mit der ATP-Bildung. Die Reaktionsraten des Aus- Austausch zu katalysieren, welcher entweder durch tausches sind zwar wesentlich kleiner als bei der Phos- Licht, durch einen Säure-Base-Wechsel oder durch phorylierung im Licht und den bisher bekannt ge- ein starkes Reduktionsmittel induziert wird. In allen wordenen ATP-ase-Reaktionen in Chloroplasten (JA- Fällen könnte angenommen werden, daß die Bildung GENDORF und URIBE 15). Die Austauschreaktion ver- eines primären, energiereichen Zustandes oder Zwi- läuft unter ähnlichen Bedingungen wie der lichtindu- schenproduktes in Chloroplasten die Voraussetzung zierte ATP — Pa-Austausch (CARMELI und AVRON 8) für den Beginn der Austauschreaktion darstellt. Die und wie die chemiosmotische Phosphorylierung. Die Ähnlichkeit im Kofaktorenbedürfnis und in der Wir- Induktion der Austauschreaktion durch den Säure- kung von Ammoniumionen als Entkoppler lassen Base-Übergang scheint analog zu ihrer Induktion vermuten, daß zwischen der durch Licht, durch ein durch Licht zu sein. Von besonderem Interesse ist Reduktionsmittel bzw. durch einen pn-Wechsel in- die Tatsache, daß audi ohne pn-Wechsel oder Licht- duzierten Austauschreaktion und der Photophospho- induktion eine nur durch DTT bewirkte Austausch- rylierung bzw. der chemiosmotischen Phosphorylie- reaktion erscheint. rung enge Zusammenhänge bestehen. Zusammenfassend kann gesagt werden, daß
Chloroplasten die Fähigkeit haben, einen ATP — Pa- Wir danken Frau M. SCHOCH für die gewissenhafte technische Mithilfe bei den Versuchen und dem Schwei- 15 A. T. JAGENDORF U. E. URIBE, in: Energy conversion by the photosynthetic apparatus, Brookhaven Symposium 19, zerischen Nationalfonds für wissenschaftliche Forschung 1967, p. 215. für die großzügige finanzielle Unterstützung.
Copper Replacement of Magnesium in the Chlorophylls and Bacteriochlorophyll
W. S. KIM
Exobiology Division, Arnes Research Center, NASA, Moffett Field, Calif.
(Z. Naturforschg. 22 b, 105-1—1061 [1967]; eingegangen am 24. Januar 1967)
Copper chelates of chlorophylls "a" and "b" and an oxidized form of bacteriochlorophyll "a" were prepared and separated by an improved method of column and thin-layer chromatography, and their physical properties and thermodynamics involved in the primary metal replacement reaction were studied. In glacial acetic acid the Mg(II) ions of the photosynthetic pigments were replaced rapidly by Cu(II) ions at 40 — 100° and profound physical changes were noted in the chelation products. Copper chelates were not fluorescent while their parent pigments and pheophy- tins were. A general lowering of absorbance and a blue shift of absorption maxima were observed with the copper complexes. The molar absorptivity values of copper chelates were determined by the metallic microtitration method and the direct analysis of chelated copper by the oxalyldihydra- zide (ODH) of copper method. In the present assay, the primary reaction of copper replacement of Mg(II) in the 3 photosynthetic pigments was the bimolecular SE2 type. The primary reaction lasted only a short time (1 — 5 min) at temperatures of 40 — 90°, and the higher the temperature, the larger the constants of the bimolecular reaction became. On longer treatment, the metal replace- ment reaction was complicated by the increasing content of pheophytin. The reaction rate con- stants became progressively smaller in the order of chlorophyll "a" — "b"-bacteriochlorophyll "a". At 70° the half lives of 20 ^M chlorophylls "a" and "b" and bacteriochlorophyll "a" for the copper replacement were 1.2, 15.2, and 117.4 minutes, respectively. Based on transition state theory, some thermodynamic constants relevant to this primary metal substitution reaction at various temper- atures were calculated, and the possible mechanism involved were discussed.
Copper chelate of chlorophyll "a" (Cu-CHL "a") * ported However, detailed methods for preparing and "b" (Cu-CHL "b") was recently prepared from the purifying these complexes are lacking, as are purified pheophytins (00s), and physicochemical comparative physical data, such as absorptivity and studies of these derivatives have already been re- thermodynamics involved in the replacement reaction
* Abbreviations will be used in the text; CHL, chlorophyll; 2 A. G. TWEET, G. L. GAINES, JR., and W. D. BELLAMY, Nature BCHL, bacteriochlorophyll "a"; Cu-CHL, copper-chelated [London] 202,696 [1964]. chlorophyll; Cu-BCHL, copper-chelate of an oxidized form 3 I. L. KUKHKEVICH and A. I. BULYAK, Urr. Khim. Zh. 31, 943 of BCHL ;
2 Fluorescence and absorbance analysis PH2 + Cu ®^PCU + 2H® For fluorometrie analysis, a Baird Atomic Fluoro- following a SE1 mechanism (here, PMg and PCu in- spec of expanded wavelength, equipped with a red-sen- dicate, respectively, the photosynthetic pigments sitive tube (RCA 7102), was used in addition to the chelated with Mg(II) and Cu(II) and PH2=0#) regular detector (IP 28). The wavelengths of excitation and (2) whether different photosynthetic pigments and emission were calibrated using a Neon Pen-ray react differently in terms of thermodynamics. lamp supplied by Baird Atomic. All the solvents used for the analysis were fluorometrie grade purchased This paper presents (1) detailed methods of pre- from the Harleco Sei. Products, Calif. paring and separating Cu-CHL "a", Cu-CHL "b" Absorption spectra of the photosynthetic pigments, and the copper chelate of an oxidized form of BCHL 00s, and copper chelates were obtained with either a (Cu-BCHL), (2) chromatography and absorbance Cary 14 or a HITACHI 139 spectrophotometer. The wavelengths as well as absorbance of the two instru- of the copper complexes in relation to the parent ments agreed closely. pigments in various solvent systems, (3) some thermodynamic quantities involved in the transition Copper chelates — preparation, isolation, state of the metal replacement, and (4) the possible and quantitation mechanism of the primary reaction of the metal re- To examine the effect of heat upon degradation, in- placement with 3 common photosynthetic pigments. tact photosynthetic pigments were heated at 100° in glacial acetic acid. After 15 minutes at 100° all 3 pig- ments were converted to 00s without noticeable degra- Materials and Methods dation as determined by TLC. Extraction and chromatography of photosynthetic Copper chelation of these pigments was easily pigments achieved by a 10 — 15 minute heating (100°) of the mixture of a pigment and copper acetate in glacial Chlorophylls "a" and "b" were extracted with ace- acetic acid with an approximate molar ratio of 1:5. tone from frozen spinach leaves ground in a blender. Single species of copper chelates with a trace amount The photosynthetic bacteria, Rhodospirillum rubrum of unreacted 00 appeared on a thin-layer plate. For (ATCC No. 277), were cultured in tripticase soy broth routine analyses, 2 — 3 mg of purified pigments were and harvested, and BCHL was extracted as previously used for the chelation. Heated samples were vacuum- described8. The acetone solutions of crude plant or dried, taken up in a small amount of cold ether and bacterial extracts were reduced to a small volume for passed through a dry-packed Adsorbosil column to re- fractional separation by column or thin-layer chromato- move the copper residues. The filtrate was reduced in graphy (TLC). volume and the copper complex was further purified
5 W. S. CAUGHEY and A. H. CORWIN, J. Amer. chem. Soc. 77, 7 J. W. BARNES and G. D. DOROUGH, J. Amer. chem. Soc. 72, 1509 [1955]. 4045 [1950]. 8 J. E. FALK and R. S. NYHOLM, in: A. ALBERT, G. M. BADGER, 8 W. S. KIM, Biochim. biophysica Acta [Amsterdam] 112, and C. W. SHOPPEE (Eds.), Current Trends in Heterocyclic 392 [1966]. Chemistry, Butterworths, London 1958, p. 130. TLC Migration relative to 00-a, sytem CHL-a 00-a Cu-CHL „a" CHL-b 00-b Cu-CHL ,,b" BCHL 00-BCHL Cu-BCHL
1 4.00 1.00 1.50 6.10 1.10 2.00 6.70 1.90 2.30 2 0.71 1.00 0.37 0.22 0.57 0.46 0 0.40 0.28 3 1.60 1.00 1.55 1.62 1.33 1.51 1.58 1.45 1.55
Table 1. Three TLC systems applied to the separation of the photosynthetic pigments, pheophytins, and copper pheohytins. System 1: Plate was coated with Kieselguhr G (0.5 ml thicknss) impregnated with 5% (v/v) triolein and the TCL was run with a mixed solvent of methanol, acetone, and water in a ratio of 6 : 2 : 1 (v/v) ; System 2: Plate was coated with Adsorbosil (0.5 mm thickness) mixed with 25% (w/v) of CaS04 and TCL run with a mixed solvent of petroleum ether (30 — 60° bp), ethyl ether, and glacial acetic acid in a 100 : 50: 1 (v/v) ratio; System 3: Plate was covered with powdered 0.5 ml thick diatomaceous earth (Gas Chrom P) and TCL run with the same solvent as System 1. by preparative TLC on various plates (Table 1). The Results bluish-green copper chelate band was eluted with ether or other solvents for further analysis. Two methods for Extraction and separation of pigments quantitation of the copper chelates were applied; the by chromatography direct analysis of the copper content by oxalyldihydra- Detailed procedures for extracting and separat- zide (ODH) and the indirect metallic microtitration of ing the photosynthetic pigments from spinach and pheophytins, a method originally described by OLIVER and RAWLINSON 9 and modified recently by KIM 10. bacteria are shown in Fig. 1, and the separation
Kinetics and thermodynamics of the metallic EXTRACTION S SEPARATION OF CHLOROPHYLLS substitution reaction .5g OF DRIED PHOTOSYNTHETIC 5 g OF FRESH OR FROZEN SPINACI BACTERIA To measure the reaction rate, tubes containing equi- GRIND IN BLENDER SUSPEND IN 100 ml GRADUALLY ADDING 250 ml molar (20 //M) pigment and copper acetate and their OF 5°C MeOH OF 5°C ACETONE acetate blanks were placed in a constant temperature bath for a definite time. A graduated 15 ml pyrex tube FILTRATION THROUGH BÜCHNER FUNNEL ON VACUUM LINE containing 20 nmole of copper acetate in 1.0 ml of gla- VACUUM DRY AND DISSOLVE CONCENTRATE TO 10 ml IN 3 ml ACETONE cial acetic acid and a tube containing only acetic acid were placed in the bath with their tops open. After 2 — 3 minutes of equilibration, 20 nmoles of pigments COLUMN CHROMATOGRAPHY dissolved in 10 //I acetone was added to each of the 2.5X20 cm COLUMN WITH "KIESELGUHR G", ELUTE WITH MeOH ACETONE: H20 = 6:2:1 V/V tubes. A timer was started simultaneously with the ad- •C f /H a a b dition of the pigment, and after a definite period of —a heating (Fig. 6), tubes were transferred quickly to an \BCHL 2 ice bath. The volume of the tubes was brought to 3.0 TTT ml at 25° by adding glacial acetic acid, and the dif- PURIFICATION OF EACH COMPONENT BY TLC ference spectra were run. The increase of absorbance ON KG, GP, ASB, PV, ETC. of the newly developing visible band of complex (650 Fig. 1. Scheme of extraction and separation of the photosyn- m/u for Cu-CHL "a", 630 m/u for Cu-CHL "b", and thetic pigments and carotenoids from spinach and bacterial 663 m/u for Cu-BCHL) was traced and the reaction cells of Rhodospirillum rubrum. C, carotenoid; 0 Table 2 it should be noted that the Axmax visible absorption peaks of all the copper complexes were Fig. 4. Absorption spectra of bacteriochlorophyll "a" and its derivatives in ether. Note the shifts of SORET band of Cu- BCHL toward red and red band toward blue and the marked reduction of absorptivity of main bands of the copper chelate relative to the parent BCHL "a" and its pheophytin. The li- gand of Cu-BCHL is an oxidized form of bacteriopheophytin. shifted toward shorter wavelengths with respect to their Mg analogues, while the 00 peaks showed a bathochromic shift. The SORET bands of Cu-CHL "a" and "b" also shifted to shorter wavelengths while the SORET band of Cu-BCHL was shifted toward the red side. Table 3 lists the main absorption bands of copper chelates and their absorptivities in some com- WAVELENGTH, mp. mon solvents. The solvent effects were observed by Fig. 2. Absorption spectra of purified chlorophyll "a" and its the changes in intensities of absorbance and the derivatives in ether. Note the blue shift of main absorption spectral position of absorption maxima; in glacial bands and reduction of absorptivity of red band of Cu-CHL "a" relative to the parent pigment, CHL "a". acetic acid the relative absorptivity values of major peaks were the smallest, and in pyridine the "red- 14 C. S. FRENCH, J. H. C. SMITH, M. I. VIRGIN, and R. L. AIRTH, shifts" of the absorption maxima were the greatest. Plant Physiol. 31, 369 [1956]. CHL-a 00 -a Cu-CHL „a" CHL-b 00-b Cu-CHL ,,b" BCHL 00-BCHL Cu-BCHL y. x V. V. X a eä oS c3 kH 7t C2 S § § § c £ § § 1 * E S E E E E c E -< £ -< "I -< c S ^ £ s £ ^ £ a c ^T ? Mr Ms Mr Mr •Mji Mr Mr Ms 430 147.1 409 151.5 414 108.3 453 192.6 434 244.0 430 112.5 358 75.2 357 120.8 425 40.5 662 112.9 667 73.1 648 58.5 645 68.3 653 47.5 626 35.6 770 98.7 750 73.1 666 35.5 Table 2. Main absorption wavelength maxima (/^max) and the millimolar absorptivity (£ mM) of 3 photosynthetic pigments, pheophytins, and copper pheophytinates in ether. Copper Ether Acetone Acetic acid Benzene Pyridine MeOH j * chelate A max |mM** •A max fmM A max |mM A max |mM •A A max £mM A A max |mM Cu-CHL „a" 414 108.3 412 108.8 412 92.5 416 104.9 422 98.7 412 96.6 648 58.5 649 58.4 650 48.5 654 58.6 655 56.6 650 55.5 CuCHL ,,b" 430 112.6 432 117.2 437 93.2 438 108.3 450 100.8 _*** — 626 35.6 627 36.7 627 29.8 630 36.7 635 34.1 — — Cu-BCHL ,,a' 1 425 40.5 428 42.0 428 31.1 432 38.0 435 39.0 — — 666 35.5 665 36.1 672 30.8 672 36.4 675 36.3 — — Table 3. Absorptivity of main absorption maxima of copper-chelated photosynthettic pigments in various solvents. * Wave- length of maximum absorption. ** Millimolar absorptivity. *** Both Cu-CHL "b" and Cu-BCHL "a" are insoluble. Determination of reaction rates the copper chelates formed. The development of visible red absorption peaks, 650 m,a for Cu-CHL Fig. 5 shows the difference spectra obtained from „a", 630 mju of Cu-CHL "b", and 663 mju Cu- 3 photosynthetic pigments reacted with equimolar BCHL, was traced for the kinetic analysis. It is (20 / 650 metallization showing negative peaks of 672 and 655 mju, respectively. Although it is not shown in the figure the difference spectrum of Cu-BCHL de- veloped a negative peak around 770 mju indicating that BCHL was still predominant over its 00. CHL"b" 65° 455 6.00 r 584/ \ 655 438V 5.50 360 5.00 0 10 20 30 40 50 60 350 450 550 650 750 WAVELENGTH, MP. TIME, sec Fig. 5. Difference absorption spectra between copper com- 5.50 r BCHL a plexes and photosynthetic pigment blanks in glacial acetic acid. Equimolar (20 /^M) mixture of a pigment and copper acetate in 3.0 ml of glacial acetic acid was heated for 10 min. at 100°. New outgrowth of absorption bands of SORET and red 5.00 peaks is evident. Note also pheophytination of CHL "a" and "b" showing negative peaks at 672 and 655 Fig. 6. Kinetics of copper chelation with 3 photosynthetic pig- drawn using the reference blank which contained ments. An equimolar (20 /uM) mixture of pigment and copper only the pigment and no copper. A prominent new acetate was heated at temperatures and for times indicated; a, initial concentration of reactants; x, concentration of pig- visible band and a SORET band were developed as ment reacted. When replacement reaction was followed for shorter time intervals (1—5 min.) at temperatures ® h 3 ffl O lO between 40 — 90° instead of 100°, the mode of me- to -h ci t^ o ö tal substitution reaction was bimolecular (SE*) and the pseudo second order rate constants were cal- oo to ® H a ai o culated from the slope of the reciprocal of unreacted pigment versus reaction time at various tempera- tures (Fig. 6). Marked differences in reaction rate H^Mt-^aooo were noted among the three pigments. The rate de- T^ T^ ^ FFL C N IO C M creased in the order: CHL "a" > "b" > BCHL. Al- though it is not indicated in Fig. 6, it was found © IC I> N I> OO o o SO © Ö id TjH Ö that a longer treatment of the reaction mixtures at so r—I I—I 35 SO higher temperatures complicated the reaction owing to the formation of 00s, and the reaction no longer 00 t- O 35 1-H o followed the second order. The half lives of 20 ^M reactants at various tem- peratures were calculated and Fig. 7 shows the cur- oq *a es © © t(5 a Tt< w © © so 35 t> © ©©©©35GOI>© © SO C Determination of thermodynamic quantities of the primary metal replacement reaction •3 f i On the basis of the transition state theory of 8 3 § § o te § 11 s •§ a a ^ ® a EYRING and using the empirical formula of ARRHE- I ^ c6 cö r"H cS 12 o o v x ce o NIUS , the activation energy, , involved in the s S M M « ^ Xg primary bimolecular reaction between the photosyn- thetic pigments and copper was determined. Fig. 8 is the plot between the logarithms of the pseudo s -„-5. =aSi 3 IqS *Q * ^u * second order rate constants, k2', and reciprocals of sN^ cc absolute temperature, l/T. From the negative slopes, values were calculated and the activation enthalpy changes, AZ/+, were determined from the simplified Metalloporphyrins chelated with closed-shell metal ions, such as Mg(II), Ca(II), Zn(II), Cd(II), Ba(II), etc., were fluorescent in the presence of a polar ligand, such as water17. The immediate quenching of the fluorescence of the photosynthetic pigments by copper replacement may then be due to the unpaired 3d electron of the copper ion. The photo-excited n electron may be paired off with the 3d electron reaching the ground state without fluo- rescing. BELLAMY and MURTY ** suggested that the two species of Cu-CHL "a" isolated from their prepara- tion might be caused by allomerization; the normal species absorbed maximally at 420 and 650 m/u in Fig. 8. Graphical determination of activation energy, for ether with an absorptivity ratio of 1.30 while the reaction of equimolar (20 ,MM) mixtures of photosynthetic allomerized form absorbed at 409 and 644 mju with pigments and copper. the ratio of 1.43. In the present experiment where intact photosynthetic pigments and copper acetate formula, E& = AH+ + RT where R is the gas con- were heated in glacial acetic acid at 100° for 10 to stant. The MAXWELL-BOLTZMANN distribution law 13 15 minutes, only a single copper chelating band was was used to calculate the fraction of activated mole- isolated from 3 different TLC systems. In ether, the cules, riß/n. The values of E& and AH^ were the smal- Cu-CHL "a" absorbed maximally at 414 and lest in the metal replacement of chlorophyll "a" and 648 mju (Table 3) and the ratio of absorptivity increased in the order: CHL ,,b" in the order: CHL "a" < "b" 15 W. S. KIM and D. D. FELLER, Federat. Proc. 25, 578 [1966]. 18 M. J. GOUTERMAN, J. chem. Physics 30, 1139 [1959]. 16 J. B. ALLISON and R. S. BECKER, J. diem. Physics 32, 1410 19 M. J. GOUTERMAN, J. chem. Physics 33, 152 3[I960]. [I960]. 20 M. J. GOUTERMAN, J. molecular Spectroscopy 6, 138 [1961]. 17 R. W. LIVINGSTON, Handbuch der Pflanzenzhysiologie, W. 21 H.FISCHER and A.STERN, Die Chemie des Pyrrols, Vol. 11,2, RUHLAND (Ed.), 1. 1960, p. 830. Akademische Verlagsgesellschaft, Leipzig 1940 b, p. 311. ** W. D. BELLAMY and N. R. MURTY, Personal communication. tra of Cu-BCHL in ether showed the SORET at 425 electrostatic attraction. Cu-CHL "a" has only one and the red maximum at 666 mju (Table 3). In view electron-seeking side chain, the 2-vinyl group, while of the general shift of the absorption peaks to the "b" has two, the 2-vinyl and 3-formyl groups. Con- blue side with both Cu-CHLs "a" and "b", it would sequently the "b" molecule would cause a greater be logical to assume that the ligand pigment of Cu- decrease in n electron densities surrounding N BCHL could be the G-2 component rather than the atoms, thus reducing the electrostatic attraction of intact BCHL. Experiments are in progress to con- the copper ion 25. This, in turn, would influence the firm this possibility. reduction of the reaction rate, the increase of AS^, The Mg ion of CHL "a" does not replace the iso- £a, AH^, Kand "s" values, and the decrease of tope 28Mg in aqueous acetone 22. The replacement of AF^ which is not spontaneous (Table 4). The lowest Mg with Cu ions in photosynthetic pigments was rate of copper replacement with BCHL cannot be easily achieved in acetic acid especially at higher explained on the basis of an electrophilic 2-acetyl temperatures (40 — 100°). While the parent pig- group. As postulated earlier, the resulting Cu-BCHL ments were easily dissociated in dilute acid, the cop- may in reality be the Cu-G-2 which may bear two per chelates were not dissociated in glacial acetic hydroxyl groups at the 3 and 4 /?-carbon positions acid, conc. HCl or even conc. H2S04 in room tem- of the porphyrin nucleus, thus giving the complex perature. Only after a prolonged reflux in the latter 3 electron-withdrawing groups in contrast to 1 in acid or wet oxidation with perchloric acid was the Cu-CHL "a" and 2 in Cu-CHL "b". However, there copper ion released. This stability difference in the is no reason to believe that the electrostatic effect is two classes of metallloporphyrins may be due pri- the only cause of rate-limiting in the replacement marily to the fact that Cu-N bond is more covalent reaction. Others, such as the geometry of planar (79%) than Mg-N bond (53%) as calculated from space of the porphyrin molecules, steric hindrance, the electronegativity values of PAULING 23. or the resonance energy encircling the 18-membered The possibility of adding more ligands in the Simpson loop, many also affect the copper in direction of the "2" axis of the metal ion to the such a way that the binding of Cu-N could be in- square d9 copper complexes of the photosynthetic fluenced in different degrees. The relatively higher pigments may well be ruled out from the reported values of AS^ obtained with the formation of Cu- fact that a repulsive force of the filled d2s orbital is BCHL appear to suggest a greater degree of struc- exerted through the "2" axis, while the interaction tural distortion of the molecule. of the porphyrin N atoms with the half-filled dxt-y1 orbital in the planar direction becomes much stron- ger 24. This 1 : 1 stoichiometry of the metal-ligand Acknowledgements binding ratio was further confirmed by the present experimental results that two sets of molar absorp- The author wishes to thank Drs. L. P. ZILL and tivity values determined by metallic microtitration R. D. JOHNSON and Mr. G. E. POLLOCK of Ames Research and the ODH copper analysis method were in good Center, who reviewed the manuscript and made many agreement. useful suggestions. Special thanks are directed to Prof. S. ARNOFF of fowa State College, Ames, Iowa, for his The metal replacement of porphyrin molecules in helpful criticism on the manuscript and stimulating solution may primarily depend upon the power of counsel. 22 S. ARONOFF, Biochim. biophysica Acta [Amsterdam] 60, 193 24 L. E. ORGEL, An Introduction to Transition-Metal Chem- [1962]. istry; Ligand-Field-Theorem, Methuen, London 1960. 23 L. PAULING, The Nature of the Chemical Bond, Cornell 25 J. E. FALK, Porphyrins and Metallophorphyrins, Elsevier Univ. Press, Ithaca 1945, N.Y. Publ. Co., New York 1964, p. 30.