If isolated reaction centers from Rb. sphaeroides or Rp. viridis are excited with a subpicosecond flash. the transfer of an electron from P* to BPh, occurs with a time constant of 3 to 4 ps [72,131,132,147,148]. The kinetics can be measured by fol- lowing the bleaching of the BPh’s absorption bands at 545 and 760 (or 800 nm for the latter in Rp. viridis) and the appearance of broad absorption bands due to BPh- and Pi at 760 and 1250 nm (1325 nm in Rp. viridis). Prior to the reduction of the BPh, P* can be detected by its broad absorption bands in the visible and near-IR regions of the spectrum, and by its stimulated emission (fluorescence) at 920 or 1000 nm. The stimulated emission from P” decays with kinetics that match the for- mation of BPh-.
The electron transfer reaction between P” and BPh is slightlyfuster at 80 K than it is at 295 K , indicating that it does not require a thermal activation energy [131].
This agrees with earlier observations that charge separation in the reaction center occurs with a high quantum yield at temperatures as low as 1 K [149].
Measurements with flashes lasting 10 to 40 ps have suggested that a transient P’BChl- state precedes t h e formation of P’BPh- [74,1.50,151]. However, the evi- dence for this conclusion has been criticized [77,152], and recent studies with higher time resolution d o not support it [131,132,133.148]. Because BChl, is located al-
most in between P and BPh, in the R p . viridis crystal structure (Fig. 4), the BChl does seem likely to play a role in the electron transfer reaction and, as discussed above, the reduction of the BPh evidently perturbs the absorption spectrum in bands that are associated with the BChl. But P'BChl- appears not to be a kinet- ically resolvable intermediate in the electron transfer process. This could mean that P'BChl- is generated from P*, but decays too quickly to be detected. However, molecular orbital calculations, together with an analysis of the reaction center's absorption spectrum, indicate that the P+BChl- charge-transfer state probably lies significantly above P* in energy [130]. It is thus not likely to be populated to a significant extent, particularly at cryogenic temperatures.
Even if the P+BChl,- charge-transfer state is not formed as a distinct inter- mediate, it probably mixes quantum mechanically with P* and with P+BPhL-. This mixing could facilitate electron tunneling from P* to the BPh by the process known as 'superexchange' [131,153]. Mixing of the excited states of BChlL with those of P could also play an important role in the reaction [130].
Spectroscopic hole-burning experiments at temperatures below 2 K [154,155]
indicate that P* may undergo relaxations on the time scale of 20 fs, which is con- siderably faster than the movement of an electron from P* to BChl, as judged from the lifetime of stimulated emission. Although a conversion from a TIT* state to a charge-transfer state has been suggested, this interpretation is not (in its simplest form) in accord with considerations that place the lowest charge-transfer state of P above the lowest TT* state in energy [130]. An alternative interpretation is that P* undergoes rapid changes in nuclear geometry. Such motions do not occur in monomeric BChl at these temperatures [ 1541, but they might be expected in the reaction center if the excited state has substantial charge-transfer character.
Like electron transfer from P* to BPh,, the movement of an electron from BPh,- to QA increases in speed with decreasing temperature. The time constant of the reaction drops from about 230 ps at 300 K to approximately 100 ps at 100 K, and then becomes essentially independent of temperature down to 4 K 1761. The back reaction between P+ and Q A - also speeds up with decreasing temperature [156-1581. Its time constant is about 100 ms at 300 K, and about 30 ms at tem- peratures below 100 K . In Ch. vinosum, electron transfer from the low-potential cytochrome to P' decreases in speed with decreasing temperature down to about 100 K, but then becomes independent of temperature [159]. The curious temper- ature-dependences of these reactions have been rationalized in terms of the effects of nuclear motions on electron tunneling [76,160-1671. Because nuclei move more slowly than electrons, the overlap of nuclear vibrational wavefunctions of the reac- tants and products is a critical factor in determining the rate of electron transfer.
Reactions that speed up with decreasing temperature generally are assumed to proceed most favorably from the lowest vibrational states of the reactants. In the case of the reaction between the cytochrome and P', motions of the tyrosine that bridges the gap between the hemes and P could be particularly important [102].
Extracting the nonheme Fe from the reaction center slows electron transfer from
QA- to QB by about a factor of 2 [168], a remarkably modest effect in view of the Fe's location between the two quinones (Fig. 4). Electron transfer from BPh,- to
QA is not affected if the Fe is replaced by Zn, but extracting the metal atom de- creases the rate of this step 50-fold, suggesting that the electric field provided by the metal is important for electron transfer to the quinone [169]. Vibrations of BPh,- o r QAp in the electrical field of the metal atom could be critical for the conversion of electronic potential energy into vibrational energy [170]. It is the large free energy change which occurs in electron transfer from BPhLp to Q A that ren- ders charge separation in the reaction center essentially irreversible (Fig. 1).
Acknowledgements
I thank Drs. J . Deisenhofer and H. Michel for providing information on the R p . viridis crystal structure and for helpful comments on the manuscript, D . Midden- dorf, C. Kirmaier, D . Holten, A . Warshel and N. Woodbury for additional help- ful discussion, and the U. S. National Science Foundation and Department of Ag- riculture for financial support.
References
1 Duyscns. L.N.M. (1954) Nature 173, 692-693.
2 Duysens, L.N.M. (1958) Brookhaven Symp. Biol. 11. 10-25.
3 Clayton, R.K. (1962) Photochem. Photohiol. I . 201-210.
4 Kuntz, I . D . . Loach. P.A. and Calvin, M. (1964) Biophys. J . 4. 227-249.
5 Parson. W.W. (1968) Biochim. Biophys. Acta 153. 24%259.
6 Norris, J . R . . Uphaus. R . A . . Crespi. H . L . and Katz, J.J. (1971) Proc. Natl. Acad. Sci. USA 68.
7 McElroy. J . D . . Fehcr. G. and Mauzerall, D.C (1972) Biochim. Biophys. Acta 267. 363-374.
8 Feher. G . . Hoff. A . J . . Isaacson. R . A . and Ackerson, L.C. (1975) Ann. N.Y. Acad. Sci. 244, 9 Fajer. J.. Davis. M . S . . Brune, D . C . . Spaulding, L . D . , Borg. D . C . and Forman. A. (1977) Brook- 10 Lendzian, F.. Lubitz, W.. Scheer, H . . Bubenzer. C. and Mobius, K. (1981) J . Am. Chem. Soc.
I 1 Wasielewski, M.R., Norris. J.R.. Crcspi. H . L . and Harper, J . (1981) J . Am. Chem. Soc. 103.
12 Lubitz. W.. Isaacson, R . A . , Abresch. E . C . and Feher, G . (1984) Proc. Natl. Acad. Sci. USA 81.
13 Cusanovich, M . A . and Kamen. M . D . (1968) Biochim. Biophys. Acta 153. 3 7 6 3 9 6 . I4 Case, G . D . and Parson, W . W . (1971) Biochim. Biophys. Acta 253. 187-202.
15 Dutton, P . L . (1971) Biochim. Biophys. Acta 226. 63-80.
16 Prince. R . C . . Leigh, J.S. and Dutton. P.L. (1976) Biochim. Biophys. Acta 440. 622-636.
17 Bruce, B . D . , Fuller. R.C. and Blankenship. R.E. (1982) Proc. Natl. Acad. Sci. USA 79. 6532-6536.
18 McElroy. J . D . . Feher. G . and Mauzerall. D.C. (1970) Biophys. J. 10, 204a.
19 Leigh, J . S . and Dutton. P.L. (1972) Biochem. Biophys. Res. Commun. 4h. 414-421.
20 Dutton, P.L. and Leigh, J.S. (1973) Biochim. Biophys. Acta 314. l7%190.
21 Feher. G . and Okamura, M . Y . (1978) in the Photosynthetic Bacteria (Clayton R . K . and Sistrom.
22 Loach, P . A . and Hall, R . L . (1972) Proc. Natl. Acad. Sci, USA 69. 78&790.
23 Imhoff. J . F . . Truper, H.G. and Pfennig. N . (1984) Int. J . Syst. Bact. 34. 340-343.
625-628.
239-259.
haven Symp. Biol. 28, 74-103.
103. 4635-4637.
7664-766s.
7792-7796.
W . R . , eds.). pp. 349-386, Plenum Press. New York.
26 Halsey, Y. and Parson, W.W. (1974) Biochim. Biophys. Acta 347, 404-416.
27 Vermeglio, A . (1977) Biochim. Biophys. Acta 459. 516-524.
28 Wraight, C . A . (1977) Biochim. Biophys. Acta 459, 525-531.
29 Wraight, C . A . (1979) Biochim. Biophys. Acta 548, 30%327.
30 Wraight, C.A. (1981) Israel J. Chem. 21, 348-354.
31 Vermeglio, A . and Clayton. R.K. (1977) Biochim. Biophys. Acta 461, 159-165.
32 Shopes, R.J. and Wraight. C.A. (1985) Biochim. Biophys. Acta 806, 348-356.
33 Dutton, P.L., Leigh. J.S. and Wraight. C . A . (1973) FEBS Lett. 36. 169-173.
34 Arata. H. and Parson, W.W. (1981) Biochitn. Biophys. Acta 638, 201-209.
35 Prince, R.C. and Dutton, P.L. (1976) Arch. Biochem. Biophys. 172, 329-334.
36 Rutherford, A.W. and Evans, M.C.W. (1980) FEBS Lett. 110, 257-261.
37 Clayton, R.K. and Straley, S.C. (1972) Biophys. J. 12, 1221-1234.
38 Slooten, L. (1972) Biochim. Biophys. Acta 275, 208-218.
39 Feher. G . , Okamura. M.Y. and McElroy, J.D. (1972) Biochim. Biophys. Acta 267. 222-226.
40 Kleinfeld. D . , Okamura, M.Y. and Feher. G. (1985) Biophys. J . 48, 849-852.
41 Lubitz, W., Abresch, E . C . , Debus, R.J., Isaacson. R . A . . Okarnura, M.Y. and Feher, G. (1985) 42 Wraight, C.A. and Stein, R. (1980) FEBS Lett. 113, 73-77.
43 Kleinfeld, D., Okamura, M.Y. and Feher, G. (1984) Biochim. Biophys. Acta 766. 126-140.
44 Mancino, L.J., Dean, D.P. and Blankenship. R . E . (1984) Biochim. Biophys. Acta 764, 4654.
45 Arata. H. (1985) Biochim. Biophys. Acta 809. 284-287.
46 Blankenship, R.E. (1984) Photochern. Photohiol. 40, 801-806.
47 Vasrnel, H . and Amesz, J. (1983) Biochim. Biophys. Acta 724, 118-122.
48 Wraight, C.A. and Clayton, R.K. (1974) Biochim. Biophys. Acta 333, 246-260.
49 Zankel, K.L., Reed, D.L. and Clayton. R.K. (1968) Proc. Natl. Acad. Sci. USA 61. 1243-1249.
50 Slooten, L. (1972) Biochim. Biophys. Acta 256, 452-466.
51 Parson, W.W. (1969) Biochim. Biophys. Acta 189, 384-396.
52 Parson, W.W.. Clayton, R.K. and Cogdell, R.J. (1975) Biochim. Biophys. Acta 387. 265-278.
53 Parson, W.W. and Monger. T.G. (1977) Brookhaven Symp. Biol. 28. 195-212.
54 Holten, D., Windsor, M.W., Parson, W.W. and Thornber. J.P. (1978) Biochim. Biophys. Acta 501, 55 Fajer. J . , Brune, D.C., Davis, M.S., Forman, A. and Spaulding, L.D. (1975) Proc. Natl. Acad.
56 Fajer, J . , Davis, M.S.. Brune, D.C., Forman. A . and Thornber. J.P. (1978) J . Am. Chem. SOC.
57 Shuvalov, V.A. and Parson, W.W. (1981) Proc. Natl. Acad. Sci. USA 78. 957-961.
58 Blankenship, R.E.. Schaafsma, T.J. and Parson. W.W. (1977) Biochim. Biophys. Acta 461,297-305.
59 Hoff, A.J., Rademaker, H . , van Grondelle, R . and Duysens. L.N.M. (1977) Biochim. Biophys.
60 Werner, H.J., Schulten, K. and Weller. A . (1978) Biochim. Biophys. Acta 502. 255-268.
61 Levanon, H. and Norris. J.R. (1978) Chem. Rev. 78, 185-198.
62 Haberkorn, R . and Michel-Beyerle, M.E. (1979) Biophys. J . 26, 489-498.
63 Hoff. A.J. (1981) Qt. Rev. Biophys. 14, 599-665.
64 Rademaker, H . and Hoff, A.J. (1981) Biophys. J. 34. 325-344.
65 Schenck, C.C., Blankenship, R.E. and Parson. W.W. (1982) Biochim. Biophys. Acta 680, 44-59.
66 Boxer. S . G . , Chidsey, C.E.D. and Roelofs, M.G. (1983) Annu. Rev. Phys. Chem. 34, 389-417.
61 Shuvalov. V.A. and Klimov, V.V. (1976) Biochim. Biophys. Acta 440, 587-599.
68 Tiede. D.M.. Prince. R.C. and Dutton, P.L. (1976) Biochim. Biophys. Acta 449. 447-467.
69 Okamura, M.Y., Isaacson, R.A. and Feher. G. (1979) Biochim. Biophys. Acta 546, 394-417.
70 Rockley, M., Windsor, M.W., Cogdell. R.J. and Parson, W.W. (1975) Proc. Natl. Acad. Sci. USA 71 Kaufmann. K.J., Dutton. P.L., Netzel. T.L.. Leigh, J.S. and Rentzepis, P.M. (1975) Science 188,
Biochim. Biophys. Acta 808. 464-469.
112-126.
Sci. USA 72, 49564960.
100, 191S1920.
Acta 460, 547-554.
72, 2251-2255.
130 1- 1304.
72 Holten, D . . Hoganson, C . , Windsor. M . W . , Schcnck. C.C., Parson. W.W., Migus. A , . Fork, R.L.
73 Peters, K.. Avouris. P. and Rentzepis. P.M. (1978) Biophys. J . 23. 207-217.
74 Shuvalov, V.A.. Klevanik. A . V . , Sharkov. A . V . . Matveetz, J.A. and Krukov. P . G . (1978) FEBS 75 Kirmaier. C . . Holten. D . and Pamon, W.W. (1985) Biochim. Biophys. Acta 810. 33-48.
76 Kirmaier. C . . Holtcn. D . and Parson, W.W. (1985) Biochim. Biophys. Acta 810. 49-61.
77 Kirmaier. C.. Holten. D. and Parson. W.W. (1985) FEBS Lett. 185, 76-82.
78 Godik, V.I. and Borisov. A . Yu. (1979) Biochini. Biophys. Acta 548, 296-308.
79 Godik. V.I. Kotova. E . A . and Borisov, A . Yu. (1982) Photobiochem. Photobiophys. 4, 219-226.
80 Woodbury, N.W. and Parson, W . W . (1984) Biochim. Biophys. Acta 767, 345-361.
X I Woodbury. N.W. and Parson. W.W. (1986) Biochim. Biophys. Acta 850. 197-210.
82 Blankenship. R.E. (1985) Photosynth. Res. 6, 317-333.
83 Nuijs. A.M.. Vasrnel, H.. Joppe. H.L.P.. Duysens, L.N.M. and Amesz. J . (1985) Biochim. Bio- 84 Brauniann, T., Vasmel, H.. Grimme. L.H and Amesz, J. (1986) Biochim. Biophys. Acta 848, S 9 l . 85 Shuvalov. V . A . . Arnesz. J . and Duysens. L.N.M. (1986) Biochim. Biophys. Acta 851. 1-5.
86 Clayton, R . K . and Wang. R.T. (1971) Methods Enzymol. 23, 696-704.
87 Noel. H.. Van d e r Rest. M . and Gingras, G . (1972) Biochim. Biophys. Acta 275. 219-230.
88 Jolchine. G. and Reiss-Husson, F. (1974) FEBS Lett. 40. 5-8.
89 Okamura. M . Y . . Steiner. L.A. and Feher. G. (1974) Biochemistry 13, 13941403.
90 Prince, R . C . . Tiede. D . M . . Thornber. J . P . and Dutton, P.L. (1977) Biochim. Biophys. Acta 462, 91 Clayton, B . J . and Clayton. R . K . (197X) Biochim. Biophys. Acta 501. 47G477.
92 Michel. H . (1982) J. Mol. Biol. 1.58. 567-572.
93 Pierson, B.K. and Thornber, J.P. (1983) Proc. Natl. Acad. Sci. USA 80, 8&84.
94 Kirrnaier. C.. Holten. D . and Blankenship. R . E . (1983) FEBS Lett. 158, 73-78.
95 Vasmel. H . . Meiburg. R . F . . Kramer. H.J.M., de Vos. L.J. and Amesz. J . (1983) Biochim. Bio- 96 Linn. L. and Thornber. J.P. (1975) Photochem. Photobiol. 22. 37-40.
97 Vasmel. H . . Swarthoff. T . , Kramer. H . J . M . and Amesz, J . (1983) Biochim. Biophys. Acta 725, 98 Allen, J . P . and Feher, G . (1984) Proc. Natl. Acad. Sci. USA 81, 4795-4799.
99 Gast, P. and Norris, J.R. (1984) FEBS Lett. 177, 277-280.
and Shank. C.V. (1980) Biochim. Biophvs. Acta 592, 461-477.
Lett. 91, 135-139.
phys. Acta 807. 24-34.
467-490.
phys. Acta 724. 333-339.
361-367.
100 Chang. C . - H . . Schiffer. M . . Tiede. D.. Smith. U . and Norris. J. (1985) J. Mol. Biol. 186, 201-203.
101 Deisenhofer. J., E p p . 0.. Miki. K . , Huber. R. and Michel, H. (1984) J . Mol. Biol. 180, 385-398.
102 Deisenhofer. J . . Epp, 0.. Miki, K.. Huher. R . and Michel. H . (1985) Nature 318. 61Pr624.
103 Youvan. D . C . . Bylina. E . J . . Alberti. M . . Mcgush. H . and Hearst, J . E . (1984) Cell 37, 949-957.
104 Wiemken. V . and Bachofen, R . (1984) FEBS Lett. 166. 155-159.
105 Case. G . D . . Parson. W . W . and Thornher. J . P . (1970) Biochim. Biophys. Acta 223. 122-128.
106 Parson. W . W . and Case. G . D . (1970) Biochim. Biophys. Acta 205, 232-245.
107 Prince. R . C . and Dutton. P.L. (1977) Biochim. Biophys. Acta 459. 57.%577.
108 Williams. J . C . , Steiner. L . A . . Ogden. R.C.. Simon. M.I. and Feher. G. (1983) Proc. Natl. Acad.
109 Williams. J.C.. Steiner. L . A . , Fehcr. G . and Simon. M.I. (1984) Proc. Natl. Acad. Sci. USA X1.
110 Michel, H.. Weyer. K.A.. Gruenberg. I f . . Dunger. I.. Oesterhelt, D . and Lottspeich, F. (1986) 111 Michel, H.. Weyer. K.A.. Gruenherg. H. and Lottspeich, F. (1985) E M B O J . 4, 1667-1672.
112 Bachmann, R.C., Gillies, K. and Takcmoto. J.Y. (1981) Biochemistry 20. 459C-4596.
113 Valkirs. G . E . and Feher. G. (1982) J . Cell B i d 95, 179-188.
114 Nabedryk. E.. Tiede. D.M.. Dutton. P.L. and Breton, J . (1982) Biochim. Biophys. Acta 682.
115 Brunisholz. R . A . . Wiemken. V . , Suter. F. and Bachofen. R . (1984) Hoppe-Seyler’s Z. Physiol.
Sci, U S A 80, 6.505-6509.
7303-7307.
E M B O J . 5. 1149-1 158.
273-280.
Chem. 365. 689-70 1.
117 Feher, G., Isaacson, R.A., McElroy, J.D., Ackerson, L.C. and Okamura, M.Y. (1974) Biochim.
118 Nam, H.K., Austin, R.H. and Dismukes, G.C. (1984) Biochim. Biophys. Acta 765, 301-308.
119 Prince, R.C. (1978) Biochim. Biophys. Acta 501, 195-207.
120 Robert, B. and Lutz, M. (1986) Biochemistry 25, 230S2309.
121 Bunker, G., Stern, E.A., Blankenship, R.E. and Parson, W.W. (1982) Biophys. J . 37, 539-551.
122 Eisenberger, D., Okamura, M.Y. and Feher, G. (1982) Biophys. J. 37, 523-538.
123 Cogdell, R.J., Parson, W.W. and Kerr, M. (1976) Biochim. Biophys. Acta 430, 83-93.
124 Vermeglio, A., Breton, J . Paillotin, G. and Cogdell, R.J. (1978) Biochim. Biophys. Acta 501, 125 Thornber, J.P., Cogdell, R.J., Seftor, R.E.B. and Webster, G.D. (1980) Biochim. Biophys. Acta 126 McGann, W.J. and Frank, H.A. (1985) Biochim. Biophys. Acta 807, 101-109.
127 Cogdell, R.J., Monger, T.G. and Parson, W.W. (1978) Biochim. Biophys. Acta 408, 189-199.
128 Knapp, E.W. and Fischer, S.F. (1985) in Antennas and Reaction Centers of Photosynthetic Bac- 129 Knapp, E.W., Fischer, S.F., Zinth, W., Sander, M., Kaiser, W., Deisenhofer, J. and Michel, H.
130 Parson, W.W., Scherz, A . and Warshel, A . (1985) in Antennas and Reaction Centers of Photo- 131 Woodbury, N.W., Becker, M., Middendorf, D. and Parson, W.W. (1985) Biochemistry 24, 132 Martin, J.-L., Breton, J., Hoff, A , , Migus, A. and Antonetti, A. (1986) Proc. Natl. Acad. Sci.
133 Shuvalov, V.A. and Parson, W.W. (1981) Biochim. Biophys. Acta 638, 5C59.
134 den Blanken, H.J. and Hoff, A.J. (1982) Biochim. Biophys. Acta 681, 365-374.
135 den Blanken, H.J. and Hoff, A.J. (1983) Chem. Phys. Lett. 98, 255-262.
136 Zinth, W., Sander, M., Dobler, J, Kaiser, W. and Michei, H. (1985) in Antennas and Reaction Centers of Photosynthetic Bacteria (Michel-Beyerle. M.E., ed.), pp. 97-102, Springer-Verlag, Berlin.
Biophys. Acta 368, 135-139.
5 14-530.
593, 60-75.
teria Michel-Beyerle, M.E. ed.), pp. 103-108, Springer-Verlag, Berlin.
(1985) Proc. Natl. Acad. Sci. USA 82, 8463-8467.
synthetic Bacteria (Michel-Beyerle, M.E.. ed.), pp. 122-130, Springer-Verlag, Berlin.
75167521.
USA 83, 957-961.
137 Breton, J. (1985) Biochim. Biophys. Acta 810, 235-245.
138 Parson, W.W. (1982) Annu. Rev. Biophys. Bioeng. 11, 57-80.
139 Eccles, J. and Honig, B. (1983) Proc. Natl. Acad. Sci. USA 80, 4959-4962.
140 Feher, G. (1971) Photochem. Photobiol. 14, 373-387.
141 Vermeglio, A. and Clayton, R.K. (1978) Biochim. Biophys. Acta 449, 500-515.
142 Shuvalov, V.A. and Asadov, A.A. (1979) Biochim. Biophys. Acta 545, 296308.
143 Hoff, A.J., Lous, E.J., Moehl, K.W. and Dijkman, J.A. (1985) Chem. Phys. Letts. 114, 39-43.
144 Robert, B., Lutz, M. and Tiede, D.M. (1985) FEBS Lett. 83, 324-330.
145 Netzel, T.L., Rentzepis, P.M., Tiede, D . M . , Prince, R.C. and Dutton, P.L. (1977) Biochim. Bio- 146 Schenk, C.C., Parson, W.W., Holten, D. and Windsor, M.W. (1981) Biochim. Biophys. Acta 635, 147 Paschenko, V.Z., Korvatovskii, B.N., Kononenko, A.A., Chamorovsky, S.K. and Rubin, A.B.
148 Breton, J., Martin, J.-L., Migus, A,, Antonetti, A. and Orszag, A. (1986) Proc. Natl. Acad. Sci.
149 Arnold, W . and Clayton, R.K. (1960) Proc. Natl. Acad. Sci. USA 46, 769-776.
150 Shuvalov, V.A. and Klevanik, A.V. (1983) FEBS Lett. 160, 51-55.
151 Shuvalov, V.A. and Duysens. L.N.M. (1986) Proc. Natl. Acad. Sci. USA 83, 1690-1694.
152 Borisov, A.Y., Danielius, R.V., Kudzmauskas, S . P . , Piskarskas, A.S., Razjivin, A.P., Sirutkai- 153 Heitele, H. and Michel-Beyerle, M.E. (1985) in Antennas and Reaction Centers of Photosynthetic
phys. Acta 460, 467-478.
383-392.
(1985) FEBS Lett. 191, 245-248.
USA 83, 5121-5125.
tis, V.A. and Valkunes, L.L. (1983) Photobiochem. Photobiophys. 6. 33-38.
Bacteria (Michel-Beyerle, M.E., ed.), pp. 25C-255, Springer-Verlag. Berlin.
154 Meech, S.R.. Hoff. A . J . and Wiersma. D . A . (1985) Chem. Phys. Lett. 121. 287-292.
155 Boxer. S.G., Lockhart, D.J. and Middendorf. T . R . (1986) Chem. Phys. Lett. 123, 4 7 6 4 8 2 . 156 Parson, W.W. (1966) Biochim. Biophys. Acta 131, 154-172.
157 McElroy. J . D . . Mauzerall. D . C . and Feher, G . (1974) Biochim. Biophys. Acta 333. 261-277.
158 Hsi. E.S.P. and Bolton. J . B . (1974) Biochim. Biophys. Acta 347, 126-153.
159 DeVault, D . and Chance. B . (19%) Biophys. J . 6, 825-847.
160 Hopfield, J . J . (1974) Proc. Natl. Acad. Sci. USA 71. 364(!-3644.
161 Jortner, J . (1976) J. Chem. Phys. 64. 486C4867.
162 Blankenship. R . E . and Parson, W.W. (1979) in Photosynthesis in Relation to Model Systems 163 Warshel. A . (1980) Proc. Natl. Acad. Sci. U S A 77, 3105-3109.
164 Sarai, A . (1980) Biochim. Biophys. Acta 589. 71-83.
165 Kakitani. T. and Kakitani, H. (1981) Biochim. Biophys. Acta 635, 49S514.
166 DeVault, D. (1984) Quantum Mechanical Tunneling in Biological Systems, Cambridge University 167 Marcus, R . and Sutin. N . (1985) Biochim. Biophys. Acta 811. 265-322.
168 Debus. R., Feher, G . and Okamura, M . Y . (1986) Biochemistry 25, 2276-2287.
164, Kirmaier. C.. Holten, D.. Debus, R.. Feher. G . and Okamura, M.Y. (1986) Proc. Natl. Acad.
170 DeVault, D. (1986) Photosynth. Res. 10. 125-137.
171 Seftor. R.E.B. and Thornber. J . P . (1084) Biochim. Biophys. Acta 764, 148-159.
172 Michel, H . , E p p , 0. and Diesenhofer. J . (1986) E M B O J . 5 . 2445-2451.
(Barber. J . . e d . ) , pp. 71-114, Elsevier. Amsterdam.
Press. Cambridge, U.K.
Sci. USA 83. 6407-641 1 .
CHAPTER 4
The primary reactions of photosystems I and I1 of algae and higher plants
P. MATHIS and A . W . RUTHERFORD
Dkpartement de Biologie, Service de Biophysique, C E N Sacluy 911 91 Gif-sur-Yvette Cedex, France
1. Introduction
In photosynthetic organisms, the 'primary reactions' fulfill the objective of con- verting the energy of light into a primary form of chemical energy which lasts for a time compatible with ordinary biochemical processes, i.e. milliseconds. In these reactions a rather large fraction. approximately 40%, of the photon energy is stored as chemical free energy. The primary reactions can be viewed from two major per- spectives. Firstly, from a photochemical point of view: pigment molecules are ex- cited to their lowest excited singlet state which reacts in an electron transfer re- action, the first step of a process of charge separation. Secondly, from a biochemical point of view: the reactions take place in highly organized complexes, the reaction centres. which are made up of several classes of molecules which cooperate in ful- filling complementary roles: architectural support, light absorption, energy trans- fer and electron transfer [ 1-31. Reaction centres are membrane-bound complexes, made of a few hydrophobic polypeptides which hold together, in a well-defined conformation, various pigments (chlorophylls and carotenoids) and redox centres (tetrapyrroles, quinones, iron-sulfur centers, etc). The reaction centres have a well- defined positioning with respect to the membrane plane.
Photosynthetic organisms have adopted a large variety of shapes, colors and liv- ing conditions. The primary reactions in all organisms, however, share a large number of basic properties, and the purple bacteria, which have been studied in great detail, can be used as a good general model system. In oxygenic photosyn- thetic organisms, for which water is the ultimate source of reducing power. there are two types of reaction centres, photosystem I and photosystem I1 (PS I and PS II), which operate in series for electron transfer (Fig. 1). This cooperation of two photoreactions is made necessary by the large energy gap for the electron to be transferred from water (Em = + 0.8 V) to the terminal electron acceptor NADP'
All oxygenic organisms, ranging from cyanobacteria to algae and higher plants, contain PS I and PS I1 reaction centres, with only minor variations in spite of their large taxonomic and ecological diversity. Small variations will not be emphasized (E," = -0.3 V).
PS 11 Cyt b/f PS I
P-700 -
LUMEN
2
Fig. 1. A simplified scheme of the photosynthetic membrane. illustrating electron transfer from water to ferredoxin, which involves three protein complexes (the PS I1 reaction centre, the Cyt b,/fcomplex, the PS I reaction centre) and two diffusible components, plastoquinone (PO pool) and plastocyanin (PC).
here and we will mainly focus on the general, functionally essential, properties.
When appropriate we will also underline the analogies with non-oxygenic photo- synthetic bacteria. Due to the extensive literature on the subject, citations will be generally restricted to articles published in the last few years and to recent reviews [4-71.
2 . Photosystem I reactions
A number of experimental properties of oxygen-evolving photosynthetic orga- nisms have been historically integrated into the concept of photosystem I reac- tions. We shall cite only four of them: (1) the stimulation of the rate of 0, evo- lution under red or green light by far-red light, above 700 nm, which is unable to induce O2 evolution by itself; (2) a small photoinduced bleaching of the absorption
P;l Pc !
' P- 700.1 P- 700
Fig. 2. A scheme of electron transfer in PS I . Redox centres are situated at their approximate timated midpoint potential Etn.
or es-
at 700 nm, which was interpreted as being due to the oxidation of a primary elec- tron donor, P-700; a free radical EPR signal, Signal 1, was attributed to P-700';
(3) the photoinduced appearance of EPR signals characteristic of iron-sulfur pro- teins, at cryogenic temperatures; (4) the ability to reduce low-potential electron acceptors such as ferredoxin or viologens; this can be done even in the presence of inhibitors of the PS I1 reactions, such as DCMU, provided an artificial electron donor is added.
A coherent interpretation for many experimental results was provided by the concept of a PS I reaction centre. This centre has now been isolated, albeit per- haps not in a definitely pure state. It is made up of a few hydrophobic polypep- tides, the primary donor (P-700), several electron acceptors (Fig. 2), and about 50 molecules of pigment (chlorophyll a and P-carotene). This composition is analo- gous to that of other types of reaction centres.
2.1. The primary donor P-700 2.1.1. Basic properties of P-700
In one of the first applications of differential absorption spectroscopy to photo- synthetic membranes, Kok [8] observed a small light-induced bleaching at 700 nm.
The bleaching can also be induced by addition of an oxidizing agent. It is now clearly associated with the photooxidation of P-700, the primary electron donor of PS I. This species cannot be isolated as a pure molecular entity. Its absorption spectrum is not known, but we know the difference spectrum due to its oxidation (i.e. P-700' minus P-700), which includes large bleachings at 700 ( k 3) nm and 430 nrn, and small positive bands at 810 and 450 nm [9]. At low temperature, large narrow bands develop at 690 nm (positive) and 680 nm (negative) [10,11]. In chlo- roplasts, P-700 is present at a ratio of one per about 400 chlorophylls. In the pur- ified PS l particles, which presumably are closest to the structure of a PS I reaction centre, there is one P-700 per about SO chlorophylls. The Em of P-700 is about +490 mV [12].
2.1.2. P-700: a chlorophyll species
The chemical nature of P-700 is difficult to establish. The absorption bleachings correspond approximately to the peaks of Chl II. which appears to be virtually the only tetrapyrrolic pigment in purified PS I particles. It has thus been assumed that P-700 is Chl a bound to a protein. A few recent results, however, may require this hypothesis to be refined. An examination of the spectroscopic and redox proper- ties of P-700 led Wasielewski et al. [ 131 to propose that P-700 could be the enol form of Chl a where enolization was of the keto ester on ring V. This has not been confirmed by chemical extraction. Extraction experiments, however, have evi- denced two other chlorophyll derivatives. A species named Chl-RC I has been iso- lated from PS I, at a nearly l i l molar ratio with P-700 and its structure shown to be a chlorinated derivative. It is not yet clear whether Chl-RC I is a native con- stituent of PS I or an extraction artefact. Chl-RC 1 has not been obtained in a re- cent chemical analysis by HPLC, which instead revealed two Chl a' per P-700 [14].