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PubMed1982

Evidence for a light-independent protochlorophyllide reductase in green barley leaves.

Evidence is presented that normal light-grown barley plants continue to synthesise chlorophyll when transferred to total darkness. This implies the existence of a light-independent protochlorophyllide reductase in barley as well as the light-dependent enzyme (NADPH protochlorophyllide oxido-reductase) involved in the phototransformation of protochlorophyllide to chlorophyllide. This finding is consistent with an earlier observation by Popov and Dilova (1969) that dark grown barley develops the ability to synthesise chlorophyll in the dark, after several hours exposure to light. Popov and Dilova's findings with etiolated barley are confirmed.

Darkness↗

PubMed1994

Biosynthetic studies on chlorophylls: from protoporphyrin IX to protochlorophyllide.

The series of reactions leading from protoporphyrin IX to protochlorophyllide have been studied over the last 15 years in the authors' laboratories at Davis and Clemson. Here, two crucial steps are emphasized, the discovery of the ATP requirement for Mg2+ chelation, and the oxidative cyclization of Mg-protoporphyrin IX monomethyl ester to protochlorophyllide. The in vitro systems for the chelation of Mg2+ and for the oxidative cyclization of Mg-protoporphyrin IX monomethyl ester both require membrane-associated and soluble heat-labile components. We speculate about the enzymological mechanisms of these important reactions, their sub-plastidic localization and the relationship of these individual steps to the broader questions of chloroplast and cell development.

Adenosine Triphosphate↗

PubMed2006

The excited-state chemistry of protochlorophyllide a: a time-resolved fluorescence study.

The excited-state processes of protochlorophyllide a, the precursor of chlorophyll a in chlorophyll biosynthesis, are studied using picosecond time-resolved fluorescence spectroscopy. Following excitation into the Soret band, two distinct fluorescence components, with emission maxima at 640 and 647 nm, are observed. The 640 nm emitting component appears within the time resolution of the experiment and then decays with a time constant of 27 ps. In contrast, the 647 nm emitting component is built up with a 3.5 ps rise time and undergoes a subsequent decay with a time constant of 3.5 ns. The 3.5 ps rise kinetics are attributed to relaxations in the electronically excited state preceding the nanosecond fluorescence, which is ascribed to emission out of the thermally equilibrated S(1) state. The 27 ps fluorescence, which appears within the experimental response of the streak camera, is suggested to originate from a second minimum on the excited-state potential-energy surface. The population of the secondary excited state is suggested to reflect a very fast motion out of the Franck-Condon region along a reaction coordinate different from the one connecting the Franck-Condon region with the S(1) potential-energy minimum. The 27 ps-component is an emissive intermediate on the reactive excited-state pathway, as its decay yields the intermediate photoproduct, which has been identified previously (J. Phys. Chem. B 2006, 110, 4399-4406). No emission of the photoproduct is observed. The results of the time-resolved fluorescence study allow a detailed spectral characterization of the emission of the excited states in protochlorophyllide a, and the refinement of the kinetic model deduced from ultrafast absorption measurements.

Avena↗

PubMed1995

The in vitro assembly of the NADPH-protochlorophyllide oxidoreductase in pea chloroplasts.

The NADPH-protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) is the major protein in the prolamellar bodies (PLBs) of etioplasts, where it catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide during chlorophyll synthesis in higher plants. The suborganellar location in chloroplasts of light-grown plants is less clear. In vitro assays were performed to characterize the assembly process of the pchlide reductase protein in pea chloroplasts. Import reactions employing radiolabelled precursor protein of the pchlide reductase showed that the protein was efficiently imported into fully matured green chloroplasts of pea. Fractionation assays following an import reaction revealed that imported protein was targeted to the thylakoid membranes. No radiolabelled protein could be detected in the stromal or envelope compartments upon import. Assembly reactions performed in chloroplast lysates showed that maximum amount of radiolabelled protein was associated to the thylakoid membranes in a thermolysin-resistant conformation when the assays were performed in the presence of hydrolyzable ATP and NADPH, but not in the presence of NADH. Furthermore, membrane assembly was optimal at pH 7.5 and at 25 degrees C. However, further treatment of the thylakoids with NaOH after an assembly reaction removed most of the membrane-associated protein. Assembly assays performed with the mature form of the pchlide reductase, lacking the transit peptide, showed that the pre-sequence was not required for membrane assembly. These results indicate that the pchlide reductase is a peripheral protein located on the stromal side of the membrane, and that both the precursor and the mature form of the protein can act as substrates for membrane assembly.

Adenosine Triphosphate↗

PubMed1992

Detection of the photoactive protochlorophyllide-protein complex in the light during the greening of barley.

A photoactive protochlorophyllide-protein complex with absorbance and fluorescence maxima at 648 and 653 nm was detected in greening barley leaves without any re-darkening. The variations of the amplitudes of the absorbance and the fluorescence of the photoactive protochlorophyllide with greening time at two different light intensities indicate a close relationship between the rate of chlorophyll synthesis and the amount of the complex during the first hours. The chlorophyllide resulting from photoreduction during greening has an absorbance maximum at 684 nm, which shifts towards a shorter wavelength within a few seconds, indicating rapid liberation of the pigment from the enzyme. We conclude that chlorophyll accumulation proceeds through continuous regeneration and phototransformation of the photoactive complex.

Darkness↗

PubMed1992

Spectrofluorometric estimation of intermediates of chlorophyll biosynthesis: protoporphyrin IX, Mg-protoporphyrin, and protochlorophyllide.

A highly sensitive spectrofluorometric method for quantitative estimation of certain precursors of chlorophyll biosynthesis from the mixtures of plant tetrapyrroles having overlapping fluorescence emission spectra is developed. At room temperature (293 degrees K) protoporphyrin IX is monitored from its emission maximum, 633 nm, when excited at 400 nm (E400/F633). Protochlorophyllide is estimated at 638 nm, while being excited at 440 nm (E440/F638). Mg-protoporphyrin+Mg-protoporphyrin monoester pool has emission around 589-592 nm. Therefore the integration value of the emission band that extends from 580 to 610 nm is taken to calibrate its concentration. This spectrofluorometric method designed for the determination of protoporphyrin IX, esterified and nonesterified Mg-protoporphyrin pool, and protochlorophyllide is far superior to available spectrophotometric methods and estimates as low as 1 nM concentration of plant pigments. As minute quantities of individual pigments can be quantitatively analyzed from their mixtures, this method eliminates analytical uncertainties due to recovery losses caused by chromatography. However, only dilute samples can be estimated by this spectrofluorometric method as the quantitative relation between fluorescence and concentration deviates from linearity at high, i.e., above 150 nM, concentrations of pigment to be quantified.

Acetone↗

PubMed2003

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.

Chlorophyll↗

PubMed2000

NADPH:protochlorophyllide oxidoreductase from Synechocystis: overexpression, purification and preliminary characterisation.

NADPH:protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory reaction in the chlorophyll biosynthetic pathway. POR from the cyanobacterium Synechocystis has been overproduced in Escherichia coli with a hexahistidine tag at the N-terminus. This enzyme (His(6)-POR) has been purified to homogeneity and a preliminary characterisation of its kinetic and substrate binding properties is presented. Chemical modification experiments have been used to demonstrate inhibition of POR activity by the thiol-specific reagent N-ethyl maleimide. Substrate protection experiments reveal that the modified Cys residues are involved in either substrate binding or catalysis.

Chlorophyllides↗

PubMed2003

Protochlorophyllide oxidoreductase: "dark" reactions of a light-driven enzyme.

The light-driven enzyme NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), a key regulatory step in the chlorophyll biosynthesis pathway. As POR is light activated, it allows intermediates in the reaction pathway to be observed by initiating catalysis with illumination at low temperatures, a technique that has recently been used to study the initial photochemistry. Here, we use low-temperature spectroscopy to show that the catalytic mechanism of POR involves two additional steps, which do not require light and have been termed the "dark" reactions. The first of these involves the conversion of the product of the initial light-driven reaction, a nonfluorescent radical species, into a new intermediate that has an absorbance maximum at 681 nm and a fluorescence peak at 684 nm. During the second dark step this species gradually blue shifts to yield the product, Chlide. The temperature dependence for each of these two processes was measured; the data revealed that these steps could only occur close to or above the "glass transition" temperature of proteins, suggesting that domain movements and/or reorganization of the protein are required for these stages of the catalytic mechanism.

Catalysis↗

PubMed2004

Identification and characterization of the product release steps within the catalytic cycle of protochlorophyllide oxidoreductase.

The chlorophyll biosynthetic enzyme protochlorophyllide reductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) into chlorophyllide (Chlide) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. POR is a light-driven enzyme, which has provided a unique opportunity to trap intermediates and identify different steps in the reaction pathway by initiating catalysis with illumination at low temperatures. In the present work we have used a thermophilic form of POR, which has an increased conformational rigidity at comparable temperatures, to dissect and study the final stages of the reaction where protein dynamics are proposed to play an important role in catalysis. Low-temperature fluorescence and absorbance measurements have been used to demonstrate that the reaction pathway for this enzyme consists of two additional "dark" steps, which have not been detected in previous studies. Product binding studies were used to show that spectroscopically distinct Chlide species could be observed and were dependent on whether the NADPH or NADP(+) cofactor was present. As a result we have been able to identify the intermediates that are observed during the latter stages of the POR catalytic cycle and have shown that they are formed via a series of ordered product release and cofactor binding events. These events involve release of NADP(+) from the enzyme and its replacement by NADPH, before release of the Chlide product has taken place. Following release of Chlide, the subsequent binding of Pchlide allows the next catalytic cycle to proceed.

Catalysis↗

PubMed2006

Solvent effects on the excited-state processes of protochlorophyllide: a femtosecond time-resolved absorption study.

The excited-state dynamics of protochlorophyllide a, a porphyrin-like compound and, as substrate of the NADPH/protochlorophyllide oxidoreductase, a precursor of chlorophyll biosynthesis, is studied by femtosecond absorption spectroscopy in a variety of solvents, which were chosen to mimic different environmental conditions in the oxidoreductase complex. In the polar solvents methanol and acetonitrile, the excited-state dynamics differs significantly from that in the nonpolar solvent cyclohexane. In methanol and acetonitrile, the relaxation dynamics is multiexponential with three distinguishable time scales of 4.0-4.5 ps for vibrational relaxation and vibrational energy redistribution of the initially excited S1 state, 22-27 ps for the formation of an intermediate state, most likely with a charge transfer character, and 200 ps for the decay of this intermediate state back to the ground state. In the nonpolar solvent cyclohexane, only the 4.5 ps relaxational process can be observed, whereas the intermediate intramolecular charge transfer state is not populated any longer. In addition to polarity, solvent viscosity also affects the excited-state processes. Upon increasing the viscosity by adding up to 60% glycerol to a methanolic solution, a deceleration of the 4 and 22 ps decay rates from the values in pure methanol is found. Apparently not only vibrational cooling of the S1 excited state is slowed in the more viscous surrounding, but the formation rate of the intramolecular charge transfer state is also reduced, suggesting that nuclear motions along a reaction coordinate are involved in the charge transfer. The results of the present study further specify the model of the excited-state dynamics in protochlorophyllide a as recently suggested (Chem. Phys. Lett. 2004, 397, 110).

Absorption↗

PubMed1999

Differential expression of genes encoding the light-dependent and light-independent enzymes for protochlorophyllide reduction during development in loblolly pine.

The expression patterns of the two distinct subfamilies of genes (designated porA and porB) encoding the light-dependent NADPH:protochlorophyllide oxidoreductases (PORs) in loblolly pine (Pinus taeda L.) were examined. Transcripts arising from both gene subfamilies were shown to be present at high levels in the cotyledons of dark-grown pine seedlings and to a lesser extent in their stems. Exposure of dark-grown seedlings to light resulted in increased levels of both porA and porB transcripts, as well as increased levels of mRNAs encoding other photosynthesis-related gene products, suggesting that they are under a common mode of regulation. Relative levels of the porA and porB transcripts were similar in seedling cotyledons and primary needles of two-month-old pine trees, whereas only porB transcripts were present at a significant level in mature secondary needles of two-year-old trees. Immunoblot analysis showed that the 37 kDa PORA protein was most abundant in dark-grown tissues, decreased dramatically upon exposure to light, but could still be detected at low levels in light-grown seedlings. In comparison, levels of the 38 kDa PORB protein were not significantly changed upon transfer of dark-grown tissues to light. While both PORA and PORB were detected in cotyledons and primary needles, only PORB could be detected in mature needles. Transcripts derived from the three plastid genes, chlL, chlN, and chlB, encoding subunits of the light-independent protochlorophyllide reductase were detected in the cotyledons and stems of dark-grown seedlings, and in mature needles. The highest levels of chlL, chlN, and chlB transcripts were detected within the top one-third of the stem and decreased gradually towards the stem/root transition zone. Correspondingly, the highest levels of light-independent chlorophyll formation took place near the top of the hypocotyl. A similar pattern of expression was observed for other photosynthesis-related gene products, including porA and porB. Our results suggest that many aspects of the light-dependent, tissue-specific and developmental regulation of POR expression first described in angiosperms were already established in the less evolutionarily advanced gymnosperms. However, unlike angiosperms, light is not the dominant regulatory factor controlling porA expression in these species.

Algal Proteins↗

PubMed2004

The jasmonate-free rice mutant hebiba is affected in the response of phyA'/phyA" pools and protochlorophyllide biosynthesis to far-red light.

Phytochrome (phy) A in its two native isoforms (phyA' and phyA") and the active (Pchlide(655)) and inactive (Pchlide(633)) protochlorophyllides were investigated by low-temperature fluorescence spectroscopy in the tips of rice (Oryza sativa L. Japonica cv Nihonmasari) coleoptiles from wild type (WT) and the jasmonate-deficient mutant hebiba. The seedlings were either grown in the dark or under pulsed (FRp) or continuous (FRc) far-red light (lambda(a) >/= 720 nm) of equal fluences. In the dark, the mutant had a long mesocotyl and a short coleoptile, whereas the situation was reversed under FR: short mesocotyl and long coleoptile, suggesting that the effect is mediated by phyA. Under these conditions the WT displayed a short coleoptile and emergence of the first leaf. In the dark, the spectroscopic and photochemical properties of phyA, its content and the proportion of its two pools, phyA' and phyA", were virtually identical between WT and hebiba. However, the total content of protochlorophyllides was higher in the mutant. Upon illumination with FRc, [phyA] declined in the WT and the ratio between phyA' and phyA" shifted towards phyA". In hebiba, the light-induced decline of [phyA] was less pronounced and the ratio between phyA' and phyA" did not shift. Moreover, in the WT, FRp stimulated the biosynthesis of Pchlide(655), whereas FRc was inhibiting. In contrast, in the mutant, both FRp and FRc stimulated the synthesis of Pchlide(655). This means that FRc caused the opposite effect in hebiba. This difference correlates with a slower photodestruction of primarily the light-labile phyA' pool in hebiba.

Cyclopentanes↗

PubMed1980

Light modulation of the activity of protochlorophyllide reductase.

Illumination of etiolated plants effects the activity of protochlorophyllide reductase (NADPH-protochlorophyllide oxidoreductase) in the plastids. Constant illumination or a 2-min light-triggering of etiolated plants leads to an approx. 80% decrease in activity of the enzyme, a change that can be reversed by returning the plants to darkness. The change in activity results from an alteration of the Vmax. rather than Km. Despite the fact that exogenous pigments effect the activity of the enzyme in vitro, no correlation could be drawn between the concentrations of pigments in vivo and activity of the enzyme.

Chlorophyll↗

PubMed2002

The extra loop distinguishing POR from the structurally related short-chain alcohol dehydrogenases is dispensable for pigment binding but needed for the assembly of light-harvesting POR-protochlorophyllide complex.

We have recently discovered a protochlorophyllide (Pchlide)-based light-harvesting complex involved in chlorophyll a biosynthesis. This complex consists of the two previously identified NADPH:protochlorophyllide oxidoreductases (PORs), PORA and PORB, their natural substrates (Pchlide b and Pchlide a, respectively), plus NADPH. These are all held together in a stoichiometry of five PORA-Pchlide b-NADPH complexes and one PORB-Pchlide a-NADPH complex in the prolamellar body of etioplasts. The assembly of this novel light-harvesting POR-Pchlide complex (LHPP) requires both the proper interaction of the PORA and PORB with their cognate substrates as well as the oligomerization of the resulting POR-pigment-NADPH ternary complexes into the native, lipid-containing structure of the etioplast. In this study, we demonstrate that the conserved extra sequence that distinguishes PORA and PORB from the structurally related short-chain alcohol dehydrogenases, is dispensable for pigment binding but needed for the assembly of LHPP. As shown by in vitro mutagenesis, deleting this extra sequence gave rise to assembly-incompetent but pigment-containing PORA and PORB polypeptides.

Alcohol Dehydrogenase↗

PubMed1995

Enzymatic product formation impairs both the chloroplast receptor-binding function as well as translocation competence of the NADPH: protochlorophyllide oxidoreductase, a nuclear-encoded plastid precursor protein.

The key enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR, EC 1.6.99.1), is a nuclear-encoded plastid protein. Its posttranslational transport into plastids of barley depends on the intraplastidic availability of one of its substrates, protochlorophyllide (PChlide). The precursor of POR (pPOR), synthesized from a corresponding full-length barley cDNA clone by coupling in vitro transcription and translation, is enzymatically active and converts PChlide to chlorophyllide (Chlide) in a light- and NADPH-dependent manner. Chlorophyllide formed catalytically remains tightly but noncovalently bound to the precursor protein and stabilizes a transport-incompetent conformation of pPOR. As shown by in vitro processing experiments, the chloroplast transit peptide in the Chlide-pPOR complex appears to be masked and thus is unable to physically interact with the outer plastid envelope membrane. In contrast, the chloroplast transit peptide in the naked pPOR (without its substrates and its product attached to it) and in the pPOR-substrate complexes, such as pPOR-PChlide or pPOR-PChlide-NADPH, seems to react independently of the mature region of the polypeptide, and thus is able to bind to the plastid envelope. When envelope-bound pPOR-PChlide-NADPH complexes were exposed to light during a short preincubation, the enzymatically produced Chlide slowed down the actual translocation step, giving rise to the sequential appearance of two partially processed translocation intermediates. However, ongoing translocation induced by feeding the chloroplasts delta-aminolevulinic acid, a precursor of PChlide, was able to override these two early blocks in translocation, suggesting that the plastid import machinery has a substantial capacity to denature a tightly folded, envelope-bound precursor protein. Together, our results show that pPOR with Chlide attached to it is impaired both in the ATP-dependent step of binding to a receptor protein component of the outer chloroplast envelope membrane, as well as in the PChlide-dependent step of precursor translocation.

Biological Transport, Active↗

PubMed1995

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.

Biological Transport↗

PubMed1986

Fluorescence properties of the envelope membranes from spinach chloroplasts. Detection of protochlorophyllide.

At 77 K, under excitation at 440 nm, two major fluorescence emission peaks were observed in envelope membranes from spinach chloroplasts at 636 and 680 nm. A narrow range of wavelengths around 440 nm and a wider range of wavelengths between 390 and 440 nm, respectively, were responsible for excitation of the 636 and 680 nm fluorescence emissions which, in marked contrast with thylakoid fluorescence emission, were devoid of any exciting components between 460 and 500 nm. In acetonic extract of envelope membranes, two fluorescence emission peaks were observed at 635 and 675 nm. After extraction of the acetonic solution by nonpolar solvents (petroleum ether or hexane), the 675 nm fluorescence emission was partitioned between the polar and nonpolar phases whereas the 635 nm fluorescence emission was solely recovered in the polar phase. All together, the results obtained suggest that envelope membranes contain low amounts of pigments having the absorption and fluorescence spectroscopic properties, together with the behavior in polar/nonpolar solvents, of protochlorophyllide and chlorophyllide. In addition, modulation of the level of fluorescence at 636 and 680 nm could be obtained by addition of NADPH to envelope membranes under illumination. The presence of protochlorophyllide in chloroplast envelope membranes together with its possible photoconversion into chlorophyllide could have major implication for the understanding of chlorophyll biosynthesis in mature chloroplasts.

Chlorophyll↗