlorophyllide (Pchlide) reductase (ChlL) that shares some structural similarity with Nitrogenase Reductase (NifH) could complement the functions of NifH in biological nitrogen fixation of Azotobacter vinelandii. Approach: Genetic complementation studies were performed to test if the chlL gene and its mutants cloned under transcriptional control of nifH promoter (nifHp) in a broad-host range low copy plasmid pBG1380 could render a Nif+ phenotype to NifH-deficient A. vinelandii strains. Results: Expression of ChlL could render Nif+ phenotype to NifH-deficient A. vinelandii only in the absence of NifM, a nif-specific PPIase essential for biogenesis of NifH. The ChlL mutants Cys95Thr and Cys129Thr were unable to substitute for NifH. Thus, the conserved cysteine ligands of [4Fe-4S] cluster in ChlL are essential for successful substitution of NifH by ChlL. Since C-termini of NifH and ChlL demonstrated the least similarity and Pro258, a substrate for the PPIase activity of NifM, is located in the C-terminus of NifH, we posited that replacing the C-terminus of NifH with that of ChlL would render NifM-independence to NifH. The NifH-ChlL chimera could support the growth of NifH- and NifM-deficient A. vinelandii in nitrogen limiting conditions implying that it has acquired NifM-independence. Conclusion/Recommendations: Collectively, these observations suggest that NifM, an evolutionarily conserved nif-specific PPIase, could have contributed to the functional divergence of biological nitrogen fixation and photosynthesis during evolution by virtue of its ability to exert opposing effects on structurally similar substrates, ChlL and NifH.

Environmental constraints defining the distribution, composition, and evolution of chlorophototrophs in thermal features of Yellowstone National Park

Chlorophotoautotrophy, the use of chlorophylls to convert light energy into chemical energy for carbon dioxide fixation, is the primary metabolic process linking the inorganic and organic carbon pools on Earth. To understand the potential effects of various environmental constraints on the evolution of chlorophototrophy better, we studied the distribution, diversity, and abundance of chlorophylls and genes involved in their synthesis along geothermal gradients in Yellowstone National Park, Wyoming. Genes involved in chlorophyll biosynthesis were constrained to temperatures of less than ~70 °C and were only detected at this elevated temperature when the pH was in the circumneutral to alkaline range. The upper temperature limit for the detection of chlL/bchL(1) and bchY(2) decreased systematically with increasingly acidic pH, an observation likely attributable to sulfide, which upon oxidation, generates acidic spring water and reduces the availability of bicarbonate the preferred source of inorganic carbon for phototrophs. Spring pH was also the best predictor of the phylogenetic diversity of chlL/bchL communities. The phylogenetic similarity of chlL/bchL genes between sites was significantly correlated with that of chlorophylls. The predominance of chlorophyll a and bacteriochlorophyll a among extracted pigments was consistent with predominance of chlL/bchL genes affiliated with the Cyanobacteria and Chloroflexiales, respectively, and might be related to the fact that the majority of these organisms are photoautotrophs. Together, these results suggest that a combination of temperature, pH, and/or sulfide influences the distribution, diversity, and evolution of chlorophotrophs and the chlorophylls that they synthesize.

クロロフィル生合成系酵素と窒素固定酵素の共通構造基盤

Dark-operative protochlorophyllide reductase (DPOR) catalyses the reduction of protochlorophyllide (Pchlide) to chlorophyllide a, which is a key step in the chlorophyll biosynthesis pathway. DPOR is a nitrogenase-like enzyme consisting of two components, BchL and BchNB, which are structurally related to nitrogenase NifH and NifDK, respectively. We determined the crystal structure of the catalytic component of DPOR, BchNB, in Pchlide-bound and Pchlide-free forms. BchNB has a novel FeS cluster (NB-cluster) coordinated uniquely by one aspartate and three cysteines. NB-cluster is located at the spatial position corresponding to an electron mediating FeS cluster, P-cluster, in nitrogenase NifDK. A Pchlide molecule found in the Pchlide-bound form is accommodated in the cavity surrounded by many hydrophobic residues. We propose a unique trans-specific reduction mechanism by comparison between the Pchlide-bound and the Pchlide-free forms. The spatial arrangement of the NB-cluster and Pchlide is almost identical to that of the P-cluster and FeMo-cofactor in nitrogenase NifDK, suggesting that a common architecture exists to reduce chemically stable multibonds of porphyrin and dinitrogen.

EPR study of 1Asp–3Cys ligated 4Fe–4S iron–sulfur cluster in NB-protein (BchN–BchB) of a dark-operative protochlorophyllide reductase complex

Dark-operative protochlorophyllide oxidoreductase, a nitrogenase-like enzyme, contains two [4Fe–4S] clusters, one in the L-protein ((BchL)2) and the other in the NB-protein ((BchN–BchB)2). The reduced NB-cluster in the NB-protein, which is ligated by 1Asp/3Cys residues, showed a broad S=3/2 electron paramagnetic resonance signal that is rather rare in [4Fe–4S] clusters. A 4Cys-ligated NB-cluster in the mutated variant BchB–D36C protein, in which the Asp36 was replaced by a Cys, gave a rhombic normal S=1/2 signal and lost the catalytic activity. The results suggest that Asp36 contributes to the low redox potential necessary to reduce protochlorophyllide.

Stress responsive gene CIPK14 is involved in phytochrome A-mediated far-red light inhibition of greening in Arabidopsis

In this study, we show that CIPK14, a stress responsive CBL-interacting protein kinase gene, is involved in phytochrome A-mediated far-red light inhibition of greening in Arabidopsis seedlings. The CIPK14-impairment mutant cipk14 grown in continuous far-red (FR) light did not show greening when exposed to white light illumination for 15 h. By contrast, the FR-grown phytochrome A null mutant phyA greened within 0.5 h of exposure to white light. Although greening of Col-4 (wild-type) was not completely abolished by FR, it exhibited a significantly decreased greening capacity compared with that of phyA. Further analyses demonstrated that the expression of protochlorophyllide reductase (POR) genes was correlated with the greening ability of the genotypes. In addition, CIPK14 appeared to be regulated by both the circadian clock and PhyA. Taken together, these results suggest that CIPK14 plays a role in PhyA-mediated FR inhibition of seedling greening, and that a Ca-related kinase may be involved in a previously undefined branch point in the phytochrome A signaling pathway.

Structure of protochlorophyllide reductase: a greening mechanism for plants in the dark

Structure of protochlorophyllide reductase: a greening mechanism for plants in the dark

AT_CHLORO, a Comprehensive Chloroplast Proteome Database with Subplastidial Localization and Curated Information on Envelope Proteins

Recent advances in the proteomics field have allowed a series of high throughput experiments to be conducted on chloroplast samples, and the data are available in several public databases. However, the accurate localization of many chloroplast proteins often remains hypothetical. This is especially true for envelope proteins. We went a step further into the knowledge of the chloroplast proteome by focusing, in the same set of experiments, on the localization of proteins in the stroma, the thylakoids, and envelope membranes. LC-MS/MS-based analyses first allowed building the AT_CHLORO database (http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/), a comprehensive repertoire of the 1323 proteins, identified by 10,654 unique peptide sequences, present in highly purified chloroplasts and their subfractions prepared from Arabidopsis thaliana leaves. This database also provides extensive proteomics information (peptide sequences and molecular weight, chromatographic retention times, MS/MS spectra, and spectral count) for a unique chloroplast protein accurate mass and time tag database gathering identified peptides with their respective and precise analytical coordinates, molecular weight, and retention time. We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). These data together with an in-depth investigation of the literature were compiled to provide accurate subplastidial localization of previously known and newly identified proteins. A unique knowledge base containing extensive information on the proteins identified in envelope fractions was thus obtained, allowing new insights into this membrane system to be revealed. Altogether, the data we obtained provide unexpected information about plastidial or subplastidial localization of some proteins that were not suspected to be associated to this membrane system. The spectral counting-based strategy was further validated as the compartmentation of well known pathways (for instance, photosynthesis and amino acid, fatty acid, or glycerolipid biosynthesis) within chloroplasts could be dissected. It also allowed revisiting the compartmentation of the chloroplast metabolism and functions.

Action Spectra of Chlorophyll a Biosynthesis in Cyanobacteria: Dark-Operative Protochlorophyllide Oxidoreductase-Deficient Mutants

Both light-dependent and light-independent (dark) protochlorophyllide (Pchlide) reductase account for catalyzing the reduction of Pchlide to chlorophyllide during the biosynthesis of Mg-tetrapyrrole pigments in cyanobacteria. To gain more insight into the interaction between the wavelength of the light and these two chlorophyll synthetic pathways in Synechocystis sp. PCC 6803, the spectral effectiveness of the formation of chlorophyll a was investigated during the regreening process in chlL(-) and chlN(-) mutants, which could not synthesize chlorophyll during growth in the dark. The action spectra showed obvious maxima around 450 nm and 650 nm, similar to those of higher plants except that the intensities of two peaks are reversed. The mRNA levels of chlL and chlN and chlorophyll a content under different wavelengths of light in the wild-type strain were also measured. The RT-PCR analysis revealed that the transcripts of chlL and chlN were up-regulated in red light but simultaneously down-regulated in green light which resulted in corresponding changes of the chlorophyll content. This fact indicates that the regulation of dark-operative protochlorophyllide oxidoreductase (DPOR) in the transcriptional level is essential for cyanobacteria to synthesize appropriate chlorophyll for acclimating in various light colour environments.

光依存型（暗所作動型）プロトクロロフィリド還元酵素のX線構造解析

光依存型（暗所作動型）プロトクロロフィリド還元酵素のX線構造解析

Crystal Structure of the L Protein of Rhodobacter sphaeroides Light-Independent Protochlorophyllide Reductase with MgADP Bound: A Homologue of the Nitrogenase Fe Protein

The L protein (BchL) of the dark-operative protochlorophyllide reductase (DPOR) from Rhodobacter sphaeroides has been purified from an Azotobacter vinelandii expression system; its interaction with nucleotides has been examined, and the X-ray structure of the protein has been determined with bound MgADP to 1.6 A resolution. DPOR catalyzes the reduction of protochlorophyllide to chlorophyllide, a reaction critical to the biosynthesis of bacteriochlorophylls. The DPOR holoenzyme is comprised of two component proteins, the dimeric BchL protein and the heterotetrameric BchN/BchB protein. The DPOR component proteins share significant overall similarities with the nitrogenase Fe protein (NifH) and the MoFe (NifDK) protein, the enzyme system responsible for reduction of dinitrogen to ammonia. Here, BchL was expressed in A. vinelandii and purified to homogeneity using an engineered polyhistidine tag. The purified, recombinant BchL was found to contain 3.6 mol of Fe/mol of BchL homodimer, consistent with the presence of a [4Fe-4S] cluster and analogous to the [4Fe-4S] cluster present in the Fe protein. The MgATP- and MgADP-induced conformational changes in BchL were examined by an Fe chelation assay and found to be distinctly different from the nucleotide-stimulated Fe release observed for the Fe protein. The recombinant BchL was crystallized with bound MgADP, and the structure was determined to 1.6 A resolution. BchL is found to share overall structural similarity with the nitrogenase Fe protein, including the subunit bridging [4Fe-4S] cluster and nucleotide binding sites. Despite the high level of structural similarity, however, BchL is found to be incapable of substituting for the Fe protein in a nitrogenase substrate reduction assay. The newly determined structure of BchL and its comparison to its close homologue, the nitrogenase Fe protein, provide the basis for understanding how these highly related proteins can discriminate between their respective functions in microbial systems where each must function simultaneously.

Structure of protochlorophyllide reductase reveals a mechanism for greening in the dark

Structure of protochlorophyllide reductase reveals a mechanism for greening in the dark

Structure of Protochlorophyllide Reductase Reveals a Mechanism for Greening in the Dark

Structure of Protochlorophyllide Reductase Reveals a Mechanism for Greening in the Dark

NB-protein (BchN–BchB) of dark-operative protochlorophyllide reductase is the catalytic component containing oxygen-tolerant Fe–S clusters

Dark-operative protochlorophyllide (Pchlide) oxidoreductase is a nitrogenase-like enzyme consisting of the two components, L-protein (BchL-dimer) and NB-protein (BchN–BchB-heterotetramer). Here, we show that NB-protein is the catalytic component with Fe–S clusters. NB-protein purified from Rhodobacter capsulatus bound Pchlide that was readily converted to chlorophyllide a upon the addition of L-protein and Mg-ATP. The activity of NB-protein was resistant to the exposure to air. A Pchlide-free form of NB-protein purified from a bchH-lacking mutant showed an absorption spectrum suggesting the presence of Fe–S centers. Together with the Fe and sulfide contents, these findings suggested that NB-protein carries two oxygen-tolerant [4Fe–4S] clusters.

NifH: Structural and Mechanistic Similarities with Proteins Involved in Diverse Biological Processes

The NifH protein is a subunit of the nitrogenase enzyme that catalyzes the reduction of atmospheric nitrogen to ammonia. This protein contains highly conserved regions including the nucleotide binding sites, metal center ligands and the Switch I and Switch II domains. A number of proteins have structural and mechanistic similarities as well as evolutionary relationships with the NifH protein, notable among them being: light independent protochlorophyllide (Pchlide) reductase (ChlL/FrxC or bChL), arsenite pump ATPase (ArsA), 2-hydroxyglutaryl dehydratase Component A (CompA) involved in glutamate degradation and MinD that functions in spatial regulation of cell division. Although involved in very diverse biological processes, these proteins share an underlying common structural framework. This review mainly focuses on the structural similarities of these proteins with the NifH protein and discusses recent reports of complementation studies involving NifH and few of the proteins mentioned.

Characterization of a Plant-like Protochlorophyllide a Divinyl Reductase in Green Sulfur Bacteria

The green sulfur bacterium Chlorobium tepidum synthesizes three types of (bacterio)chlorophyll ((B)Chl): BChl a(P), Chl a(PD), and BChl c(F). During the synthesis of all three molecules, a C-8 vinyl substituent is reduced to an ethyl group, and in the case of BChl c(F), the C-8(2) carbon of this ethyl group is subsequently methylated once or twice by the radical S-adenosylmethionine enzyme BchQ. The C. tepidum genome contains homologs of two genes, bchJ (CT2014) and CT1063, that are highly homologous to genes, bchJ and AT5G18660, and that have been reported to encode C-8 vinyl reductases in Rhodobacter capsulatus and Arabidopsis thaliana, respectively. To determine which gene product actually encodes a C-8 vinyl reductase activity, the bchJ and CT1063 genes were insertionally inactivated in C. tepidum. All three Chls synthesized by the CT1063 mutant of C. tepidum have a C-8 vinyl group. Using NADPH but not NADH as reductant, recombinant BciA reduces the C-8 vinyl group of 3,8-divinyl-protochlorophyllide in vitro. These data demonstrate that CT1063, renamed bciA, encodes a C-8 divinyl reductase in C. tepidum. The bchJ mutant produces detectable amounts of Chl a(PD), BChl a(P), and BChl c(F), all of which have reduced C-8 substituents, but the mutant cells secrete large amounts of 3,8-divinyl-protochlorophyllide a into the growth medium and have a greatly reduced BChl c(F) content. The results suggest that BchJ may play an important role in substrate channeling and/or regulation of Chl biosynthesis but show that it is not a vinyl reductase. Because only some Chl-synthesizing organisms possess homologs of bciA, at least two types of C-8 vinyl reductases must occur.

Nitrogenase Fe protein-like Fe–S cluster is conserved in L-protein (BchL) of dark-operative protochlorophyllide reductase from Rhodobacter capsulatus

Dark-operative protochlorophyllide reductase (DPOR) in bacteriochlorophyll biosynthesis is a nitrogenase-like enzyme consisting of L-protein (BchL-dimer) as a reductase component and NB-protein (BchN–BchB-heterotetramer) as a catalytic component. Metallocenters of DPOR have not been identified. Here we report that L-protein has an oxygen-sensitive [4Fe–4S] cluster similar to nitrogenase Fe protein. Purified L-protein from Rhodobacter capsulatus showed absorption spectra and an electron paramagnetic resonance signal indicative of a [4Fe–4S] cluster. The activity quickly disappeared upon exposure to air with a half-life of 20 s. These results suggest that the electron transfer mechanism is conserved in nitrogenase Fe protein and DPOR L-protein.

Differential Operation of Dual Protochlorophyllide Reductases for Chlorophyll Biosynthesis in Response to Environmental Oxygen Levels in the Cyanobacterium Leptolyngbya boryana

Most oxygenic phototrophs, including cyanobacteria, have two structurally unrelated protochlorophyllide (Pchlide) reductases in the penultimate step of chlorophyll biosynthesis. One is light-dependent Pchlide reductase (LPOR) and the other is dark-operative Pchlide reductase (DPOR), a nitrogenase-like enzyme assumed to be sensitive to oxygen. Very few studies have been conducted on how oxygen-sensitive DPOR operates in oxygenic phototrophic cells. Here, we report that anaerobic conditions are required for DPOR to compensate for the loss of LPOR in cyanobacterial cells. An LPOR-lacking mutant of the cyanobacterium Leptolyngbya boryana (formerly Plectonema boryanum) failed to grow in high light conditions and this phenotype was overcome by cultivating it under anaerobic conditions (2% CO(2)/N(2)). The critical oxygen level enabling the mutant to grow in high light was determined to be 3% (v/v). Oxygen-sensitive Pchlide reduction activity was successfully detected as DPOR activity in cell-free extracts of anaerobically grown mutants, whereas activity was undetectable in the wild type. The content of two DPOR subunits, ChlL and ChlN, was significantly increased in mutant cells compared with wild type. This suggests that the increase in subunits stimulates the DPOR activity that is protected efficiently from oxygen by anaerobic environments, resulting in complementation of the loss of LPOR. These results provide important concepts for understanding how dual Pchlide reductases operate differentially in oxygenic photosynthetic cells grown under natural environments where oxygen levels undergo dynamic changes. The evolutionary implications of the coexistence of two Pchlide reductases are discussed.

The First Catalytic Step of the Light-driven Enzyme Protochlorophyllide Oxidoreductase Proceeds via a Charge Transfer Complex

In chlorophyll biosynthesis protochlorophyllide reductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide) to chlorophyllide, providing a rare opportunity to trap and characterize catalytic intermediates at low temperatures. Moreover, the presence of a chlorophyll-like molecule allows the use of EPR, electron nuclear double resonance, and Stark spectroscopies, previously used for the analysis of photosynthetic systems, to follow catalytic events in the active site of POR. Different models involving the formation of either radical species or charge transfer complexes have been proposed for the initial photochemical step, which forms a nonfluorescent intermediate absorbing at 696 nm (A696). Our EPR data show that the concentration of the radical species formed in the initial photochemical step is not stoichiometric with conversion of substrate. Instead, a large Stark effect, indicative of charge transfer character, is associated with A696. Two components were required to fit the Stark data, providing clear evidence that charge transfer complexes are formed during the initial photochemistry. The temperature dependences of both A696 formation and NADPH oxidation are identical, and we propose that formation of the A696 state involves hydride transfer from NADPH to form a charge transfer complex. A catalytic mechanism of POR is suggested in which Pchlide absorbs a photon, creating a transient charge separation across the C-17-C-18 double bond, which promotes ultrafast hydride transfer from the pro-S face of NADPH to the C-17 of Pchlide. The resulting A696 charge transfer intermediate facilitates transfer of a proton to the C-18 of Pchlide during the subsequent first "dark" reaction.

Characterization of Three Genes Encoding the Subunits of Light-Independent Protochlorophyllide Reductase in Chlorella protothecoides CS-41

Light is necessary for hydrogenation of the D ring of protochlorophyllide leading to chlorophyllide formation in higher plants (light-dependent pathway), but it is not essential in phototrophic bacteria (dark pathway). Both pathways, however, occur in some algae, mosses, ferns, and gymnosperms, and each chloroplast genome of these organisms contains three genes, chlL, chlN, and chlB, encoding the three subunits of light-independent protochlorophyllide reductase (LIPOR) required for protochlorophyllide reduction in the dark. In this study, the three LIPOR genes chlL, chlN, and chlB were cloned from the chloroplast of Chlorella protothecoides CS-41 (CSIRO), which grew heterotrophically with considerable chlorophyll yield. Phylogenetic analysis of ChlL/BchL showed that C. protothecoides CS-41 and Chlorella vulgaris C-27 were closely related. Alignment of their amino acid sequences demonstrated that the conserved domains, including the ATP-binding motif and the Fe-S binding motif in the three subunits, were similar to those in nitrogenases. The three-dimensional structural model of ChlL revealed a hypothetical Fe-S center for redox control. Results from RT-PCR amplification indicated that the chlL gene in C. protothecoides contained a 951-bp intron, and the splicing catalytic core structure was similar to that of the light-regulated intron in the psbA gene of Chlamydomonas. The three genes were expressed in E. coli BL21. The sizes of ChlL, ChlN, and ChlB were estimated to be 38, 49, and 58 kDa, respectively, based on the SDS-PAGE analysis.

A Second Nitrogenase-like Enzyme for Bacteriochlorophyll Biosynthesis: RECONSTITUTION OF CHLOROPHYLLIDE a REDUCTASE WITH PURIFIED X-PROTEIN (BchX) AND YZ-PROTEIN (BchY-BchZ) FROM RHODOBACTER CAPSULATUS

In most photosynthetic organisms, the chlorin ring structure of chlorophyll a is formed by the reduction of the porphyrin D-ring by the dark-operative nitrogenase-like enzyme, protochlorophyllide reductase (DPOR). Subsequently, the chlorin B-ring is reduced in bacteriochlorophyll biosynthesis to form a bacteriochlorin ring structure. Phenotypic analysis of mutants lacking one of three genes, bchX, bchY, or bchZ, which show significant sequence similarity to the structural genes of nitrogenase, suggests that a second nitrogenase-like enzyme is involved in the chlorin B-ring reduction. However, there is no biochemical evidence for this. Here, we report the reconstitution of chlorophyllide a reductase (COR) with purified proteins. Two Rhodobacter capsulatus strains that overexpressed Strep-tagged BchX and BchY were isolated. Strep-tagged BchX was purified as a single polypeptide, and BchZ was co-purified with Strep-tagged BchY. When BchX and BchY-BchZ components were incubated with chlorophyllide a, ATP, and dithionite under anaerobic conditions, chlorophyllide a was converted to a new pigment with a Qy band of longer wavelength at 734 nm (P734) in 80% acetone. The formation of P734 was dependent on ATP and dithionite. High performance liquid chromatography and mass spectroscopic analysis indicated that P734 is 3-vinyl bacteriochlorophyllide a, which is formed by the B-ring reduction of chlorophyllide a. These results demonstrate that the B-ring of chlorin is reduced by a second nitrogenase-like enzyme and that the sequential actions of two nitrogenase-like enzymes, DPOR and COR, convert porphyrin to bacteriochlorin. The evolutionary implications of nitrogenase-like enzymes to determine the ring structure of (bacterio)chlorophyll pigments are discussed.