ACTION SPECTRUM for FERRICYANIDE PHOTOREDUCTION and REDOX POTENTIAL of CHLOROPHYLL 683* by TAKEKAZU Horiot and ANTHONY SAN PIETRO

1226 BIOCHEMISTRY: HORIO AND SAN PIETRO PROC. N. A. S.

ment had taken place, the centrifugation carried out at 00 did not reverse. This specific binding appears to he the initial and sometimes rate-limliting step in amino acid polymerization. The author would like to express his indebtedness to Dr. Fritz Lipmann, in whose laboratory this investigation was carried out, for his support and encouragement. * This work was supported in part by a grant from the National Science Foundation, and represents a portion of the thesis submitted by the author to the faculty of The Rockefeller Institute in partial fulfillment of the requirements for the degree of Doctor of Philosophy. t Present address: Biological Laboratories, Harvard University. 1 Spyrides, G. J., and F. Lipmann, these PROCEEDINGS, 48, 1977 (1962). 2 Barondes, S. H., and M. W. Nirenberg, Science, 138, 810 (1962). 3 Gilbert, W., J. Mol. Biol., 6, 374 (1963). 4Spyrides, G. J., Ph. D. dissertation, The Rockefeller Institute (1963). 5 Conway, T. W., these PROCEEDINGS, 51, 1216 (1964). 6 Nakamoto, T., and F. Lipmann, Federation Proc., 23, 219 (1964). 7Lubin, M., Biochim. Biophys. Acta, 72, 345 (1963). 8 Nakamoto, T., T. W. Conway, J. E. Allende, G. J. Spyrides, and F. Lipmann, in Synthesis and Structure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 227. 9 Kaji, A., and H. Kaji, Biochem. Biophys. Res. Commun, 13, 186 (1963). 10 Cannon, M., R. Krug, and W. Gilbert, J. Mol. Biol., 7, 360 (1963).

ACTION SPECTRUM FOR FERRICYANIDE PHOTOREDUCTION AND REDOX POTENTIAL OF CHLOROPHYLL 683* BY TAKEKAZU HORIOt and ANTHONY SAN PIETRO

CHARLES F. KETTERING RESEARCH LABORATORY, YELLOW SPRINGS, OHIO Communicated by Martin D. Kamen, April 27, 1964 Nearly thirty years ago, Hill1-4 discovered that chloroplasts isolated from green leaves liberate molecular oxygen upon illumination when an appropriate hydrogen acceptor (Hill oxidant) is provided. It is now generally accepted that this reaction represents the photochemical step in photosynthesis. Meanwhile, the possible photochemical function of the pigments associated with chloroplasts, namely, the chlorophylls and carotenoids, has been examined by a number of investigators.5-'0 Chen measured the action spectrum for photoreduction of 2,6-dichlorophenol indophenol by chloroplasts from Swiss chard.11 The action spectrum for this reaction coincided well with the action spectrum for photosynthetic oxygen evolution as well as with the absorption spectrum of the chloroplasts. Action spectra for NADP photoreduction and the coupled phosphorylation, measured by San Pietro et al.,12 Black et al., 13 and Jagendorf et al.14 coincided with the absorption spectrum of the chloroplasts. We have studied the action spectra for the photoreduction of ferricyanide and the coupled phosphorylation and found that the resultant action spectra differ from the absorption spectrum of the chloroplasts. In addition, we have determined the redox potential of the chlorophyll component with an absorption maximum at 683 mtu. Downloaded by guest on September 30, 2021 VOL. 51, 1964 BIOCHEMISTRY: HORIO AND SAN PIETRO 1227

Materials and Methods.-Washed chloroplasts were prepared from fresh spinach leaves according to the method of Keister et al.15 or of Stiller and Vennesland.16 Similar results were obtained with either chloroplast preparation. Chlorophyll content of chloroplasts was determined by the method of Arnon. 17 Absorption spectra were measured with a Cary model 14 spectrophotometer. In some experiments, the spectrophotometer was equipped with a Cary scatter attachment. Photoreduction of ferricyanide and the coupled phosphorylation were measured by the method of Stiller and Vennesland16 except that the amount of radioactive orthophosphate (Pi32) incorporated into ATP was determined according to the method of Nielsen and Lehninger'8 as modified by Avron.'9 Components of the reaction mixture were 0.05 Al Tris-HCl buffer, pH 8, 0.035 N NaCl, 0.002 Al potas- sium ferricyanide, 0.005 M 1\gCl2, 0.0025 M ADP, 0.0025 M Pi32, and spinach chloroplasts (equivalent to approximately 20 ,ug/ml of chlorophyll) final vol- ume, 2 ml. Reactions were carried out at 150 in the light or dark for 3 min in capped cylindrical cuvettes (2 cm in diameter, 1 cm optical path). Reactions were terminated by the addition of 0.50 ml of ice-cold 20 per cent trichloroacetic acid solution, followed by centrifugation. The supernatant solutions were used to determine the change in absorption at 420 my and the amount of Pi32 incorporated into ATP. Photosynthetic pyridine nucleotide reductase (PPNR) was purified from spinach leaves as described by San Pietro.20 The purification was carried through the Dowex-Bentonite step20 and the protein concentrated by adsorption on and elution from DEAE cellulose. The ratio of absorbance at 330 m/A to that at 280 m/A of the PPNR was about 0.65. The redox potential of the chlorophyll component with an absorption maximum at 683 mui was determined according to the method of Davenport and Hill.21 Experimental Results.-Photoreduction of ferricyanide: The action spectra for both the photoreduction of ferricyanide and the coupled phosphorylation are very similar (Fig. 1), but significantly different from the absorption spectrum of the chloroplasts (Fig. 2). It is known that as chloroplasts age they lose photophos- phorylative activity more rapidly than they do photoreductive activity. The action spectrum for ferricyanide photoreduction was essentially the same regardless of the age of the chloroplasts. The ratios of activity with 650 mp illumination to that with 680 m1A illumination (Fig. 1) were 1.01 and 0.93, respectively, for ferricyanide photoreduction and the coupled phosphorylation with fresh chloroplasts, and 1.19 for ferricyanide photoreduction with aged, nonphotophosphorylating chloroplasts. Effect offerricyanide on absorption spectrum of spinach chloroplasts: With spinach chloroplasts, the absorption maximum at 679 mMu decreases upon the addition of ferricyanide and shifts to a shorter wavelength (Fig. 3), i.e., toward 670 m/u. The difference spectrum, "+ none" minus "+ ferricyanide," indicated that only the component possessing an absorption maximum at 683 m/u was bleached (Fig. 4). Relative to the ferri-ferrocyanide system, the redox potential of the component ab- sorbing at 683 mp was determined to be approximately +0.55 volt at pH 8. Photoreduction of PPNR: The anaerobic photoreduction of PPNR in the presence of ascorbate is shown in Figure 5. When the photoreduced PPNR was re- Downloaded by guest on September 30, 2021 1228 BIOCHEMISTRY: HORIO AND SAN PIETRO PROC. N. A. S.

. . . . 100100/Q 0.13 901 8g0 REDUCTION 0.8.

>70 -

I- 60 .,;s0 A ATP FORMATION 0.6. N50i Z40- 0.5 0F0.4- 20-

10 0.3.

600 650 700 0.1 . WAVELENGTH (mya) FIG. 1.-Action spectra for the 0.1 photoreduction of ferricyanide and the coupled phosphorylation. ° . .O Monochromatic light was ob- -°Goo Leo 70700 5 tained from a 1-meter grating WENWAVELENGTH (uy) monochromator (Bausch and Lomb) with a Sylvania Sun Gun FIG. 2.-Absorption spectrum as light source. The action spectra of spinach chloroplasts. Chloro- were determined with equal inci- plasts (equivalent to 12.5 jg per dent light energy (104 ergs/cm2/ ml of chlorophyll) in 0.05 M Tris- sec). At the wavelength of 660 HC1 buffer, pH 8, containing 0.01 mg, the reaction rates were 166 M KCL. The spectrophotometer ,umoles of ferricyanide reduced and was equipped with the scatter at- 67 umoles of ATP formed per hr tachment. per mg of chlorophyll.

0.8 0.705~~~~/ 0.6

z. 0.4U4M A. 0.3 T

0.2 z 0.1 0.~~~~~~~~~~~~~~~~bUi. 600 650 700...... WAVELENGTH Amy) 550 600 650 700 WAVELENGTH (mp) FIG. 3.-Effect of ferricyanide on absorption spectrum of spinach FIG. 4.-Dlfference spectrum of chloroplasts. Chloroplasts (equiv- spinach with and alent to 20 ,ug per ml of chloro- without ferricyanide.chloroplaste Experi- phyll) in 0.05 M Tris-HCl buffer, mental conditions as for Fig. 3 pH 8, containing 0.01 M KC1. except that the amount of The absorption spectra were meas- plasts present was equivalentchtoro-to 40 ured everv 10 min after the addi- Mg per ml of chlorophyll. tion of ferricyanide (0.29 M). Downloaded by guest on September 30, 2021 VOL. 51, 1964 BIOCHEMISTRY: HORIO AND SAN PIETRO 1229

0.9 .I..

0.8 (C)+NADP IN DARK (D) FIG. 5.-Photoreduction of PPNR and reoxidation by NADP. 0.7 _ The reaction mixture, containing ORIGINALOXIDIZED PPNR (A) chloroplasts equivalent to 3.6 Ag 0.6 V per ml of chlorophyll, 0.05 M ascorbate, PPNR, and buffer, was prepared in a Thunberg-type cu- - Ecu vette. To provide anaerobic con- A\\\A ditions, the cuvette was evacuated 0.4 -. and flushed with argon several

times and finally filled with argon. 0.3 - (A) prior to illumination; (B) after 5 min in light (approximately 0.2 . PPNR PHOTOREDUCED 1000 ft-c); (C) after25or30minPATLY() in light; (D) NADP (500 JM) oP COMPLETEALLY( added to (C). C .

o0..I..I..0.... 300 350 I 400...I.|450 500 560 600 WAVELENGTH (Cm) oxidized by NADP in the presence of chloroplasts in the dark, there was complete restoration of the original spectrum of the protein and the appearance of NADPH. In six experiments, the ratio for the number of moles of photoreduced PPNR re- quired to reduce one mole of NADP varied from 1.85 to 2.37. This stoichiometry is in excellent agreement with the data of Whatley et al.22 and Fry et al.21 Discussion.-The action spectrum for a photosynthetic or photochemical reaction with chloroplasts should provide information as to which pigment(s) is in- volved and, possibly, to what extent. Theoretically, the action spectrum should parallel the absorption spectrum of the substance responsible for the absorption of light. Certain aspects of some action spectra for photosynthetic reactions with chloroplasts are summarized in Table 1. These action spectra appear to be di- TABLE 1 SUMMARY OF ACTION SPECTRA FOR SOME PHOTOSYNTHETIC REACTIONS WITH CHLOROPLASTS Peak, Ratio of activity at Source Reaction mA 650 my and 680 mp* Reference Spinach NADP photoreduction 675 0.68 12 Spinach NADP photoreduction 675 0.67 13 Swiss chard Photoreduction of 2,6-dichlorophenol 675 0.57 11 indophenol Spinach Photoreduction of ferricyanide 660t 1.01 670$ 1.19 Swiss chard Endogenous oxygen evolution 650 1.11 24 Swiss chard Oxygen evolution with ferricyanide 680 0.88 24 Spinach Photophosphorylation with NADP 680 0.50 13 Spinach Photophosphorylation with phenazine 680 0.30 13 methosulfate Spinach Photophosphorylation with ferricyanide 660t 0.93 * The ratio of absorbance at 650 mp to that at 680 my for spinach chloroplasts is 0.49 (Fig. 2) and for Swiss chard chloroplasts is 0.57." The wavelength of the absorption maximum is 679 my for spinach chloroplasts and 675 mp for Swiss chard chloroplasts. t Freshly prepared chloroplasts. t Aged chloroplasts. visible into two types: one type, e.g., photoreduction of NADP or 2,6-dichlorophenol indophenol, is in close agreement with the absorption spectrum of the chloroplasts; the second type, e.g., photoreduction of ferricyanide and the coupled phosphorylation, is not. Downloaded by guest on September 30, 2021 1230 BIOCHEMISTRY: HORIO AND SAN PIETRO PROC. N. A. S.

Using chloroplasts from Swiss chard (Beta vuigaris L. var. cicla), Fork24 has re- cently measured the action spectra for photosynthetic oxygen evolution with and without added ferricyanide. These action spectra are basically similar to those described herein for the photoreduction of ferricyanide and the coupled phosphorylation (Fig. 1). According to French,25 green plants and algae usually contain two different kinds of chlorophyll a whose main absorption maxima are at approximately 682 m/A (Ca 682) and 670 m~i (Ca 670). Brown and French.26 demonstrated that Ca 682 could be transformed into Ca 670 and that in mature green plants, both Ca 682 and Ca 670 were present in almost equal amounts. Thus, only a single absorption maximum was observed in the region of 675-680 mwA. Spinach chloroplasts exhibit an absorption maximum at 679 my and a definite shoulder around 650 m/A. It is conceivable that spinach chloroplasts possess Ca 670 and chlorophyll b (Cb 650) in addition to Ca 683. The latter is bleached by ferricyanide and appears to have a redox potential (+0.55 volt at pH 8) more reducing than that of the other chlorophylls which are unaffected by ferricyanide. The current view is that photosynthesis requires two photochemical reactions driven by functionally discrete systems termed the "accessory pigment system" and "the long wavelength chlorophyll a system." Ca 670 and Cb 650 are included in the former and Ca 683 in the latter system. The photoreduction of NADP and the coupled phosphorylation by chloroplasts is thought to require the participation of both systems operating in series. From the data presented herein (Fig. 1) and that reported by Fork,24 it appears that the photoreduction of ferricyanide and the coupled phosphorylation by chloroplasts requires only the "accessory pigment or short wavelength system." The simplest explanation of this result is that ferricyanide oxidizes some component in the chloroplast whose oxidation, under physio- logical conditions, requires the absorption of light by the "long wavelength chlorophyll a system." Thus, ferricyanide would essentially substitute for the "long wavelength chlorophyll a system" and its photoreduction by chloroplasts would re- (uire only the participation of the "accessory pigment or short wavelength system." Sumrnary.-The action spectra for the photoreduction of ferricyanide and the coupled phosphorylation by spinach chloroplasts have been determined and differ from the absorption spectrum of the chloroplasts. In addition, the redox potential of Ca 683 has been measured and is +0.55 volt at pH 8.

The authors are indebted to Dr. Leo P. Vernon for many helpful suggestions and discussions. * Contribution no. 154 of the Charles F. Kettering Research Laboratory. This research was supported in part by a grant (GM-10129-01) from the National Institutes of Health, IT.S. Public Health Service. t Permanent address: Division of Enzymology, Institute for Protein Research, Osaka Uni- versity, Joan-cho 36, Kita-ku, Osaka, Japan. 1 Hill, R., Nature, 139, 881 (1937). 2 Hill, R., Proc. Roy. Soc. (London), 127B, 192 (1939). 3 Hill, R., and R. Scarisbrick, Proc. Roy. Soc. (London), 129B, 238 (1940). 4 Hill, R., and R. Scarisbrick, Nature, 146, 61 (1940). 5 Dutton, H. J., and W. M. Manning, Am. J. Botany, 28, 516 (1941). 6 Emerson, R., and C. M. Lewis, J. Gen. Physiol., 25, 579 (1942). 7 Emerson, R., and C. M. Lewis, Am. J. Botany, 30, 165 (1943). Downloaded by guest on September 30, 2021 VOL. 51, 1964 BIOCHEMISTRY: MAJERUS ET AL. 1231

8 French, C. S., and G. S. Rabideau, J. Gen. Physiol., 28, 329 (1945). 9 French, C. S., J. Gen. Physiol., 21, 71 (1937). 10 Thomas, J. B., Biochim. Biophys. Acta, 5, 186 (1950). "Chen, S. L., Plant Physiol., 27, 35 (1952). 12 San Pietro, A., S. B. Hendricks, J. Giovanelli, and F. E. Stolzenbach, Science, 128, 845 (1958). 13 Black, C. C., J. F. Turner, M. Gibbs, D. W. Krogmann, and S. A. Gordon, J. Biol. Chem., 237, 580 (1962). 14 Jagendorf, A. T., S. B. Hendricks, M. Avron, and M. Evans, Plant Physiol., 33, 72 (1958). 15 Keister, D. L., A. San Pietro, and F. E. Stolzenbach, Arch. Biochemt. Biophys., 98, 235 (1962). 16 Stiller, M., and B. Vennesland, Biochim. Biophys. Acta, 60, 562 (1962). 17 Arnon, D. I., Plant Physiol., 24, 1 (1949). 18 Nielsen, S. O., and A. L. Lehninger, J. Biol. Chem., 215, 555 (1955). 19Avron, M., Biochim. Biophys. Acta, 40, 257 (1960). 0 San Pietro, A., in Methods in Enzymology, ed. S. P. Colowick and N. 0. Kaplan (New York: Academic Press, 1963), vol. 6, p. 439. 21 Davenport, H. E., and R. Hill, Proc. Roy. Soc. (London), 139B, 327 (1952). 22 Whatley, F. R., K. Tagawa, and D. I. Arnon, these PROCEEDINGS, 49,266 (1963). 23 Fry, K. T., R. A. Lazzarini, and A. San Pietro, these PROCEEDINGS, 50, 652 (1963). 24 Fork, D. C., Plant Physiol., 38, 323 (1963). 2 French, C. S., in Light and Life, ed. W. 1). McElroy and B. Glass (Baltimore: Johns Hopkins Press, 1961), p. 447. 2Brown, J. S., and C. S. French, Plant Physiol., 34, 305 (1959).

THE ACYL CARRIER PROTEIN OF FATTY ACID SYNTHESIS: PURIFICATION, PHYSICAL PROPERTIES, AND SUBSTRATE BINDING SITE BY PHILIP W. IAIAJERUS, ALFRED W. ALBEirS, AND P. RoY VAGELOS LABORATORY OF BIOCHEMISTRY, NATIONAL HEART INSTITUTE, BETHESDA, MARYLAND Communtinicated by Konrad Bloch, April 30, 196J64 The de novo synthesis of long-chain saturated fatty acids proceeds by the following over-all reaction:

Acetyl-CoA + 7 malonyl CoA + 14 TPNII + 14 H+ -- palmitate + 14 TPN+ + 8 CoA + 7 C02 + 6 H20. (1) The fact that the Coenzyme A intermediates of the 3-oxidation pathway are not intermediates ill the synthesis of fatty acids from malonyl CoA in several systems,1- in addition to the finding by Lynein2 4 that the product of the condensation of acetyl CoA and malonyl CoA is protein-bound acetoacetate in the yeast synthetase suggested that all of the intermediates in fatty acid synthesis might be protein- bound. As shown by Goldman et al.,5-7 the product of the condensation of acetyl CoA and malonyl CoA in the E. coli system is acetoacetate which is bound to a heat- stable protein in thiolester linkage. This protein has been called Enzyme II in earlier publications. In the presence of TPNH and a crude E. coli fraction, aceto- acetyl-Enzyme II is converted to butyryl-Enzyme II.7 'Furthermore, both of these compoumids are enzylnatically converted to long-chain fatty acids and therefore have been proposed as intermediates in long-chain fatty acid synthesis.6' Downloaded by guest on September 30, 2021