Chlorophyll Biosynthesis: Various Chlorophyllides as Exogenous Substrates for Chlorophyll Synthetase Jürgen Benz and Wolfhart Rüdiger Botanisches Institut, Universität München, Menziger Str. 67, D-8000 München 19
Z. Naturforsch. 36 c, 51 -5 7 (1981); received October 10, 1980
Dedicated to Professor Dr. H. Merxmüller on the Occasion of His 60th Birthday
Chlorophyllides a and b, Protochlorophyllide, Bacteriochlorophyllide a, 3-Acetyl-3-devinylchlo- rophyllide a, Pyrochlorophyllide a, Pheophorbide a The esterification of various chlorophyllides with geranylgeranyl diphosphate was investigated as catalyzed by the enzyme chlorophyll synthetase. The enzyme source was an etioplast membrane fraction from etiolated oat seedlings ( Avena sativa L.). The following chlorophyllides were prepared from the corresponding chlorophylls by the chlorophyllase reaction: chlorophyllide a (2) and b (4), bacteriochlorophyllide a (5), 3-acetyl-3-devinylchlorophyllide a (6), and pyro chlorophyllide a (7). The substrates were solubilized with cholate which reproducibly reduced the activity of chlorophyll synthetase by 40-50%. It was found that the following compounds were good substrates for chlorophyll synthetase: chlorophyllide a and b, 3-acetyl-3-devinylchloro- phyllide a, and pyrochlorophyllide a. Only a poor or no reaction was found with protochloro phyllide, pheophorbide a, and bacteriochlorophyllide. This difference of reactivity was not due to distribution differences of the substrates between solution and pelletable membrane fraction. Furthermore, no interference between good and poor substrate was detected. Structural features necessary for chlorophyll synthetase substrates were discussed.
Introduction Therefore no exogenous 2 was applied. The only substrate was 2 formed by photoconversion of endo The last steps of chlorophyll a (Chi a) biosynthe genous Protochlide (1) in the etioplast membrane. sis are the photoconversion of Protochlide (1) to Thus the substrate source was limited with regard to Chlide a (2) and the subsequent esterification to substrate nature and amount. Furthermore, chloro Chi a. The latter reaction is catalyzed by the re phyll synthetase activity could only be determined cently detected enzyme chlorophyll synthetase [1]. in illuminated membranes which left the question This enzyme needs - besides Chlide a - tetraprenyl open whether the enzyme had been activated by alcohols plus ATP or the diphosphate derivatives of light or not. tetraprenyl alcohols as substrates. The best sub We describe here experiments with exogenous strate is geranylgeranyl diphosphate (GGPP) which chlorophyllides in which we adopted the system of leads to Chi aoG (2 a). This is then stepwise hydro Griffiths [4] for solubilization of Protochlide with genated via Chi aH2GG and Chi aH4GG to Chi aP (2 b) cholate. This enabled us to vary the substrate con in vitro [2] and in vivo [3]. centration and to test the following compounds as A problem of previous experiments with chloro possible substrates: pheophorbide a (3), chlorophyll phyll synthetase was the limited source of the ide b (4), bacteriochlorophyllide a (5), 3-acetyl-3- second substrate, Chlide a (2). This substrate is devinylchlorophyllide a (6), and pyrochlorophyllide a barely water-soluble. Solvents (e. g. acetone) or de (7). Furthermore the experiments were carried out tergents ( e. g. triton X 100) which can solubilize 2 with etioplast membranes without any illumination. destroy the activity to chlorophyll synthetase [1],
Materials and Methods Abbreviations: Chi, chlorophyll; Chlide, chlorophyllide; Protochi, protochlorophyll; Protochlide, protochlorophylli Preparation of Protochlide (1): The shoots (120 g de; GGPP, geranylgeranyl diphosphate; subscripts for chlo rophylls and pheophytins containing GG, geranylgeraniol; fresh weight) of 7.5 days old etiolated oat seedlings H2GG, dihydrogeranylgeraniol; H4GG, tetrahydrogeranyl- (Avena sativa L. var. Parzival, Baywa, München) geraniol; P, phytol. were harvested in dim-green safelight and frozen in Reprint requests to Professor Dr. Wolfhart Rüdiger. liquid nitrogen for 2 minutes. The frozen tissue was 0341-0382/81/0100-0051 $01.00/0 then homogenized (Waring blender) and exhaus- 52 J. Benz and W. Rüdiger • Chlorophyll Biosyntheses
NADPH ,H
'chlorophyll synthetase GG PP
NADPH, H
2b 2a
5 6 7 J. Benz and W. Rüdiger ■ Chlorophyll Biosyntheses 53 tively extracted with acetone (total volume 500 ml). acetyl-3-devinyl-chlorophyllide a (6) and pyrochlo- The acetone solution was concentrated in vacuo to rophyllide a (7) (yield 10-20%), respectively. about 250 ml and mixed with 100 ml diethyl ether. Chlorophyll synthetase test: Broken etioplasts were Then water was added until the phases separated. prepared from 7.5 days old, etiolated oat seedlings The diethyl ether phase was washed with water as previously described [1] but using ultrasonic (2-3 times 100 ml) and diluted with 100 ml petro power (1x5 sec) in 0.05 m Hepes buffer (pH 7.2) for leum benzene (b. p. 40-60 °C). Extraction into lysis of etioplasts. Cofactors were added as pre methanol/NH3 and reextraction into diethyl ether viously described [1] but omitting NADPH if not were performed according to Griffiths [4], otherwise stated. The respective chlorophyllide sub strate in the diethyl ether stock solution was, after Preparation of Chlide a (2) and Chlide b (4): addition of the same volume petroleum benzene and Leaves from Heracleum lanatum (70 g fresh weight) 0.1vol. 90% methanol containing 0.1% sodium cholate were frozen in liquid nitrogen and exhaustively ex (Sigma, München), transferred into aqueous cholate- tracted with acetone (total volume 1000 ml). The containing solution according to Redlinger and Apel solid residue (acetone powder) was dried at room [9]. A ratio of 6 nmol chlorophyllide: 1 mg cholate: temperature under air (14 g dry weight) and used 1 ml incubation volume was maintained in all ex for extraction of chlorophyllase (see below). The periments. GGPP was added in 18-25 fold molar acetone solution was mixed with diethylether, excess over the exogenous chlorophyllide substrate. washed with water, dried over sodium sulfate and The mixture was incubated in the dark (without evaporated. The residue was dissolved in petroleum illumination!) for 60 min at 28 °C. Chlorophyll pig benzene (b. p. 40-60 °C). Chlorophylls were isolat ments were extracted, demetalled to pheophytins ed by chromatography on a powder sugar column and analyzed by HPLC as previously described [1]. according to Hager [5] with petroleum benzene con For calculation of pigment concentrations, spectra taining 0-1% n-propanol. Yield 10 mg Chi a (2b), were run in diethylether. The following molar ex 3.5 mg Chi b (4 b). tinction coefficients (1 • mmol-1 • cm-1) were calcu For the chlorophyllase reaction, 1 g acetone pow lated from data of French [10]: Protochlorophyll- der of Heracleum lanatum (see above) were ex (ide) e624 = 35.6; protopheophytin/protopheophor- tracted with 15 ml 0.02 m citrate buffer (pH 7.2) con bide e565 = 22.0; Chl(ide) a £662 = 90.2; pheophytina taining 0.5 M NaCl and 0.001 NaN3 according to £667 = 55.5; Chl(ide) b £644 = 56.3; pheophytin b £655 Bacon and Holden [6]. The chlorophyllase solution = 37.3. was mixed with 2.2 mg of the respective chlorophyll Further molar extinction coefficient were taken (2 b or 4 b) in 15 ml acetone and incubated 2.5 h at from [7]: 3-acetyl-3-devinylchlorophyll(ide) a £676 = 30 °C in the dark. The reaction was stopped by 65.2; 3-acetyl-3-devinyl-pheophytin £682 = 39.1; bac- addition of 40 ml acetone. The pigments were then teriochl(ide) a £770 = 96.0; bacteriopheophytin a £750 extracted into ethyl acetate. This solution was evap = 60.5. orated in vacuo and the residue dissolved in diethyl Pigment fractions isolated by HPLC were hydro ether/petroleum benzene (1:1, v/v). The chloro- lyzed with methanolic KOH [1], The alcohol was phyllide (2 or 4) was purified according to Griffiths analyzed by gas chromatography on a OV 101 capil [4], Yield 10-25%. lary column (25 m), carrier 0.8 atü He, splitless, in Further substrates: Pheophorbide a (3) was pre jector temperature 130 °C, column temperature pared from Chlide a by demetallation with hydro 180 °C. chloric acid [3]. Bacteriochlorophyll a (from Rhodo- pseudomonas spheroides), 3-acetyl-3-devinylchloro- Results phyll a obtained from bacteriochlorophyll a by de hydrogenation with dichlorodicyanobenzoquinone The low solubility of Chlide in aqueous buffers [7] and pyrochlorophyll obtained from Chi a by de- did not allow the direct application of the previously carboxymethylation in pyridine [8], were gifts from used [1] buffer system for exogenous Chlide and Dr. H. Scheer, München. The hydrolysis catalyzed similar substrates. Griffiths [4] and Redlinger and by chlorophyllase (see above) yielded the corres Apel [9] solubilized Protochlide with cholate. Chlide a ponding substrates bacteriochlorophyllide a (5) 3- (2) and similar substrates can be solubilized with 54 J. Benz and W. Rüdiger • Chlorophyll Biosyntheses cholate as well. We therefore investigated at first the influence of cholate upon the esterification of endo 40- genous Chlide a (/. e. the pigment present in the illu minated etioplast membrane [1]) (Table I). In ac cordance with the data of Griffiths [4], cholate did not significantly reduce the phototransformation of 30- Protochlide to Chlide. Esterification of endogenous -O Chlide was, however, inhibited by cholate. Interest 0) ingly, the relative inhibition of esterification was of (V the same order of magnitude (40-60%) with exoge
Substrate applied Esterified
[nmol] [nmol] [%]
Chlide a 36 18.9 52 -1- pheophorbide a 13 Chlide a 23 10.1 44 + bacteriochlide a 10 0.75 7.5
The low esterification of pheophorbide a contrary retention time(min) to Chlide a caused the question whether the distri Fig. 2. High Performance Liquid Chromatogram of the bution of both pigments would be different between main fractions of esterified pigments after removal of mag the (pelletable) membrane fraction and the (cholate nesium. 1 = 3-acetyl-3-devinyl-pheophytin a< 3G, ^max = 680 containing) solution. Griffiths [4] had determined nm; 2 =pheophytin boG, '-max = 655 nm; 3 = pheophytin aoG, /'-max = 667 nm; 1 — 3 as mixture (----- ); 4 = pyropheo- the distribution of Protochlide between such a phytin aoG, /max = 667 nm ( ------). membrane fraction and the cholate solution but did 56 J. Benz and W. Rüdiger • Chlorophyll Biosyntheses not give a time dependency for the distribution. Our or the corresponding diphosphates, less efficiently results show (Table III) that the bulk of Chlide a with triprenyl alcohol and not with a diprenyl and pheophorbide a was found in the membrane alcohol or other alcohols whereas chlorophyllase fraction soon after the application. The difference in which does not use diphosphates has no preference esterification is therefore not due to a distribution as to the structure of the alcohol. Chlorophyll effect. synthetase has a higher specificity than chlorophyl For a further comparison we mixed Chlide a (as a lase for the second substrate, too. All substrates good substrate) with pheophorbide a or bacterio- listed in Table II (except Protochlide) had been chlorophyllide a, respectively (as poor substrates). prepared by the action of chlorophyllase on the Analysis of the esterified products revealed (Table corresponding chlorophylls. The back reaction must IV) that esterification of Chlide a was not inhibited therefore also be catalyzed by chlorophyllase with by the poor substrates and that the low esterification the proper substrate concentrations. Chlorophyll of bacteriochlorophyllide a was not influenced by synthetase on the contrary shows nearly no reaction the presence of Chlide a. with bacteriochlorophyllide a and pheophorbide a. The biosynthesis of bacteriochlorophyll in photo synthetic bacteria is probably mediated by a chloro Discussion phyll synthetase of different specificity which has One important aspect of the present experiments still to be detected. Rhodopseudomonas spheroides was the esterification of Chlide in the dark. So far mutants which accumulate bacteriochlorophyllide [1, 3, 11], esterification in vivo and in vitro had only and lack the esterification step [13] are possibly de been studied in illuminated membranes because the ficient in this enzyme. Pheophytin a which is photoconversion of Protochlide to Chlide a was an probably present in the reaction center of obligatory precondition for those studies. We used photosystem I [14] is probably formed by de- here etioplast membranes which had not received metallation of Chi a rather than by ester light at any time. This was controlled by the pre ification of pheophorbide a (Table II). Interest sence of only Protochl(ide) with no trace of Chl- ingly, pheophorbide a shows about the same dis (ide) a in the membranes. The esterification of tribution between solution and membrane as exogenous Chlide a with chlorophyll synthetase of Chlide a (Table III). We do not yet know anything these membranes (Fig. 1, Tables II and IV) means about eventual receptor proteins and the distribu that no (direct or indirect) light activation is neces tion of these substrates within the membrane. But sary for this enzyme activity. It can furthermore be because pheophorbide a and bacteriochlorophyllide a concluded that the binding site for Chlide (at do not interfere with the esterification of Chlide a chlorophyll-synthetase) is different from the binding (Table IV), the most simple explanation would be that site for Protochlide (at protochlorophyllide-reduc- the latter substrate is specifically bound to chloro tase [4] because the presence of Protochlide in the phyll synthetase whereas the former both are not. membrane did not inhibit the synthetase reaction. Within this model, structural features for the Another aspect of the present work was the com binding to chlorophyll synthetase can be deduced. parison of substrate specificity of chlorophyll syn Essential structural elements for the substrate are the thetase and chlorophyllase. The latter enzyme has central magnesium (results of substrate 3 versus 2), repeatedly been discussed as biosynthetic enzyme the hydrogenated ring D (1 versus 2), and the un responsible for esterification of chlorophyllide (re saturated ring B (5 versus 6) whereas modification view see [12]). The previous observation that chloro of side chains has nearly no effect upon the binding phyll synthetase is sensitive against organic solvents to chlorophyll synthetase (2, 4, 6, 7). We consider 6 and detergents (contrary to chlorophyllase [1]) was and 7 as artificial substrates which have only inter extended here to the mild detergent cholate (Table est for comparative structural considerations. The I). Interestingly, the esterification in the presence of reaction with chlorophyllide b (4) may be of physio cholate was even somewhat better with exogenous logical importance because it has been postulated (Fig. 1) than with endogenous Chlide (Table I). It that the oxidation of the 7-methyl side chain (lead has already been pointed out [1] that chlorophyll ing from the chlorophyll a to the b series) occurs synthetase acts only with tetraprenyl alcohols + ATP prior to the esterification reaction [15]. J. Benz and W. Rüdiger • Chlorophyll Biosyntheses 57
Acknowledgement We thank the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg for support of this work.
[1] W. Rüdiger, J. Benz, and C. Guthoff, Eur. J. Biochem. [10] C. S. French, Handbuch der Pflanzenphysiologie, 109,193-200(1980). (W. Ruhland, ed.), Bd. 5/1,252-297 (1960). [2] J. Benz, Ch. Wolf, and W. Rüdiger, Plant Sei. Letters [11] H. Kasemir and G. Prehm, Planta 132, 291-295 19,225-230(1980). (1976). [3] S. Schoch, U. Lempert, and W. Rüdiger, Z. Pflanzen- [12] L. Bogorad, Chemistry and Biochemistry of Plant physiol. 83,427-436 (1977). Pigments, (T. W. Goodwin, ed.), 2nd edn. V ol. 1, [4] W. T. Griffiths, Biochem. J. 174,681-692 (1978). pp. 64-148, Academic Press, London 1976. [5] A. Hager, Planta 48,552-621 (1957). [13] A. E. Brown, F. A. Eiserling, and J. Lascelles, Plant [6] M. F. Bacon and M. Holden, Phytochemistry 9, Physiol. 50, 743-746 (1972). 115-125 (1970). [14] L. Anderson and H. Egneus, Physiol. Plant. 47,11-14 [7] J. R. L. Smith and M. Calvin, J. Amer. Chem. Soc. (1979). 88,4500-4506(1966). [15] H. Oelze-Karow and H. Mohr, Photochem. Photobiol. [8] F. C. Pennington, H. H. Strain, W. A. Svec, and J. J. 27,189-193 (1978). Katz, J. Amer. Chem. Soc. 86,1416-1426 (1964). [9] T. E. Redlinger and K. Apel, Arch. Biochem. Biophys. 200,253-260(1980).