Structure of ADP-aluminium fluoride-stabilized protochlorophyllide oxidoreductase complex.
ABSTRACT: Photosynthesis uses chlorophylls for the conversion of light into chemical energy, the driving force of life on Earth. During chlorophyll biosynthesis in photosynthetic bacteria, cyanobacteria, green algae and gymnosperms, dark-operative protochlorophyllide oxidoreductase (DPOR), a nitrogenase-like metalloenzyme, catalyzes the chemically challenging two-electron reduction of the fully conjugated ring system of protochlorophyllide a. The reduction of the C-17=C-18 double bond results in the characteristic ring architecture of all chlorophylls, thereby altering the absorption properties of the molecule and providing the basis for light-capturing and energy-transduction processes of photosynthesis. We report the X-ray crystallographic structure of the substrate-bound, ADP-aluminium fluoride-stabilized (ADP·AlF(3)-stabilized) transition state complex between the DPOR components L(2) and (NB)(2) from the marine cyanobacterium Prochlorococcus marinus. Our analysis permits a thorough investigation of the dynamic interplay between L(2) and (NB)(2). Upon complex formation, substantial ATP-dependent conformational rearrangements of L(2) trigger the protein-protein interactions with (NB)(2) as well as the electron transduction via redox-active [4Fe-4S] clusters. We also present the identification of artificial "small-molecule substrates" of DPOR in correlation with those of nitrogenase. The catalytic differences and similarities between DPOR and nitrogenase have broad implications for the energy transduction mechanism of related multiprotein complexes that are involved in the reduction of chemically stable double and/or triple bonds.
Project description:Photosynthesis converts solar energy to chemical energy using chlorophylls (Chls). In a late stage of biosynthesis of Chls, dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR), a nitrogenase-like enzyme, reduces the C17 = C18 double bond of Pchlide and drastically changes the spectral properties suitable for photosynthesis forming the parental chlorin ring for Chl a. We previously proposed that the spatial arrangement of the proton donors determines the stereospecificity of the Pchlide reduction based on the recently resolved structure of the DPOR catalytic component, NB-protein. However, it was not clear how the two-electron and two-proton transfer events are coordinated in the reaction. In this study, we demonstrate that DPOR initiates a single electron transfer reaction from a [4Fe-4S]-cluster (NB-cluster) to Pchlide, generating Pchlide anion radicals followed by a single proton transfer, and then, further electron/proton transfer steps transform the anion radicals into chlorophyllide (Chlide). Thus, DPOR is a unique iron-sulphur enzyme to form substrate radicals followed by sequential proton- and electron-transfer steps with the protein folding very similar to that of nitrogenase. This novel radical-mediated reaction supports the biosynthesis of Chl in a wide variety of photosynthetic organisms.
Project description:Chlorophylls (Chls) play pivotal roles in energy absorption and transduction and also in charge separation in reaction centers in all photosynthetic organisms. In Chl biosynthesis steps, only a step for the enzymatic reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) is mediated by both nuclear- and chloroplast-encoded genes in land plants. Many plants encode the genes for light-dependent Pchlide reductase (LPOR) and light-independent Pchlide reductase (DPOR) in the nucleus and chloroplast genome, respectively. During the diversification of land plants, the reduction step of Pchlide to Chlide has become solely dependent on LPOR, and the genes for DPOR have been lost from chloroplast genome. It remains unclear why DPOR persists in some land plants, how they were eliminated from chloroplast genomes during the diversification of land plants, and under what environmental conditions DPOR was required. We demonstrate that DPOR is functional in liverwort (Marchantia polymorpha L.) and plays an important role in Chl biosynthesis. Having established a plastid transformation system in liverwort, we disrupted chlB, which encodes a subunit of DPOR in the M. polymorpha chloroplast genome. Morphological and Chl content analysis of a chlB mutant grown under different photoperiods revealed that DPOR is particularly required for Chl biosynthesis under short-day conditions. Our findings suggest that an environmental condition in the form of photoperiod is an important factor that determines the loss or retention of chloroplast-encoded genes mediating Pchlide reduction to Chlide.
Project description:Nitrogenase-like light-independent protochlorophyllide oxidoreductase (DPOR) is involved in chlorophyll biosynthesis. Bacteriochlorophyll formation additionally requires the structurally related chlorophyllide oxidoreductase (COR). During catalysis, homodimeric subunit BchL(2) or ChlL(2) of DPOR transfers electrons to the corresponding heterotetrameric catalytic subunit, (BchNB)(2) or (ChlNB)(2). Analogously, subunit BchX(2) of the COR enzymes delivers electrons to subunit (BchYZ)(2). Various chimeric DPOR enzymes formed between recombinant subunits (BchNB)(2) and BchL(2) from Chlorobaculum tepidum or (ChlNB)(2) and ChlL(2) from Prochlorococcus marinus and Thermosynechococcus elongatus were found to be enzymatically active, indicating a conserved docking surface for the interaction of both DPOR protein subunits. Biotin label transfer experiments revealed the interaction of P. marinus ChlL(2) with both subunits, ChlN and ChlB, of the (ChlNB)(2) tetramer. Based on these findings and on structural information from the homologous nitrogenase system, a site-directed mutagenesis approach yielded 10 DPOR mutants for the characterization of amino acid residues involved in protein-protein interaction. Surface-exposed residues Tyr(127) of subunit ChlL, Leu(70) and Val(107) of subunit ChlN, and Gly(66) of subunit ChlB were found essential for P. marinus DPOR activity. Next, the BchL(2) or ChlL(2) part of DPOR was exchanged with electron-transferring BchX(2) subunits of COR and NifH(2) of nitrogenase. Active chimeric DPOR was generated via a combination of BchX(2) from C. tepidum or Roseobacter denitrificans with (BchNB)(2) from C. tepidum. No DPOR activity was observed for the chimeric enzyme consisting of NifH(2) from Azotobacter vinelandii in combination with (BchNB)(2) from C. tepidum or (ChlNB)(2) from P. marinus and T. elongatus, respectively.
Project description:Bacteriochlorophyll a biosynthesis requires the stereo- and regiospecific two electron reduction of the C7-C8 double bond of chlorophyllide a by the nitrogenase-like multisubunit metalloenzyme, chlorophyllide a oxidoreductase (COR). ATP-dependent COR catalysis requires interaction of the protein subcomplex (BchX)2 with the catalytic (BchY/BchZ)2 protein to facilitate substrate reduction via two redox active iron-sulfur centers. The ternary COR enzyme holocomplex comprising subunits BchX, BchY, and BchZ from the purple bacterium Roseobacter denitrificans was trapped in the presence of the ATP transition state analog ADP·AlF4(-). Electron paramagnetic resonance experiments revealed a [4Fe-4S] cluster of subcomplex (BchX)2. A second [4Fe-4S] cluster was identified on (BchY/BchZ)2. Mutagenesis experiments indicated that the latter is ligated by four cysteines, which is in contrast to the three cysteine/one aspartate ligation pattern of the closely related dark-operative protochlorophyllide a oxidoreductase (DPOR). In subsequent mutagenesis experiments a DPOR-like aspartate ligation pattern was implemented for the catalytic [4Fe-4S] cluster of COR. Artificial cluster formation for this inactive COR variant was demonstrated spectroscopically. A series of chemically modified substrate molecules with altered substituents on the individual pyrrole rings and the isocyclic ring were tested as COR substrates. The COR enzyme was still able to reduce the B ring of substrates carrying modified substituents on ring systems A, C, and E. However, substrates with a modification of the distantly located propionate side chain were not accepted. A tentative substrate binding mode was concluded in analogy to the related DPOR system.
Project description:A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), catalyzed by dark-operative protochlorophyllide oxidoreductase (DPOR). DPOR is made of electron donor (BchL) and acceptor (BchNB) component proteins. BchNB is further composed of two subunits each of BchN and BchB arranged as an α<sub>2</sub>β<sub>2</sub> heterotetramer with two active sites for substrate reduction. Such oligomeric architectures are found in several other electron transfer (ET) complexes, but how this architecture influences activity is unclear. Here, we describe allosteric communication between the two identical active sites in <i>Rhodobacter sphaeroides</i> BchNB that drives sequential and asymmetric ET. Pchlide binding to one BchNB active site initiates ET from the pre-reduced [4Fe-4S] cluster of BchNB, a process similar to the deficit spending mechanism observed in the structurally related nitrogenase complex. Pchlide binding in one active site is recognized <i>in trans</i> by an Asp-274 from the opposing half, which is positioned to serve as the initial proton donor. A D274A variant DPOR binds to two Pchlide molecules in the BchNB complex, but only one is bound productively, stalling Pchlide reduction in both active sites. A half-active complex combining one WT and one D274A monomer also stalled after one electron was transferred in the WT half. We propose that such sequential electron transfer in oligomeric enzymes serves as a regulatory mechanism to ensure binding and recognition of the correct substrate. The findings shed light on the functional advantages imparted by the oligomeric architecture found in many electron transfer enzymes.
Project description:During (bacterio)chlorophyll biosynthesis of many photosynthetically active organisms, dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the two-electron reduction of ring D of protochlorophyllide to form chlorophyllide. DPOR is composed of the subunits ChlL, ChlN, and ChlB. Homodimeric ChlL(2) bearing an intersubunit [4Fe-4S] cluster is an ATP-dependent reductase transferring single electrons to the heterotetrameric (ChlN/ChlB)(2) complex. The latter contains two intersubunit [4Fe-4S] clusters and two protochlorophyllide binding sites, respectively. Here we present the crystal structure of the catalytic (ChlN/ChlB)(2) complex of DPOR from the cyanobacterium Thermosynechococcus elongatus at a resolution of 2.4 A. Subunits ChlN and ChlB exhibit a related architecture of three subdomains each built around a central, parallel beta-sheet surrounded by alpha-helices. The (ChlN/ChlB)(2) crystal structure reveals a [4Fe-4S] cluster coordinated by an aspartate oxygen alongside three cysteine ligands. Two equivalent substrate binding sites enriched in aromatic residues for protochlorophyllide substrate binding are located at the interface of each ChlN/ChlB half-tetramer. The complete octameric (ChlN/ChlB)(2)(ChlL(2))(2) complex of DPOR was modeled based on the crystal structure and earlier functional studies. The electron transfer pathway via the various redox centers of DPOR to the substrate is proposed.
Project description:Recent studies highlight the diversity and significance of marine phototrophic microorganisms such as picocyanobacteria, phototrophic picoeukaryotes, and bacteriochlorophyll- and rhodopsin-holding phototrophic bacteria. To assess if freshwater ecosystems also harbor similar phototroph diversity, genes involved in the biosynthesis of bacteriochlorophyll and chlorophyll were targeted to explore oxygenic and aerobic anoxygenic phototroph composition in a wide range of lakes. Partial dark-operative protochlorophyllide oxidoreductase (DPOR) and chlorophyllide oxidoreductase (COR) genes in bacteria of seven lakes with contrasting trophic statuses were PCR amplified, cloned, and sequenced. Out of 61 sequences encoding the L subunit of DPOR (L-DPOR), 22 clustered with aerobic anoxygenic photosynthetic bacteria, whereas 39 L-DPOR sequences related to oxygenic phototrophs, like cyanobacteria, were observed. Phylogenetic analysis revealed clear separation of these freshwater L-DPOR genes as well as 11 COR gene sequences from their marine counterparts. Terminal restriction fragment length analysis of L-DPOR genes was used to characterize oxygenic aerobic and anoxygenic photosynthesizing populations in 20 lakes differing in physical and chemical characteristics. Significant differences in L-DPOR community composition were observed between dystrophic lakes and all other systems, where a higher proportion of genes affiliated with aerobic anoxygenic photosynthetic bacteria was observed than in other systems. Our results reveal a significant diversity of phototrophic microorganisms in lakes and suggest niche partitioning of oxygenic and aerobic anoxygenic phototrophs in these systems in response to trophic status and coupled differences in light regime.
Project description:Dark-operative protochlorophyllide oxidoreductase (DPOR) is a key enzyme to produce chlorophyll in the dark. Among photosynthetic eukaryotes, all three subunits chlL, chlN, and chlB are encoded by plastid genomes. In some gymnosperms, two codons of chlB mRNA are changed by RNA editing to codons encoding evolutionarily conserved amino acid residues. However, the effect of these substitutions on DPOR activity remains unknown. We first prepared cyanobacterial ChlB variants with amino acid substitution(s) to mimic ChlB translated from pre-edited mRNA. Their activities were evaluated by measuring chlorophyll content of dark-grown transformants of a chlB-lacking mutant of the cyanobacterium Leptolyngbya boryana that was complemented with pre-edited mimic chlB variants. The chlorophyll content of the transformant cells expressing the ChlB variant from the fully pre-edited mRNA was only one-fourth of the control cells. Co-purification experiments of ChlB with Strep-ChlN suggested that a stable complex with ChlN is greatly impaired in the substituted ChlB variant. We then confirmed that RNA editing efficiency was markedly greater in the dark than in the light in cotyledons of the black pine Pinus thunbergii. These results indicate that RNA editing on chlB mRNA is important to maintain appropriate DPOR activity in black pine chloroplasts.
Project description:Plant photosynthesis relies on the capacity of chlorophylls and carotenoids to absorb light. One of the roles of carotenoids is to harvest green-blue light and transfer the excitation energy to the chlorophylls. The corresponding dynamics were investigated here for the first time, to our knowledge, in the CP26 and CP24 minor antenna complexes. The results for the two complexes differ substantially. In CP26 fast transfer (80 fs) occurs from the carotenoid S(2) state to chlorophylls a absorbing at 675 and 678 nm, whereas transfer from the hot S(1) state to the lowest energy chlorophylls is observed in <1 ps. In CP24, energy transfer from the S(2) state leads in 80 fs to the population of chlorophylls b and high-energy chlorophylls a absorbing at 670 nm, whereas the low-energy chlorophylls a are populated only in several picoseconds. The results suggest that CP26 has a structural and functional organization similar to that of LHCII, whereas CP24 differs substantially from the other Lhc complexes, especially regarding the lutein L1 binding domain. No energy transfer from the carotenoid S(1) state to chlorophylls was observed in either complex, suggesting that this state is energetically below the chlorophyll Qy state and therefore may play a role in the quenching of chlorophyll excitations.
Project description:There exists a wealth of means of efficient utilization of solar energy in nature, with photosynthesis of chlorophylls as a prime example. Separately, artificially structured plasmonic materials are versatile in light harvesting and energy conversion. Using a simple and scalable design of near-percolating silver nanostructures, we demonstrate that the light-harvesting efficiency of chlorophylls can be drastically enhanced by tuning the plasmon frequency of the constituent silver nanoparticles to coincide with the maximal photon flux of sunlight. In particular, we show that the photon upconversion efficiency can be readily enhanced by over 20 folds, with the room-temperature fluorescence quantum yield increased by a factor of 2.63. The underlying mechanism for the upconversion enhancement is attributed to a one-electron-per-photon anti-Stokes process, involving absorption of a characteristic phonon mode of the chlorophylls. These findings suggest that chlorophylls can serve as molecular building blocks for high-efficiency light harvesting and solar energy conversion.