In vitro-mutagenesis of NADPH:protochlorophyllide oxidoreductase B: two distinctive protochlorophyllide binding sites participate in enzyme catalysis and assembly

NADPH:protochlorophyllide oxidoreductase (POR) B is a key enzyme for the light-induced greening of etiolated angiosperm plants. It is nucleus-encoded, imported into the plastids posttranslationally, and assembled into larger light-harvesting POR:protochlorophyllide complexes termed LHPP (Reinbothe et al., Nature 397:80–84, 1999). An in vitro-mutagenesis approach was taken to study the role of the evolutionarily conserved Cys residues in pigment binding. Four Cys residues are present in the PORB of which two, Cys276 and Cys303, established distinct pigment binding sites, as shown by biochemical tests, protein import studies, and in vitro-reconstitution experiments. While Cys276 constituted the Pchlide binding site in the active site of the enzyme, Cys303 established a second, low affinity pigment binding site that was involved in the assembly and stabilization of imported PORB enzyme inside etioplasts.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Sub-etioplast localization of the enzyme NADPH: protochlorophyllide oxidoreductase

The complete nucleotide sequence of the influenza A/PR/8/34 nucleoprotein gene was determined after cloning for dsDNA copy in pBR322. The nucleoprotein gene is 1517 nucleotides long of which 1446 nucleotides code for 482 amino acids. The calculated amino acid composition is in good agreement with those published for influenza A nucleoprotein genes. The amino acid sequence of the nucleoprotein contains clusters of basic amino acids and proline, a property shared with other nucleic-acid-associated proteins like Semliki forest virus nucleocapsid protein, VP1 protein of polyoma virus and Simian virus 40, and the core antigen of hepatitis B virus. The described nucleoprotein structure brings the number of sequenced genes of influenza A/PR/8/34 to five out of eight genes.     

Purification of the enzyme NADPH: protochlorophyllide oxidoreductase.

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.     

ATP-driven reduction by dark-operative protochlorophyllide oxidoreductase from Chlorobium tepidum mechanistically resembles nitrogenase catalysis

During chlorophyll and bacteriochlorophyll biosynthesis in gymnosperms, algae, and photosynthetic bacteria, dark-operative protochlorophyllide oxidoreductase (DPOR) reduces ring D of aromatic protochlorophyllide stereospecifically to produce chlorophyllide. We describe the heterologous overproduction of DPOR subunits BchN, BchB, and BchL from Chlorobium tepidum in Escherichia coli allowing their purification to apparent homogeneity. The catalytic activity was found to be 3.15 nmol min(-1) mg(-1) with K(m) values of 6.1 microm for protochlorophyllide, 13.5 microm for ATP, and 52.7 microm for the reductant dithionite. To identify residues important in DPOR function, 21 enzyme variants were generated by site-directed mutagenesis and investigated for their metal content, spectroscopic features, and catalytic activity. Two cysteine residues (Cys(97) and Cys(131)) of homodimeric BchL(2) are found to coordinate an intersubunit [4Fe-4S] cluster, essential for low potential electron transfer to (BchNB)(2) as part of the reduction of the protochlorophyllide substrate. Similarly, Lys(10) and Leu(126) are crucial to ATP-driven electron transfer from BchL(2). The activation energy of DPOR electron transfer is 22.2 kJ mol(-1) indicating a requirement for 4 ATP per catalytic cycle. At the amino acid level, BchL is 33% identical to the nitrogenase subunit NifH allowing a first tentative structural model to be proposed. In (BchNB)(2), we find that four cysteine residues, three from BchN (Cys(21), Cys(46), and Cys(103)) and one from BchB (Cys(94)), coordinate a second inter-subunit [4Fe-4S] cluster required for catalysis. No evidence for any type of molybdenum-containing cofactor was found, indicating that the DPOR subunit BchN clearly differs from the homologous nitrogenase subunit NifD. Based on the available data we propose an enzymatic mechanism of DPOR.     

Ultrafast enzymatic reaction dynamics in protochlorophyllide oxidoreductase

The reaction catalyzed by the light-driven enzyme protochlorophyllide oxidoreductase (POR) has been initiated with a 50-fs laser pulse. We show that the catalytic mechanism, involving proton and hydride transfers, proceeds with time constants of 3 ps and 400 ps. It is known that catalysis by POR involves thermally excited protein dynamics; our results show that these molecular motions occur on an ultrafast timescale.     

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.     

POR C of Arabidopsis thaliana: a third light- and NADPH-dependent protochlorophyllide oxidoreductase that is differentially regulated by light

During the sequencing of the genome of Arabidopsis thaliana a gene has been identified that encodes a novel NADPH-protochlorophyllide oxidoreductase (POR)-like protein (accession number AC 002560). This protein has been named POR C. We have expressed the POR C protein in Escherichia coli and have determined its in vitro activity. POR C shows the characteristics of a light-dependent and NADPH-requiring POR similar to POR A and POR B. The expression of the POR C gene differs markedly from that of the POR A and POR B genes. In contrast to the POR A and POR B mRNAs, the POR C mRNA has been shown previously to accumulate only after the beginning of illumination. In light-adapted mature plants only POR B and POR C mRNAs were detectable. The amounts of both mRNAs show pronounced diurnal rhythmic fluctuations. While the oscillations of POR B mRNA are under the control of the circadian clock, those of POR C mRNA are not. Another difference between POR B and POR C was found in seedlings that were grown under continuous white light. The concentration of POR C mRNA rapidly declined and soon dropped beyond the limit of detection, after these seedlings were transferred to the dark. On the other hand, POR B mRNA was unaffected by this light/dark shift. When seedlings were exposed to different light intensities, the amounts of POR B mRNA remained the same, while POR A and POR C mRNAs were modulated in an inverse way by these light intensity changes. POR A mRNA was still detectable in seedlings grown under low light intensities but disappeared at higher light intensities, while the mRNA concentration of POR C rose with increasing light intensities. These different responses to light suggest that the functions of the three PORs of Arabidopsis are not completely redundant, but may allow the plant to adapt its needs for chlorophyll biosynthesis more selectively by using preferentially one of the three enzymes under a given light regime.     

Substrate-dependent transport of the NADPH:protochlorophyllide oxidoreductase into isolated plastids.

The key regulatory enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR), is a nuclear-encoded plastid protein. Its post-translational transport into plastids is determined by its substrate. The precursor of POR (pPOR) is taken up and processed to mature size by plastids only in the presence of protochlorophyllide (Pchlide). In etioplasts, the endogenous level of Pchlide saturates the demands for pPOR translocation. During the light-induced transformation of etioplasts into chloroplasts, the Pchlide concentration declined drastically, and isolated chloroplasts rapidly lost the ability to import the precursor enzyme. The chloroplasts' import capacity for the pPOR, however, was restored when their intraplastidic level of Pchlide was raised by incubating the organelles in the dark with delta-aminolevulinic acid, a common precursor of tetrapyrroles. Additional evidence for the involvement of intraplastidic Pchlide in regulating the transport of pPOR into plastids was provided by experiments in which barley seedlings were grown under light/dark cycles. The intraplastidic Pchlide concentration in these plants underwent a diurnal fluctuation, with a minimum at the end of the day and a maximum at the end of the night period. Chloroplasts isolated at the end of the night translocated pPOR, whereas those isolated at the end of the day did not. Our results imply that the Pchlide-dependent transport of the pPOR into plastids might be part of a novel regulatory circuit by which greening plants fine tune both the enzyme and pigment levels, thereby avoiding the wasteful degradation of the imported pPOR as well as photodestruction of free Pchlide.     

Prolamellar bodies formed by cyanobacterial protochlorophyllide oxidoreductase in Arabidopsis

In angiosperms, chlorophyll biosynthesis is light dependent. A key factor in this process is protochlorophyllide oxidoreductase (POR), which requires light to catalyze the reduction of protochlorophyllide to chlorophyllide. It is believed that this protein originated from an ancient cyanobacterial enzyme that was introduced into proto‐plant cells during the primary symbiosis. Here we report that PORs from the cyanobacteria Gloeobacter violaceus PCC7421 and Synechocystis sp. PCC6803 function in plastids. First, we found that the G. violaceus POR shows a higher affinity to its substrate protochlorophyllide than the Synechocystis POR but a similar affinity to plant PORs. Secondly, the reduced size of prolamellar bodies caused by a knockdown mutation of one of the POR genes, PORA, in Arabidopsis could be complemented by heterologous expression of the cyanobacterial PORs. Photoactive protochlorophyllide in the etioplasts of the complementing lines, however, was retained at a low level as in the parent PORA knockdown mutant, indicating that the observed formation of prolamellar bodies was irrelevant to the assembly of photoactive protochlorophyllide. This work reveals a new view on the formation of prolamellar bodies and provides new clues about the function of POR in the etioplast–chloroplast transition.     

Overexpression of protochlorophyllide oxidoreductase C regulates oxidative stress in Arabidopsis

Light absorbed by colored intermediates of chlorophyll biosynthesis is not utilized in photosynthesis; instead, it is transferred to molecular oxygen, generating singlet oxygen ((1)O(2)). As there is no enzymatic detoxification mechanism available in plants to destroy (1)O(2), its generation should be minimized. We manipulated the concentration of a major chlorophyll biosynthetic intermediate i.e., protochlorophyllide in Arabidopsis by overexpressing the light-inducible protochlorophyllide oxidoreductase C (PORC) that effectively phototransforms endogenous protochlorophyllide to chlorophyllide leading to minimal accumulation of the photosensitizer protochlorophyllide in light-grown plants. In PORC overexpressing (PORCx) plants exposed to high-light, the (1)O(2) generation and consequent malonedialdehyde production was minimal and the maximum quantum efficiency of photosystem II remained unaffected demonstrating that their photosynthetic apparatus and cellular organization were intact. Further, PORCx plants treated with 5-aminolevulinicacid when exposed to light, photo-converted over-accumulated protochlorophyllide to chlorophyllide, reduced the generation of (1)O(2) and malonedialdehyde production and reduced plasma membrane damage. So PORCx plants survived and bolted whereas, the 5-aminolevulinicacid-treated wild-type plants perished. Thus, overexpression of PORC could be biotechnologically exploited in crop plants for tolerance to (1)O(2)-induced oxidative stress, paving the use of 5-aminolevulinicacid as a selective commercial light-activated biodegradable herbicide. Reduced protochlorophyllide content in PORCx plants released the protochlorophyllide-mediated feed-back inhibition of 5-aminolevulinicacid biosynthesis that resulted in higher 5-aminolevulinicacid production. Increase of 5-aminolevulinicacid synthesis upregulated the gene and protein expression of several downstream chlorophyll biosynthetic enzymes elucidating a regulatory net work of expression of genes involved in 5-aminolevulinicacid and tetrapyrrole biosynthesis.     

Isolation and characterization of photoactive complexes of NADPH:protochlorophyllide oxidoreductase from wheat

A photoactive substrate-enzyme complex of the NADPH:protochlorophyllide oxidoreductase (POR; EC 1. 3. 1. 33) was purified from etiolated Triticum aestivum L. by gel chromatography after solubilization of prolamellar bodies by dodecyl-maltoside. Irradiation by a 1-ms flash induced the phototransformation of protocholorophyllide a (Pchlide) with −196 °C absorbance and emission maxima at 640 and 643 nm, respectively. The apparent molecular weight of this complex was 112 ± 24 kDa, which indicates aggregation of enzyme subunits. By lowering the detergent concentration in the elution buffer, a 1080 ± 250-kDa particle was obtained which displayed the spectral properties of the predominant form of photoactive Pchlide in vivo (−196 °C absorbance and fluorescence maxima at 650 and 653 nm). In this complex, POR was the dominant polypeptide. Gel chromatography in the same conditions of an irradiated sample of solubilized prolamellar bodies indicated rapid disaggregation of the complex after Pchlide phototransformation. High performance liquid chromatographic analysis of the POR complexes obtained using two detergent concentrations indicates a possible association of zeaxanthin and violaxanthin with the photoactive complex. Received: 25 February 1998 / Accepted: 8 June 1998     

Laser excitation studies of the product release steps in the catalytic cycle of the light-driven enzyme, protochlorophyllide oxidoreductase

The latter stages of the catalytic cycle of the light-driven enzyme, protochlorophyllide oxidoreductase, have been investigated using novel laser photoexcitation methods. The formation of the ternary product complex was initiated with a 6-ns laser pulse, which allowed the product release steps to be kinetically accessed for the first time. Subsequent absorbance changes associated with the release of the NADP+ and chlorophyllide products from the enzyme could be followed on a millisecond timescale. This has facilitated a detailed kinetic and thermodynamic characterization for the interconversion of all the various bound and unbound product species. Initially, NADP+ is released from the enzyme in a biphasic process with rate constants of 1210 and 237 s(-1). The rates of both phases show a significant dependence on the viscosity of the solvent and become considerably slower at higher glycerol concentrations. The fast phase of this process exhibits no dependence on NADP+ concentration, suggesting that conformational changes are required prior to NADP+ release. Following NADP+ release, the NADPH rebinds to the enzyme with a maximum rate constant of approximately 72 s(-1). At elevated temperatures (>298 K) chlorophyllide is released from the enzyme to yield the free product with a maximum rate constant of 20 s(-1). The temperature dependencies of the rates of each of these steps were measured, and enthalpies and entropies of activation were calculated using the Eyring equation. A comprehensive kinetic and thermodynamic scheme for these final stages of the reaction mechanism is presented.     

Nicotinamide is a specific inhibitor of dark-operative protochlorophyllide oxidoreductase,a nitrogenase-like enzyme,from Rhodobacter capsulatus

Dark-operative protochlorophyllide oxidoreductase (DPOR) is a nitrogenase-like enzyme consisting of two components, L-protein as a reductase component and NB-protein as a catalytic component. Elucidation of the crystal structures of NB-protein (Muraki et al., Nature 2010, 465: 110–114) has enabled us to study its reaction mechanism in combination with biochemical analysis. Here we demonstrate that nicotinamide (NA) inhibits DPOR activity by blocking the electron transfer from L-protein to NB-protein. A reaction scheme of DPOR, in which the binding of protochlorophyllide (Pchlide) to the NB-protein precedes the electron transfer from the L-protein, is proposed based on the NA effects.     

Chlorophylls of the c family: absolute configuration and inhibition of NADPH:protochlorophyllide oxidoreductase

Using circular dichroism (CD) spectroscopy, the stereochemistry at C-13(2) of members of the chlorophyll (Chl) c family, namely Chls c(1), c(2), c(3) and [8-vinyl]-protochlorophyllide a (Pchlide a) was determined. By comparison with spectra of known enantiomers, all Chl c members turned out to have the (R) configuration, which is in agreement with considerations drawn from chlorophyll biosynthesis. Except for a double bond in the side chain at C-17, the chemical structure of Chl c(1) is identical with Pchlide a, the natural substrate of the light-dependent NADPH:protochlorophyllide oxidoreductase (POR). Thus, lack of binding to the active site due to the wrong configuration at C-13(2), which had been proposed previously, cannot be an explanation for inactivity of Chl c in this enzymic reaction. Our results show rather that Chl c(1) is a competitive inhibitor for this enzyme, tested with Pchlide a and Zn-protopheophorbide a (Zn-Ppheide a) as substrates.     

Substrate recognition of nitrogenase-like dark operative protochlorophyllide oxidoreductase from Prochlorococcus marinus

Chlorophyll and bacteriochlorophyll biosynthesis requires the two-electron reduction of protochlorophyllide a ringDbya protochlorophyllide oxidoreductase to form chlorophyllide a. A light-dependent (light-dependent Pchlide oxidoreductase (LPOR)) and an unrelated dark operative enzyme (dark operative Pchlide oxidoreductase (DPOR)) are known. DPOR plays an important role in chlorophyll biosynthesis of gymnosperms, mosses, ferns, algae, and photosynthetic bacteria in the absence of light. Although DPOR shares significant amino acid sequence homologies with nitrogenase, only the initial catalytic steps resemble nitrogenase catalysis. Substrate coordination and subsequent [Fe-S] cluster-dependent catalysis were proposed to be unrelated. Here we characterized the first cyanobacterial DPOR consisting of the homodimeric protein complex ChlL(2) and a heterotetrameric protein complex (ChlNB)(2). The ChlL(2) dimer contains one EPR active [4Fe-4S] cluster, whereas the (ChlNB)(2) complex exhibited EPR signals for two [4Fe-4S] clusters with differences in their g values and temperature-dependent relaxation behavior. These findings indicate variations in the geometry of the individual [4Fe-4S] clusters found in (ChlNB)(2). For the analysis of DPOR substrate recognition, 11 synthetic derivatives with altered substituents on the four pyrrole rings and the isocyclic ring plus eight chlorophyll biosynthetic intermediates were tested as DPOR substrates. Although DPOR tolerated minor modifications of the ring substituents on rings A-C, the catalytic target ring D was apparently found to be coordinated with high specificity. Furthermore, protochlorophyllide a, the corresponding [8-vinyl]-derivative and protochlorophyllide b were equally utilized as substrates. Distinct differences from substrate binding by LPOR were observed. Alternative biosynthetic routes for cyanobacterial chlorophyll biosynthesis with regard to the reduction of the C8-vinyl group and the interconversion of a chlorophyll a/b type C7 methyl/formyl group were deduced.     

The protochlorophyllide holochrome of barley (Hordeum vulgare L.). Phytochrome-induced decrease of translatable mRNA coding for the NADPH: protochlorophyllide oxidoreductase

During the illumination of dark-grown barley plants light induces a rapid decrease of a translatable mRNA which codes for a polypeptide of Mr 44000. This component was identified as a precursor of the NADPH:protochlorophyllide oxidoreductase. The precursor has an Mr larger than the authentic protein by approximately 8000. The light-induced change in the level of translatable mRNA can be induced by a 15-s red-light pulse followed by 5 h of darkness. The red-light effect is reversed by a subsequent far-red-light treatment. It is concluded that the light-induced decline of translatable mRNA for the NADPH:protochlorophyllide oxidoreductase is controlled by phytochrome. The significance of this finding for present concepts of light-dependent control of chloroplast development and chlorophyll synthesis is discussed.     

Origin and evolution of the light-dependent protochlorophyllide oxidoreductase (LPOR) genes

Abstract: Light‐dependent NADPH‐protochlorophyllide oxidoreductase (LPOR) is a nuclear‐encoded chloroplast protein in green algae and higher plants which catalyzes the light‐dependent reduction of protochlorophyllide to chlorophyllide. Light‐dependent chlorophyll biosynthesis occurs in all oxygenic photosynthetic organisms. With the exception of angiosperms, this pathway coexists with a separate light‐independent chlorophyll biosynthetic pathway, which is catalyzed by light‐independent protochlorophyllide reductase (DPOR) in the dark. In contrast, the light‐dependent function of chlorophyll biosynthesis is absent from anoxygenic photosynthetic bacteria. Consequently, the question is whether cyanobacteria are the ancestors of all organisms that conduct light‐dependent chlorophyll biosynthesis. If so, how did photosynthetic eukaryotes acquire the homologous genes of LPOR in their nuclear genomes? The large number of complete genome sequences now available allow us to detect the evolutionary history of LPOR genes by conducting a genome‐wide sequence comparison and phylogenetic analysis. Here, we show the results of a detailed phylogenetic analysis of LPOR and other functionally related enzymes in the short chain dehydrogenase/reductase (SDR) family. We propose that the LPOR gene originated in the cyanobacterial genome before the divergence of eukaryotic photosynthetic organisms. We postulated that the photosynthetic eukaryotes obtained their LPOR homologues through endosymbiotic gene transfer.     

Site-directed mutagenesis of Tyr-189 and Lys-193 in NADPH: protochlorophyllide oxidoreductase from Synechocystis

NADPH:protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory reaction in the chlorophyll biosynthetic pathway. Sequence comparisons have revealed that POR is a member of the short-chain alcohol dehydrogenase family of enzymes. A tyrosine and a lysine residue are conserved throughout all members of this family, and are proposed to be within the active site. This present study describes how site-directed mutagenesis has been used to change Tyr-189 to Phe and Lys-193 to Arg in the Synechocystis POR enzyme. The mutant enzymes were produced with a His tag in Escherichia coli and subsequently purified on a Ni(2+)-affinity column. The two mutations resulted in inactive enzymes, indicating that both residues are crucial for activity. The K(d) value for NADPH binding to the K193R mutant was significantly higher than for the wild-type enzyme, suggesting that the affinity for NADPH has also been reduced.