Protochlorophyllide b does not occur in barley etioplasts

Barley (Hordeum vulgare L.) etioplasts were isolated, and the pigments were extracted with acetone. The extract was analyzed by HPLC. Only protochlorophyllide a and no protochlorophyllide b was detected (limit of detection < 1% of protochlorophyllide a). Protochlorophyllide b was synthesized starting from chlorophyll b and incubated with etioplast membranes and NADPH. In the light, photoconversion to chlorophyllide b was observed, apparently catalyzed by NADPH :protochlorophyllide oxidoreductase. In darkness, reduction of the analogue zinc protopheophorbide b to zinc 7-hydroxy-protopheophorbide a was observed, apparently catalyzed by chlorophyll b reductase. We conclude that protochlorophyllide b does not occur in detectable amounts in etioplasts, and even traces of it as the free pigment are metabolically unstable. Thus the direct experimental evidence contradicts the idea by Reinbothe et al. (Nature 397 (1999) 80-84) of a protochlorophyllide b-containing light-harvesting complex in barley etioplasts.     

Pigment-free NADPH:protochlorophyllide oxidoreductase from Avena sativa L. Purification and substrate specificity.

The enzyme NADPH:protochlorophyllide oxidoreductase (POR) is the key enzyme for light-dependent chlorophyll biosynthesis. It accumulates in dark-grown plants as the ternary enzyme-substrate complex POR-protochlorophyllide a-NADPH. Here, we describe a simple procedure for purification of pigment-free POR from etioplasts of Avena sativa seedlings. The procedure implies differential solubilization with n-octyl-beta-D-glucoside and one chromatographic step with DEAE-cellulose. We show, using pigment and protein analysis, that etioplasts contain a one-to-one complex of POR and protochlorophyllide a. The preparation of 13 analogues of protochlorophyllide a is described. The analogues differ in the side chains of the macrocycle and in part contain zinc instead of the central magnesium. Six analogues with different side chains at rings A or B are active substrates, seven analogues with different side chains at rings D or E are not accepted as substrates by POR. The kinetics of the light-dependent reaction reveals three groups of substrate analogues with a fast, medium and slow reaction. To evaluate the kinetic data, the molar extinction coefficients in the reaction buffer had to be determined. At concentrations above 2 mole substrate/mole enzyme, inhibition was found for protochlorophyllide a and for the analogues.     

Photoactive pigment—enzyme complexes of chlorophyll precursor in plant leaves

This review summarizes contemporary data on structure and function of photoactive pigment--enzyme complexes of the chlorophyll precursor that undergoes photochemical transformation to chlorophyllide. The properties and functions of the complex and its principal components are considered including the pigment (protochlorophyllide), the hydrogen donor (NADPH), and the photoenzyme protochlorophyllide oxidoreductase (POR) that catalyzes the photochemical production of chlorophyllide. Chemical variants of the chlorophyll precursor are described (protochlorophyllide, protochlorophyll, and their mono- and divinyl forms). The nature and photochemical activity of spectrally distinct native protochlorophyllide forms are discussed. Data are presented on structural organization of the photoenzyme POR, its substrate specificity, localization in etioplasts, and heterogeneity. The significance of different POR forms (PORA, PORB, and PORC) in adaptation of chlorophyll biosynthesis to various illumination conditions is considered. Attention is paid to structural and functional interactions of three main constituents of the photoactive complex and to possible existence of additional components associated with the pigment-enzyme complex. Historical aspects of the problem and the prospects of further investigations are outlined.     

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.     

The regulation of NADPH-protochlorophyllide oxidoreductases A and B in green barley plants kept under a diurnal light/dark cycle

In etiolated barley (Hordeum vulgare L.) seedlings the light-induced accumulation of chlorophyll is controlled by two light-dependent NADPH-proto-chlorophyllide oxidoreductase (POR; EC 1.6.99.1) enzymes. While the concentration of one of these enzymes (POR A) and its mRNA rapidly decline during illumination, the second POR protein (POR B) and its mRNA remain at an approximately constant level during the transition from dark growth to the light. These results may suggest that only one of the enzymes, POR B, operates throughout the greening process and in light-adapted mature plants while the second enzyme, POR A, is active only in etiolated seedlings at the beginning of illumination. The fate of the two POR proteins and their mRNAs in fully green plants, however, has not been studied yet. In the present work we determined changes in the level of POR A and POR B proteins and mRNAs in green barley plants kept under a diurnal 12 h light/12 h dark cycle. In green barley plants, not only POR B is present but also trace amounts of POR A continue to reappear transiently at the end of a night period and seem to be involved in the synthesis and accumulation of chlorophyll at the beginning of each day.Abbreviations Chl chlorophyll - Chlide chlorophyllide - Lhcb light-harvesting chlorophyll a/b protein - Pchlide protochlorophyllide - POR NADPH-protochlorophyllide oxidoreductase Dedicated to Horst Senger on the occasion of his 65th birthday.We thank Dr. Dieter Rubli for photography and Renate Langjahr for typing. This work was supported by the Swiss National Science Foundation and the ETH-Zürich.     

Characterization of SOC1's Central Role in Flowering by the Identification of Its Upstream and Downstream Regulators

Chlorophyll b is synthesized by the oxidation of a methyl group on the B ring of a tetrapyrrole molecule to a formyl group by chlorophyllide a oxygenase (CAO). The full-length CAO from Arabidopsis (Arabidopsis thaliana) was overexpressed in tobacco (Nicotiana tabacum) that grows well at light intensities much higher than those tolerated by Arabidopsis. This resulted in an increased synthesis of glutamate semialdehyde, 5-aminolevulinic acid, magnesium-porphyrins, and chlorophylls. Overexpression of CAO resulted in increased chlorophyll b synthesis and a decreased chlorophyll a/b ratio in low light-grown as well as high light-grown tobacco plants; this effect, however, was more pronounced in high light. The increased potential of the protochlorophyllide oxidoreductase activity and chlorophyll biosynthesis compensated for the usual loss of chlorophylls in high light. Increased chlorophyll b synthesis in CAO-overexpressed plants was accompanied not only by an increased abundance of light-harvesting chlorophyll proteins but also of other proteins of the electron transport chain, which led to an increase in the capture of light as well as enhanced (40%-80%) electron transport rates of photosystems I and II at both limiting and saturating light intensities. Although the quantum yield of carbon dioxide fixation remained unchanged, the light-saturated photosynthetic carbon assimilation, starch content, and dry matter accumulation increased in CAO-overexpressed plants grown in both low- and high-light regimes. These results demonstrate that controlled up-regulation of chlorophyll b biosynthesis comodulates the expression of several thylakoid membrane proteins that increase both the antenna size and the electron transport rates and enhance carbon dioxide assimilation, starch content, and dry matter accumulation.     

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.     

With chlorophyll pigments from prolamellar bodies to light-harvesting complexes

The biosynthetic chain leading from 5-aminolevulinic acid to chlorophyll is localised to the plastid. Many of the enzymes are nuclear-encoded. NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33) is one such enzyme which is encoded by two different genes and can exist in an A and a B form. Its import into the plastid seems to be facilitated when protochlorophyllide is present in the chloroplast envelope. Within the plastid the reductase is assembled to thylakoids or prolamellar bodies. The specific properties of the reductase together with the specific properties of the lipids present in the etioplast inner membranes promote the formation of the three-dimensional regular network of the prolamellar bodies. The reductase forms a ternary complex with protochlorophyllide and NADPH that gives rise to different spectral forms of protochlorophyllide. Light transforms protochlorophyllide into chlorophyllide and this photoreaction induces a conformational change in the reductase protein which leads to a process of disaggregation of enzyme, pigment aggregates and membranes, which can be followed spectroscopically and with electron microscopy. The newly formed chlorophyllide is esterified by a membrane-bound nuclear-encoded chlorophyll synthase and the chlorophyll molecule is then associated with proteins into active pigment protein complexes in the photosynthetic machinery.     

Kinetic characterisation of the light-driven protochlorophyllide oxidoreductase (POR) from Thermosynechococcus elongatus.

The light-driven enzyme NADPH:protochlorophyllide oxidoreductase (POR) catalyses the reduction of the C17-C18 double bond of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), which is a key regulatory step in the chlorophyll biosynthesis pathway. POR from the thermophilic cyanobacterium Thermosynechococcus elongatus is an attractive system for following the reaction and in the present work we have carried out a detailed steady state kinetic characterisation of this enzyme. The thermophilic POR was shown to have maximal activity at approximately 50 degrees C, which is similar to the growth temperature of the organism. The V(max) was calculated to be 0.53 microM min(-1) and the K(m) values for NADPH and Pchlide were 0.013 microM and 1.8 microM, respectively. The binding properties for both substrates as well as the NADP(+) product have been analysed by using fluorescence emission measurements, which have allowed the dissociation constants for binding to be calculated. These results represent the first steady state kinetic characterisation of a thermophilic version of POR.     

Chloroplast biogenesis: [4-vinyl] chlorophyllide a reductase is a divinyl chlorophyllide a-specific, NADPH-dependent enzyme.

Some properties of [4-vinyl] chlorophyllide a reductase are described. This enzyme converts divinyl chlorophyllide a to monovinyl chlorophyllide a. The latter is the immediate precursor of monovinyl chlorophyll a, the main chlorophyll in green plants. [4-Vinyl] chlorophyllide a reductase plays an important role in daylight during the conversion of divinyl protochlorophyllide a to monovinyl chlorophyll a. [4-Vinyl] chlorophyllide a reductase was detected in isolated plastid membranes. Its activity is strictly dependent on the availability of NADPH. Other reductants such as NADH and GSH were ineffective. The enzyme appears to be specific for divinyl chlorophyllide a, and it does not reduce divinyl protochlorophyllide a to monovinyl protochlorophyllide a. The conversion of divinyl protochlorophyllide a to monovinyl protochlorophyllide a has been demonstrated in barley and cucumber etiochloroplasts and appears to be catalyzed by a [4-vinyl] protochlorophyllide a reductase [Tripathy, B.C., & Rebeiz, C.A. (1988) Plant Physiol. 87, 89-94]. On the basis of reductant requirements and substrate specificity, it is possible that two different 4-vinyl reductases may be involved in the reduction of divinyl protochlorophyllide a and divinyl chlorophyllide a to their respective 4-ethyl analogues.     

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.     

Distinct roles for light-dependent NADPH:protochlorophyllide oxidoreductases (POR) A and B during greening in higher plants

Light-dependent NADPH:protochlorophyllide oxidoreductase (POR), a nuclear-encoded plastid-localized enzyme, catalyzes the photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in higher plants, algae and cyanobacteria. Angiosperms require light for chlorophyll (Chl) biosynthesis and have recently been shown to contain two POR-encoding genes, PorA and PorB , that are differentially regulated by light and developmental state. PorA expression rapidly becomes undetectable after illumination of etiolated seedlings, whereas PorB expression persists throughout greening and in adult plants. In order to study the in vivo functions of Arabidopsis POR A and POR B we have abolished the expression of PorA through the use of the phytochrome A-mediated far-red high irradiance response. Wild-type seedlings grown in continuous far-red light (cFR) display the morphology of white light (WL)-grown seedlings, but contain only traces of Chl and do not green upon transfer to WL. This cFR-induced greening defect correlates with the absence of PorA mRNA, the putative POR A protein, phototransformable Pchlide-F₆₅₅, and with strongly reduced POR enzymatic activity in plant extracts. In contrast, a cFR-grown phyA mutant expresses the PorA gene, accumulates Chl and visibly greens in WL. Furthermore, constitutive overexpression of POR A in cFR-grown transgenic Arabidopsis wild-type seedlings restores Chl accumulation and WL-induced greening. These data demonstrate that POR A is required for greening and that the availability of POR A limits Chl accumulation during growth in cFR. POR B apparently provides a means to sustain light-dependent Chl biosynthesis in fully greened, mature plants in the absence of phototransformable Pchlide-F₆₅₅.     

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.     

Identification of NADPH:protochlorophyllide oxidoreductases A and B: a branched pathway for light-dependent chlorophyll biosynthesis in Arabidopsis thaliana.

Illumination releases the arrest in chlorophyll (Chl) biosynthesis in etiolated angiosperm seedlings through the enzymatic photoreduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), the first light-dependent step in chloroplast biogenesis. NADPH: Pchlide oxidoreductase (POR, EC 1.3.1.33), a nuclear-encoded plastid-localized enzyme, mediates this unique photoreduction. Paradoxically, light also triggers a drastic decrease in the amounts of POR activity and protein before the Chl accumulation rate reaches its maximum during greening. While investigating this seeming contradiction, we identified two distinct Arabidopsis thaliana genes encoding POR, in contrast to previous reports of only one gene in angiosperms. The genes, designated PorA and PorB, by analogy to the principal members of the phytochrome photoreceptor gene family, display dramatically different patterns of light and developmental regulation. PorA mRNA disappears within the first 4 h of greening, whereas PorB mRNA persists even after 16 h of illumination, mirroring the behavior of two distinct POR protein species. Experiments designed to help define the functions of POR A and POR B demonstrate exclusive expression of PorA in young seedlings and of PorB both in seedlings and in adult plants. Accordingly, we propose the existence of a branched light-dependent Chl biosynthesis pathway in which POR A performs a specialized function restricted to the initial stages of greening and POR B maintains Chl levels throughout angiosperm development.     

Identification of the Main Species of Tetrapyrrolic Pigments in Envelope Membranes from Spinach Chloroplasts

The chlorophyll precursors protochlorophyllide and chlorophyllide were identified in purified envelope membranes from spinach (Spinacia oleracea) chloroplasts. This was shown after pigment separation by high performance liquid chromatography (HPLC) using specific fluorescence detection for these compounds. Protochlorophyllide and chlorophyllide concentrations in envelope membranes were in the range of 0.1 to 1.5 nmol/mg protein. Chlorophyll content of the envelope membranes was extremely low (0.3 nmol chlorophyll a/mg protein), but the molar ratios of protochlorophyllide and chlorophyllide to chlorophyll were 100 to 1000 times higher in envelope membranes than in thylakoid membranes. Therefore, envelope tetrapyrrolic pigments consist in large part (approximately one-half) of nonphytylated molecules, whereas only 0.1% of the pigments in thylakoids are nonphytylated molecules. Clear-cut separation of protochlorophyllide and chlorophyllide by HPLC allowed us to confirm the presence of a slight protochlorophyllide reductase activity in isolated envelope membranes from fully developed spinach chloroplasts. The enzyme was active only when envelope membranes were illuminated in the presence of NADPH.     

Etioplast differentiation in arabidopsis: both PORA and PORB restore the prolamellar body and photoactive protochlorophyllide-F655 to the cop1 photomorphogenic mutant.

The etioplast plastid type of dark-grown angiosperms is defined by the accumulation of the chlorophyll (Chl) precursor protochlorophyllide (Pchlide) and the presence of the paracrystalline prolamellar body (PLB) membrane. Both features correlate with the presence of NADPH:Pchlide oxidoreductase (POR), a light-dependent enzyme that reduces photoactive Pchlide-F655 to chlorophyllide and plays a key role in chloroplast differentiation during greening. Two differentially expressed and regulated POR enzymes, PORA and PORB, have recently been discovered in angiosperms. To investigate the hypothesis that etioplast differentiation requires PORA, we have constitutively overexpressed PORA and PORB in the Arabidopsis wild type and in the constitutive photomorphogenic cop1-18 (previously det340) mutant, which is deficient in the PLB and Pchlide-F655. In both genetic backgrounds, POR overexpression increased PLB size, the ratio of Pchlide-F655 to nonphotoactive Pchl[ide]-F632, and the amount of Pchlide-F655. Dramatically, restoration of either PORA or PORB to the cop1 mutant led to the formation of etioplasts containing an extensive PLB and large amounts of photoactive Pchlide-F655.     

POR - import and membrane association of a key element in chloroplast development

The development of proplastids or etioplasts to chloroplast is visualized by the accumulation of chlorophyll in leaves of higher plants. The biosynthesis of chlorophyll includes a light-dependent reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide). This light-dependent step is catalysed by the nucleus-encoded NADPH:Pchlide oxidoreductase (POR, EC 1.6.99.1). POR is active within plastids and therefore has to be translocated over the plastid envelope membranes. The import of chloroplast proteins seems to follow a general import pathway using translocons at the outer and inner envelope membrane. POR cross-linking to Toc75, one of the major translocon components at the outer envelope membrane, indicates its use of the general import pathway. However, since variations exist within the so-called general import pathway one has to consider previous data suggesting a novel totally Pchlide-dependent import pathway of one POR isoform, PORA. The suggested Pchlide dependency of POR import is discussed since recent observations contradict this idea. In the stroma the POR transit peptide is cleaved off and the mature POR protein is targeted to the plastid inner membranes. The correct and stable association of POR to the membrane requires the cofactor NADPH. Functional activity of POR calls for formation of an NADPH–Pchlide–POR complex, a formation that probably takes place after the membrane association and is dependent on a phosphorylation reaction.     

On the nature of the two pathways in chlorophyll formation from protochlorophyllide

The kinetics of formation of esterified chlorophyll in etiolated barley (Hordeum vulgare L.) leaves after illumination with a single flash was studied. It was found that after partial (14–24%) and after full photoreduction of protochlorophyllide, the same quantity of esterified products appear during the first 5 s after the flash. The rest of formed chlorophyllide was esterified in a slow process during at least 30 min at 15 °C. The product of fast esterification can be correlated with ‘short-wavelength’ chlorophyll, characterized by a fluorescence emission peak at 673–675 nm. This is the only chlorophyll form detectable within 20 s after partial (14%) photoconversion, and it appears at the same time as the shoulder of the chlorophyll(ide) fluorescence after full photoconversion. The main product after full photoconversion shows a fluorescence at 689 nm shifting in darkness within 15 s to 693 nm and then within 30 min to 682 nm (Shibata shift). The slow esterification proceeds with similar kinetics as the Shibata shift. We propose that the fast esterification of only part of total chlorophyllide after full photoconversion of protochlorophyllide in etiolated leaves reflects the restricted capacity of the esterifying system. The slow esterification of the residual chlorophyllide may be time-limited by its release from protochlorophyllide oxidoreductase, by disaggregation of prolamellar bodies and by diffusion of tetraprenyl diphosphates towards chlorophyll synthase. 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.