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-b-dglucoside 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.
Chlorophyll synthetase catalyzes the last step of chlorophyll biosynthesis, namely prenylation (esterification) of chlorophyllide with phytyl diphosphate or geranylgeranyl diphosphate. During investigation of various chlorophyllide derivatives as potential substrates we observed lower esterification with increasing percentages of chlorophyllide a' in epimeric mixtures of chlorophyllides a and a: To avoid epimerization during esterification, we studied the reaction in detail with model compounds [zinc-1 32(R)-methoxy-pheophorbide a and zinc-1 3'(S)-rnethoxy-pheophorbide a, zinc-1 32(R)-methoxy-pyropheophorbide a and zinc-chlorin e,-l3', 1 5'-dimethylester]. We conclude that compounds which have the 13'-carbomethoxy group at the same side of the macrocycle as the propionic side chain of ring D are neither substrates nor competitive inhibitors. Only compounds having the 132-carbomethoxy group at the opposite site are substrates for the enzyme. Naturally occuring chlorophyll a ' must be formed by epimerization after esterification.Chlorophyll a' [a-'prime', 13Z(S)-chlorophyll a ] has been known since 1942 (Strain and Manning, 1942) as a byproduct of isolation of chlorophyll a [13'(R)-chlorophyll a]. Due to the easy epimerization of chlorophyll at C-13' (Hynninen, 1991) it is generally believed that it is formed from chlorophyll a during the extraction procedure. However, increasing evidence has accumulated during the last decade that chlorophyll a' is a natural constituent of higher plants and cyanobacteria (Watanabe et al., 1985 a,b;Kobayashi et al., 1988). Investigations on pigment composition of Chlamydomonas reinhardtii (Maroc and Tremolieres, 1990) and of P700-enriched chloroplasts of higher plants (Maeda et al., 1992) revealed that two chlorophyll a' molecules are situated in the core of photosystem I. Furthermore, the presence of two bacteriochlorophyll g 'molecules in the reaction center of heliobacteria was also described (Kobayashi et al., 1990(Kobayashi et al., , 1991. The question now arises, at which stage of the biosynthetic pathway of the chlorophylls is the prime pigment synthesized, especially whether it is formed before or after esterification of chlorophyllide a.Chlorophyll synthetase catalyzes prenylation of chlorophyllides with geranylgeranyl diphosphate (GerGerP,) or phytyl diphosphate (PhyP,), the last step of chlorophyll biosynthesis (Rudiger et al., 1980). This step is essential for translation and accumulation of chlorophyll a apoproteins (Eichacker et al., 1990(Eichacker et al., , 1992 and probably for stable assembly also for other components of the thylakoid membrane (Paulsen et al., 1990; Rudiger 1992 Rudiger , 1993. Chlorophyll synthetase catalyzes prenylation not only of chlorophyllide a, but also of chlorophyllide b and some modified derivatives (Benz and Rudiger, 1981 and Rudiger, 1992;Vezitskii and Sherbakov, 1987). During our studies on the substrate specificity of chlorophyll synthetase, we observed fractions of chlorophyllide a with a greatly reduced ability for esterificat...
Stabilization of chlorophyll a-binding apoproteins P700, CP47, CP43, D2, and D1 against proteolytic degradation has been investigated through in vitro synthesis of chlorophyll a or Zn-pheophytin a in intact etioplasts from barley. Stabilization of the apoproteins was dependent on the concentration of chlorophyll a or Znpheophytin a. Zn-pheophytin a was superior to chlorophyll a with respect to the concentration of pigment required for an equal yield of the stabilized chlorophyll a protein CP47, CP43, and P700 and for the total yield of chlorophyll a proteins. Zn-pheophytin a was most efficient for stabilizing CP47 and, at an increased concentration, efficient for stabilizing CP43, P700, and D1. Stabilization of apoproteins was highest after de novo synthesis of 90 -300 pmol of Zn-pheophytin a or of about 400 -600 pmol of chlorophyll a/4.2 ؋ 10 7 etioplasts. The yield of stabilized chlorophyll proteins decreased at higher concentrations of Zn-pheophytin a, but was unaffected by higher concentrations of chlorophyll a.The biogenesis of higher plant photosystems I and II requires assembly of nuclear-and plastid-encoded apoproteins with cofactors (e.g. chlorophyll, carotenoid, heme, quinone, iron, and manganese) within the inner plastid membrane system. Chlorophyll a (Chl) 1 is the key chromophore for higher plants to carry out the photosynthetic light reactions and is known to regulate the accumulation of the nuclear-and plastid-encoded apoproteins of the photosystems (1-4).Etioplasts isolated from 4-day-old, dark-grown barley are ideal to study the Chl-dependent accumulation of plastid-encoded photosystem proteins. Etioplasts in barley are formed from proplastids, during early primary leaf and plastid development, which proceeds uninhibited in the absence of light (5, 6).In the dark, etioplasts do not synthesize Chl and neither accumulate plastid-encoded Chl a-binding proteins (Chl aP) (7, 8) nor nuclear-encoded Chl a/b-binding apoproteins (1, 2), although they accumulate protochlorophyllide (Pchlide), a Chl precursor. When plants are illuminated, Pchlide is reduced to chlorophyllide (Chlide) in the plastid by protochlorophyllide oxidoreductase (9) in a light-and NADPH-dependent reaction. Illumination leads to disintegration of the prolamellar body and its dispersal into the primary lamellar layers of the prothylakoid membrane (10). Chlide is esterified with geranylgeranylpyrophosphate (GGPP) to yield Chl GG in a lightindependent enzymatic step catalyzed by chlorophyll synthase (11-13). In addition to the prenylation of the natural substrates Chlide a and b, chlorophyll synthase prenylates modified tetrapyrrol derivatives (14). Pentacoordinate metals (e.g. magnesium or zinc) are accepted as central atoms of the tetrapyrrole substrate, whereas metal-free pheophorbides or typical tetracoordinate central atoms (e.g. copper, nickel) do not act as a substrate for the enzyme (15). In isolated plastids Chl formation is accompanied by the accumulation of the plastid-encoded Chl-binding apoproteins (8) and assembly of the...
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 6 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 I -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.z 1999 Federation of European Biochemical Societies.
A preparation of prolamellar bodies from wheat etioplasts was used as a source for NADPH-protochlorophyllide oxidoreductase (pchlide reductase). The enzyme was solubilized with Triton X-100 after reduction of the endogenous photoconvertible protochlorophyllide a to chlorophyllide a by saturating illumination. Protochlorophylls a and b, protochlorophyllide a and zinc protopheophorbide b were added to the soluble enzyme preparation to determine if they were reduced in the dark or in the light. None of the compounds were reduced (with NADPH) in the dark; however, light-dependent reduction was demonstrated with protochlorophyllide a and zinc protopheophorbide b. The yield was approximately 50% for both substrates. Photoreduction did not occur with the esterified protochlorophylls a and b. Photoreduction of zinc protopheophorbide b, the zinc analogue of protochlorophyllide b, is the first demonstration of the reduction of a chlorophyll-b-related compound by pchlide reductase.
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