Fast phototransformation of the 636 nm-emitting protochlorophyllide form in epicotyls of dark-grown pea (Pisum sativum)

The phototransformation of protochlorophyllide forms was studied in epicotyls of dark-germinated pea (Pisum sativum L. cv. Zsuzsi) seedlings. Middle segments were illuminated with white or 632.8 nm laser flash or continuous light at room temperature and at −15°C. At low light intensities, photoreduction could be distinguished from bleaching. 77 K fluorescence emission spectra were measured, difference spectra of illuminated and non-illuminated samples were calculated and/or the spectra were deconvoluted into Gaussian components. The 629 nm-emitting protochlorophyllide form, P629 (Pxxx where xxx is the fluorescence emission maximum), was inactive. For short-period (2–100 ms) and/or low-intensity (0.75–1.5 µmol m⁻² s⁻¹) illumination, particularly with laser light, the transformation of P636 into the 678 nm-emitting chlorophyllide form, C678 (Cxxx where xxx is the fluorescence emission maximum), was characteristic. This process was also found when the samples were cooled to −15°C. The transformation of P644 into C684 usually proceeded in parallel with the process above as a result of the strong overlap of the excitation bands of P636 and P644. The Shibata shift of C684 into a short-wavelength form, C675–676, was observed. Long-period (20–600 s) and/or high-intensity (above 10 µmol m⁻² s⁻¹) illumination resulted in the parallel transformation of P655 into C692. These results demonstrate that three flash-photoactive protochlorophyllide forms function in pea epicotyls. As a part of P636 is flash photoactive, its protochlorophyllide molecule must be bound to the active site of a monomer protein unit [Böddi B, Kis-Petik K, Kaposi AD, Fidy J, Sundqvist C (1998) The two short wavelength protochlorophyllide forms in pea epicotyls are both monomeric. Biochim Biophys Acta 1365: 531–540] of the NADPH:protochlorophyllide oxidoreductase (EC 1.3.1.33). Dynamic interconversions of the protochlorophyllide forms into each other, and their regeneration, were also found, which are summarized in a scheme.     

Aggregation of the 636 nm emitting monomeric protochlorophyllide form into flash-photoactive, oligomeric 644 and 655 nm emitting forms in vitro

Artificial formation of flash-photoactive oligomeric protochlorophyllide complexes was found in etiolated pea (Pisum sativum L. cv. Zsuzsi) epicotyl homogenates containing glycerol (40% v/v) and sucrose (40% m/v). The 77 K fluorescence emission spectra indicated that the ratio of the 644 and 655 nm emitting forms to the 636 nm form increased during 3 to 5-day incubation in the dark at -14 degrees C. Electron micrographs showed the presence of well-organized prolamellar bodies in the homogenates. The same phenomena were found when the homogenates were frozen into liquid nitrogen and thawed to room temperature in several cycles. Similar treatments of intact epicotyl pieces caused significant membrane destructions. In homogenates, the in vitro produced 644 and 655 nm emitting protochlorophyllide forms were flash-photoactive; the extent of phototransformation increased compared to that in native epicotyls. The newly appeared 692 nm chlorophyllide band showed a blue shift (similar to the Shibata shift in leaves), however this process took place only partially due to the effect of the isolation medium. These results prove that the in vitro accumulated 644 and 655 nm protochlorophyllide forms were produced from the flash-photoactive 636 nm emitting monomeric NADPH:protochlorophyllide oxidoreductase units via aggregation, in connection with structure stabilization properties of glycerol and sucrose.     

Aggregation of the 636 nm emitting monomeric protochlorophyllide form into flash-photoactive, oligomeric 644 and 655 nm emitting forms in vitro

Artificial formation of flash-photoactive oligomeric protochlorophyllide complexes was found in etiolated pea (Pisum sativum L. cv. Zsuzsi) epicotyl homogenates containing glycerol (40% v/v) and sucrose (40% m/v). The 77 K fluorescence emission spectra indicated that the ratio of the 644 and 655 nm emitting forms to the 636 nm form increased during 3 to 5-day incubation in the dark at −14 °C. Electron micrographs showed the presence of well-organized prolamellar bodies in the homogenates. The same phenomena were found when the homogenates were frozen into liquid nitrogen and thawed to room temperature in several cycles. Similar treatments of intact epicotyl pieces caused significant membrane destructions. In homogenates, the in vitro produced 644 and 655 nm emitting protochlorophyllide forms were flash-photoactive; the extent of phototransformation increased compared to that in native epicotyls. The newly appeared 692 nm chlorophyllide band showed a blue shift (similar to the Shibata shift in leaves), however this process took place only partially due to the effect of the isolation medium.These results prove that the in vitro accumulated 644 and 655 nm protochlorophyllide forms were produced from the flash-photoactive 636 nm emitting monomeric NADPH:protochlorophyllide oxidoreductase units via aggregation, in connection with structure stabilization properties of glycerol and sucrose.     

ADP/ATP and protein phosphorylation dependence of phototransformable protochlorophyllide in isolated etioplast membranes

The effects of modulated ADP/ATP and NADPH/NADP⁺ ratios, and of protein kinase inhibitors, on the in vitro reformation of phototransformable protochlorophyllide, i.e. the aggregated ternary complexes between NADPH, protochlorophyllide, and NADPH-protochlorophyllide oxidoreductase (POR, EC 1.3.1.33), in etioplast membranes isolated from dark-grown wheat (Triticum aestivum) were investigated. Low temperature fluorescence emission spectra (–196 °C) were used to determine the state of the pigments. The presence of spectral intermediates of protochlorophyllide and the reformation of phototransformable protochlorophyllide were reduced at high ATP, but favoured by high ADP. Increased ADP level partly prevented the chlorophyllide blue-shift. The protein kinase inhibitor K252a prevented reformation of phototransformable protochlorophyllide without showing any effect on the chlorophyllide blue-shift. Addition of NADPH did not overcome the inhibition. The results indicate that protein phosphorylation plays a role in the conversion of the non-phototransformable protochlorophyllide to POR-associated phototransformable protochlorophyllide. The possible presence of a plastid ADP-dependent kinase, the activity of which favours the formation of PLBs, is discussed. Reversible protein phosphorylation is suggested as a regulatory mechanism in the prolamellar body formation and its light-dependent dispersal by affecting the membrane association of POR. By the presence of a high concentration of phototransformable protochlorophyllide, prolamellar bodies can act as light sensors for plastid development. The modulation of plastid protein kinase and protein phosphatase activities by the NADPH/NADP⁺ ratio is suggested. This revised version was published online in June 2006 with corrections to the Cover Date.     

Activation volumes of processes linked to the phototransformation of protochlorophyllide determined by fluorescence spectroscopy at high pressure

The photochemical activity of NADPH:protochlorophyllide oxidoreductase (POR) was studied in etiolated wheat (Triticum aestivum, L., cult. MV17) leaf homogenates. The kinetics of the transformation of protochlorophyllide into chlorophyllide was detected by fluorescence intensity changes at 690 nm (formation of chlorophyllide) and 655 nm (decay of protochlorophyllide) at 20 degrees C, excited at 440 nm while the pressure was varied between 0.1 and 400 MPa. Both kinetics could be fitted by two exponentials and the reaction rates were pressure-dependent. A model was suggested based on the comparison of the two kinetics. Reaction rates of the processes occurring during the prototransformation were determined in function of pressure. The evaluation yielded the activation volume as 1.7 ml mol(-1), which corresponds with the formation of one H-bond/molecule.     

Protochlorophyllide and POR development in dark‐grown plants with different proportions of short‐wavelength and long‐wavelength protochlorophyllide spectral forms†

The effect of leaf developmental age on the protochlorophyllide (Pchlide) spectral forms and the expression of messenger RNA (mRNA) encoding NADPH‐protochlorophyllide oxidoreductase (POR) were investigated. Four plant species, maize, wheat, pea and the lip1 mutant of pea, known to have different composition of the spectral forms of Pchlide, were used. In very young plants short‐wavelength Pchlide with a fluorescence emission at 631 nm was dominating. Long‐wavelength Pchlide fluorescing mainly around 655 nm increased during development, which led to a relative decrease of the short‐wavelength forms. During ageing of the leaves, the short‐wavelength forms slightly increased again. The different proportions of short‐ and long‐wavelength Pchlide spectral forms were, however, found to vary with the developmental stage in a species specific pattern. The steady‐state level of POR mRNA and the amount of the POR protein were similar in species dominated by short‐wavelength forms and in species dominated with long‐wavelength forms. Even if POR is necessary for the formation of the long‐wavelength Pchlide form it is not the only limiting factor for formation of long‐wavelength Pchlide forms in mature plants.     

Differential action of Hg2+ and Cd2+ on the phycobilisomes and chlorophyll a fluorescence,and photosystem II dependent electron transport in the cyanobacterium Anabaena flos-aquae

The effect of equimolar concentrations of Hg²⁺ and Cd²⁺ on the whole cell absorption spectra, absorption spectra of the extracted phycocyanin (PC) and fluorescence emission spectra of phycobilisomes (PBS) was investigated in the cells of Anabaena flos-aquae. The PC component of the PBS was found to be extremely sensitive to the Hg²⁺ rather than the Cd²⁺ ions. Further, the results showed that Hg²⁺ and Cd²⁺ induced decrease in the rate of Hill activity (H₂O - DCPIP) was partially restored by the electron donor NH₂OH, not by the diphenyl carbazide. Similarly, chlorophyll a fluorescence emission in the presence of metals showed that addition of NH₂OH could effectively reverse the metal induced alterations in the fluorescence emission intensity. These results, together, suggested that Hg²⁺ and Cd²⁺ caused damage to the photosystems (PS) II reaction center. However, a relatively higher stimulation of the chlorophyll a emission at 695 nm with a red shift of 4.0 nm in the presence of Hg²⁺, and Cd²⁺ induced preferential decrease in the emission intensity at 676 nm as compared with the peak at 695 nm were indicative of the differential action of Hg²⁺ and Cd²⁺ on the PS II.     

Preferential regeneration of the NADPH: protochlorophyllide oxidoreductase oligomer complexes in pea epicotyls after bleaching

The regeneration and stability of the NADPH:protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) enzyme complexes were studied in bleached epicotyls of 9-day-old dark-germinated pea ( Pisum sativum L. cv. Zsuzsi) seedlings. Middle segments were illuminated with 1300 µmol m⁻² s⁻¹photon flux density (PFD) white light and subsequently incubated in total darkness for 4–24 h at 24°C. Almost the full amount of protochlorophyllide (Pchlide) was degraded after 60 min illumination. The preferential regeneration of the 655 nm emitting Pchlide form was observed after 4 h dark incubation; the accumulation of the short-wavelength Pchlide form—dominating in epicotyls of dark-grown seedling—required 18–24 h dark. The Pchlide content of bleached samples was around 2.5% of that of the etiolated samples; after 4 h of dark incubation this value increased to 4–7%. Polyacrylamide gel electrophoresis and western blot showed that the amount of the POR protein decreased to about 50% during bleaching; after 4 h regeneration it reached almost the same level as that of dark-grown samples. We concluded that much more POR protein compared with Pchlide pigment remained stable during bleaching and the non-destroyed POR units were able to form preferentially oligomers during the dark-regeneration which could collect de novo synthesized Pchlide into 655 nm emitting complexes. These data indicate the high stability of the POR protein in pea epicotyls and the importance of the molecular environment in stimulating the aggregation of POR units.     

Disintegration of the prolamellar body structure at high concentrations of Hg2+

The effects of high concentrations of Hg (2+) (10 (-2) M and 10 (-3) M) were investigated on the ultrastructure and on the light-induced transformation of isolated prolamellar bodies (PLBs) of dark-grown wheat leaves. Our earlier work on wheat leaf homogenates ( , Plant Biology 6, 358 - 368) showed that, depending on the concentration, Hg (2+) reacts with protochlorophyllide, NADPH and the NADPH : protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) enzyme and induces disaggregation of the macrodomain structure of this latter. Spectroscopic analyses confirmed that 15 min incubation with 10 (-2) M Hg (2+) at 4 degrees Celsius completely inhibited the activity of POR also in isolated PLBs. Ultrastructural investigations revealed the loosening of the PLB structure in the Hg (2+)-treated sample, i.e., intensive vesicle formation on the surface of the PLB membranes. The hexagonal geometry of the inner lattice was not disturbed, however, the unit cell size significantly increased. The disruption of the PLB membranes upon irradiation was studied after 40 min incubation with 10 (-3) M Hg (2+) at 4 degrees Celsius and a subsequent irradiation for 40 min at 20 degrees Celsius. Equimolar concentrations (10 (-3) M) of NADPH and Hg (2+) were added to the samples 10 min prior or after the addition of Hg (2+). Our results suggest that Hg (2+) accelerates the disruption of the PLB membranes and that NADPH can only partially prevent this process. These membrane transformations were similar to those observed in the initial steps of the Shibata shift of control samples.     

The influence of glycerol and chloroplast lipids on the spectral shifts of pigments associated with NADPH:protochlorophyllide oxidoreductase from Avena sativa L.

Dark-grown angiosperm seedlings lack chlorophylls, but accumulate protochlorophyllide a complexed with the light-dependent enzyme NADPH:protochlorophyllide oxidoreductase. Previous investigators correlated spectral heterogeneity of in vivo protochlorophyllide forms and a shift of chlorophyllide forms from 680 to 672 nm (Shibata shift) occurring after irradiation, with intact membrane structures which are destroyed by solubilization. We demonstrate here that the various protochlorophyllide forms and the Shibata shift which disappear upon solubilization are restored if the reconstituted complex is treated with plastid lipids and 80% (w/v) glycerol. We hypothesize that the lipids can form a cubic phase and that this is the precondition in vitro and in vivo for the observed spectral properties before and after irradiation.     

Participation of Free Radicals in Photoreduction of Protochlorophyllide to Chlorophyllide in an Artificial Pigment–Protein Complex

The primary stages of protochlorophyllide phototransformation in an artificially formed complex containing heterologously expressed photoenzyme protochlorophyllide-oxidoreductase (POR), protochlorophyllide, and NADPH were investigated by optical and ESR spectroscopy. An ESR signal (g = 2.002; H = 1 mT) appeared after illumination of the complex with intense white light at 77 K. The ESR signal appeared with simultaneous quenching of the initial protochlorophyllide fluorescence, this being due to the formation of a primary non-fluorescent intermediate. The ESR signal disappeared on raising the temperature to 253 K, and a new fluorescence maximum at 695 nm belonging to chlorophyllide simultaneously appeared. The data show that the mechanism of protochlorophyllide photoreduction in the complex is practically identical to the in vivo mechanism: this includes the formation of a short-lived non-fluorescent free radical that is transformed into chlorophyllide in a dark reaction.     

Hg(2+) reacts with different components of the NADPH : protochlorophyllide oxidoreductase macrodomains

The molecular background of Hg (2+)-induced inhibition of protochlorophyllide (Pchlide) photoreduction was investigated in homogenates of dark-grown wheat leaves. Our earlier work showed that 15 min incubation with 10 (-2) M Hg (2+) completely inhibits the activity of NADPH : Pchlide oxidoreductase ( ). Detailed analysis of spectra recorded at 10 K indicated the appearance of emission bands at 638 and 650 nm, which are characteristic for NADP (+)-Pchlide complexes. Fluorescence emission spectra recorded with different excitation wavelengths, fluorescence lifetime measurements and the analysis of acetone extractions revealed that Hg (2+) can also react directly with Pchlide, resulting in protopheophorbide formation. At 10 (-3) M Hg (2+), the phototransformation was complete but the blue shift of the chlorophyllide emission band speeded up remarkably. This indicates oxidation of the NADPH molecules that have a structural role in keeping together the etioplast inner membrane components. We suggest a complex model for the Hg (2+) effect: depending on concentration it can react with any components of the NADPH : Pchlide oxidoreductase macrodomains.     

Analysis of protochlorophyllide reaccumulation in the phytochrome chromophore-deficient aurea and yg-2 mutants of tomato by in vivo fluorescence spectroscopy

The aurea and yellow-green-2 (yg-2) mutants of tomato (Solanum lycopersicum) are unable to synthesize the phytochrome chromophore from heme resulting in a block of this branch of the tetrapyrrole pathway. We have previously shown that these mutants also exhibit an inhibition of protochlorophyllide (Pchlide) synthesis and it has been hypothesised that this is due to feedback inhibition by heme on the synthesis of 5-aminolevulinic acid (ALA). In this study we have investigated Pchlide reaccumulation in cotyledons from etiolated wild-type (WT), aurea and yg-2 seedlings using low-temperature fluorescence spectroscopy. WT cotyledons showed two characteristic Pchlide emission maxima at 630 nm (F630) and 655 nm (F655) respectively, while the aurea and yg-2 mutants contained only phototransformable Pchlide F655. Following a white-light flash to WT cotyledons, reaccumulation of phototransformable Pchlide F655 in the first 30 min was absolutely dependent on the presence of Pchlide F630 before the flash. Reaccumulation of Pchlide F630 was not apparent until at least 2 h after the phototransformation. In contrast, Pchlide F630 never accumulated in aurea cotyledons. The relative rates of both Pchlide F655 and total Pchlide synthesis were approximately twice as high in WT compared to aurea. Measurement of ALA synthesis capacity during this period showed that the reduced rate of Pchlide reaccumulation in aurea was due to an inhibition at this step of the pathway. In addition, feeding of ALA resulted in a substantial and equal increase of non-phototransformable Pchlide in both WT and aurea indicating that aurea cotyledons are capable of accumulating high levels of Pchlide that is not associated to the active site of NADPH:Pchlide oxidoreductase (POR). The implications of these results for the mechanism of inhibition of Pchlide synthesis in phytochrome chromophore-deficient mutants and the role of non-phototransformable Pchlide F630 during plastid development are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photoconversion of long-wavelength protochlorophyll native form Pchl 682/672 into chlorophyll Chl 715/696 in Chlorella vulgaris B-15

By spectral methods, the final stages of chlorophyll formation from protochlorophyll (ide) were studied in heterotrophic cells of Chlorella vulgaris B-15 mutant, where chlorophyll dark biosynthesis is inhibited. It was shown that during the dark cultivation, in the mutant cells, in addition to the well-known protochlorophyll (ide) forms Pchlide 655/650, Pchl(ide) 640/635, Pchl(ide) 633/627, a long-wavelength protochlorophyll form is accumulated with fluorescence maximum at 682 nm and absorption maximum at 672 nm (Pchl 682/672). According to the spectra measured in vivo and in vitro, illumination of dark grown cells leads to the photoconversion of Pchl 682/672 into the stable long wavelength chlorophyll native form Chl 715/696. This reaction was accompanied by well-known photoreactions of shorter-wavelength Pchl (ide) forms: Pchlide 655/650Chlide 695/684 and Pchl (ide) 640/635Chl (ide) 680/670. These three photoreactions were observed at room temperature as well as at low temperature (203–233 K).Abbreviations Chl chlorophyll - Chlide chlorophyllide - Pchlide protochlorophyllide - Pchl protochlorophyll - PS I RC Photosystem I reaction centres. Abbreviations for native pigment forms: the first number after the pigment symbol corresponds to maximum position of low-temperature (77 K) fluorescence band (nm), second number to maximum position of long-wavelength absorption band     

Prevention by Ce3+ of DNA destruction caused by Hg2+ in fish intestine

The interactions between Hg²⁺, Ce³⁺, and the mixuure of Ce³⁺ and Hg²⁺, and DNA from fish intestine in vitro were investigated by using absorption spectrum and fluorescence emission spectrum. The ultraviolet absorption spectra indicated that the addition of Hg²⁺, Ce³⁺, and the mixture of Ce³⁺ and Hg²⁺ to DNA generated an obviously hypochromic effect. Meanwhile, the peak of DNA at 205.2 nm blue-shifted and at 258.2 nm red-shifted. The size of the hypochromic effect and the peak shift of DNA by metal ion treatments was Hg²⁺>Hg²⁺+Ce³⁺>Ce³⁺. The fluorescence emission spectra showed that with the addition of Hg²⁺, Ce³⁺, and the mixture of Ce³⁺ and Hg²⁺ the emission peak at about 416.2 nm of DNA did not obviously change, but the intensity reduced gradually and the sequence was Hg²⁺>Hg²⁺+Ce²⁺>Ce³⁺. Hg²⁺, Ce³⁺, and the mixture of Ce³⁺ and Hg²⁺ had 1.12, 0.19, and 0.41 binding sites to DNA, respectively; the fluorescence quenching of DNA caused by the metal ions all attributed to static quenching. The binding constants (K _A ) of binding siees were 8.98×10⁴ L/mol and 1.02×10⁴ L/mol, 5.12×10⁴ L/mol and 1.10×10³ L/mol, 6.66×10⁴ L/mol and 2.36×10³ L/mol, respectively. The results showed that Ce³⁺ could relieve the destruction of Hg²⁺ on the DNA structure.     

Catalytic function of a novel protein protochlorophyllide oxidoreductase C of Arabidopsis thaliana

In Arabidopsis thaliana Por C has been identified only on sequence homology to that of por A and por B. To demonstrate its catalytic function Arabidopsis thaliana protochlorophyllide oxidoreductase C gene (por c) that codes for the mature part of POR C protein having 335 amino acids was expressed in Escherchia coli cells. The POR C enzyme in the presence of NADPH and protochlorophyllide when incubated in dark formed a ternary complex. When it was excited at 433 nm, it had a fluorescence emission peak at 636 nm. After illumination with actinic cool white fluorescent light, a peak at 673 nm due to chlorophyllide gradually increased with concomitant decrease of 636 nm emission, demonstrating the gradual phototransformation of protochlorophyllide to chlorophyllide. The significance of differential por gene expression in light and dark among different species is discussed.     

Formation of the Pigment Apparatus in Etiolated Barley Leaves under the Influence of Levulinic Acid

The effects of levulinic acid (LA) on the synthesis of pigments and the membrane system of etioplasts were studied in etiolated leaves of barley (Hordeum vulgare L.). Growing in the solution of LA during a six-day period, which started one day after the soaking of seeds, resulted in a retardation of leaf growth, more than a twofold decrease in the level of carotenoids and protochlorophyllide (Pd) in the leaf tissue, and suppression of the synthesis of long-wave form of Pd₆₅₅; these effects depended on the LA concentration. In etioplasts isolated from the seedlings treated with 50 M LA and containing predominantly a short-wave form of Pd with a peak of fluorescence at 632 nm (–196°C), there was a membrane fraction whose location in the sucrose density gradient was identical to that of prolamellar bodies (PLB) in the control plants. The content of Pd and carotenoids in this fraction calculated on a protein basis was 2.46 and 1.3 times lower than in control seedlings, while the relative content of Pd oxidoreductase (POR) essentially did not change. Thus, the suppression of Pd synthesis did not affect translocation of POR from the cytoplasm to the membranes of etioplasts. In the PLB membranes, there was no transfer of energy from the molecules of lipophilic fluorescent probe pyrene (excitation at 337, 278, and 296 nm) to Pd; however, under pigment deficiency, the production therein of pyrene excimer form at the expense of energy transfer from protein tryptophanyls (excitation at 278 and 296 nm) became more efficient, which indicated changes in protein–lipid interactions. The obtained results suggest that the short-wave form of Pd₆₃₂ accumulating in etioplasts under the suppressed synthesis of tetrapyrroles is not a free pigment.     

Tyr275 and Lys279 stabilize NADPH within the catalytic site of NADPH:protochlorophyllide oxidoreductase and are involved in the formation of the enzyme photoactive state

Fluorescence spectroscopic and kinetic analysis of photochemical activity, cofactor and substrate binding, and enzyme denaturation studies were performed with highly purified, recombinant pea NADPH:protochlorophyllide oxidoreductase (POR) heterologously expressed in Escherichia coli. The results obtained with an individual stereoisomer of the substrate [C8-ethyl-C13(2)-(R)-protochlorophyllide] demonstrate that the enzyme photoactive state possesses a characteristic fluorescence maximum at 646 nm that is due to the presence of specific charged amino acids in the enzyme catalytic site. The photoactive state is converted directly into an intermediate having fluorescence at 685 nm in a reaction involving direct hydrogen transfer from the cofactor (NADPH). Site-directed mutagenesis of the highly conserved Tyr275 (Y275F) and Lys279 (K279I and K279R) residues in the enzyme catalytic pocket demonstrated that the presence of these two amino acids in the wild-type POR considerably increases the probability of photoactive state formation following cofactor and substrate binding by the enzyme. At the same time, the presence of these two amino acids destabilizes POR and increases the rate of enzyme denaturation. Neither Tyr275 nor Lys279 plays a crucial role in the binding of the substrate or cofactor by the enzyme. In addition, the presence of Tyr275 is absolutely necessary for the second step of the protochlorophyllide reduction reaction, "dark" conversion of the 685 nm fluorescence intermediate and the formation of the final product, chlorophyllide. We propose that Tyr275 and Lys279 participate in the proper coordination of NADPH and PChlide in the enzyme catalytic site and thereby control the efficiency of the formation of the POR photoactive state.     

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