bacterial photosynthesis. (magnetic field effect/photosynthetic reaction centers/optically detected magnetic resonance/radical ion pair state in photosynthesis).
Proc. NatL Acad. Sci. USA Vol. 78, No. 6, pp. 3305-3307, June 1981 Chemistry
Magnetic resonance spectroscopy of the primary state, PF, of bacterial photosynthesis (magnetic field effect/photosynthetic reaction centers/optically detected magnetic resonance/radical ion pair state in photosynthesis)
M. K. BOWMANt, D. E. BUDILO, G. L. CLOSSt§, A. G. KOSTKAt, C. A.
WRAIGHT$, AND J.
R. Nomist
tChemistry Division, Argonne National Laboratory, Argonne, Illinois 60439; §Department of Chemistry, and tDepartment of Biophysics and Theoretical Biology, University of Chicago, Chicago, Illinois 60637; and IDepartment of Botany, University of Illinois, Urbana, Illinois 61801
Contributed by Gerhard L. Closs, March 13, 1981
ABSTRACT We have obtained the magnetic resonance spectrum of the radical pair state pF by using reaction yield detected magnetic resonance spectroscopy. The magnetic resonance spectrum is quite sensitive to the local environment of pF. The data place limits on the lifetime of triplet pF and the distance of charge separation.
(24). Optical measurements of the pR yield upon recombination show large magnetic field effects and have become one method of studying the radical pair state. In addition, we have found by optical measurements that the pR yield also is influenced by resonant microwaves applied to the radical pair. This is a convenient method to record the EPR spectrum of pF.
Most of the transient states involved in the primary events of photosynthesis are paramagnetic, making magnetic resonance and magnetic field effects two convenient methods of studying the photosynthetic apparatus. Magnetic resonance spectros'copy has been used extensively to help identify the radical intermediates and products of photosynthesis. In addition, the magnetic resonance spectrum and its electron spin polarization and the magnetic field dependence of various reaction products have yielded some information about electron-electron dipolar interactions between radicals and about the exchange interactions that are so important for electron transfer. As attempts are made to see increasingly earlier intermediates, experimental difficulties arise due to the steadily decreasing lifetimes ofthese states. For example, in bacterial photosynthetic reaction centers, one of the earliest states is a radical pair consisting of the cation of the primary donor, P+, and the anion of a bacteriopheophytin intermediate acceptor, I- (1, 2). From the EPR spectrum of this radical pair, both the electron exchange interaction, J, and the electron-electron dipolar interaction (and hence distance of separation) can in principle be determined. However, the short lifetime of this radical pair state, pF (3) (which even in blocked reaction centers is only 10 nsec) has prevented its direct observation by conventional EPR techniques, and interactions within pF have been studied only indirectly by optical spectroscopy (3-10) or through magnetic field effects (11-16). We report here the observation ofthe magnetic resonance spectrum of this radical pair state pF at physiological temperatures by using reaction yield detected magnetic resonance (RYDMAR) spectroscopy (17-21). This radical pair state forms within about 5 psec (3, 4, 10) and decays within 200 psec or about 10 nsec (7, 8), depending on whether or not further electron transfer is blocked. When the transfer of an electron from I- to the iron-ubiquinone acceptor (22, 23), Fe-Q, is prevented by removal or chemical reduction of the quinone, charge recombination of a triplet radical pair produces the lowest excited triplet state of p, pR (7, 8, 24-30), while recombination of a singlet radical pair produces both P and I in their singlet ground states (15, 24). The relative yields of pR and ground state P depend strongly on the electron spin dynamics of the radical pair state (31-33), pF which in turn is influenced by any applied magnetic field
EXPERIMENTAL Reaction centers were purified from Rhodopseudomonas sphaeroides R-26 chromatophores by the method of Stein and Wraight (unpublished) by extraction with 0.45% lauryldimethylamine oxide (LDAO) in 0.1 M NaCl, fractionation in solution with 30% and 50% saturated (NH4)2SO4, and DEAE-cellulose column chromatography. Quinone-free samples were prepared according to Okamura et al. (27); all solutions used in the extraction were kept anaerobic. Samples to be reduced with sodium dithionite were obtained by substituting 0.1% Triton X-100 for 0.03% LDAO in the dialyses and column chromatography. Oxygen was carefully excluded from the EPR samples during their preparation. An EPR flat cell containing a sample solution with an absorbance of approximately 1 at 870 nm was placed in a Varian E234 optical transmission cavity. A weak probing beam of light from a tungsten filament was focused through the sample and onto the slit of a monochromator. The transmitted light was detected by either a photomultiplier with S-1 response or a photodiode. Actinic pulses (10-20 mJ) were supplied by a frequency-doubled MY-32 neodymium yttrium-aluminum garnet laser operating at 20 Hz. The magnetic field was supplied by a current-regulated H4VW electromagnet from Walker Scientific (Worcester, MA) placed around the EPR cavity. The relative amount of pR formed by decay of the pF produced by the laser pulse was measured from the difference in transmission of the probing beam immediately before and about 5 Asec after the laser pulse, using a Princeton Applied Research 162 boxcar integrated with a pair of 164 gated integrators. The output ofthe boxcar integrator was digitized and stored by a Nicolet 1070 signal averager, which also furnished the ramp that swept the magnetic field. A Central Microwaves (St. Charles, MO) CME420 Gunn diode oscillator generated microwaves at about 9.2 GHz which were amplified to 2 kW by a Litton 624 pulsed traveling wave tube amplifier. Microwave pulses with 1. 6-,sec duration centered on the laser pulse were applied to the microwave cavity. A rotary vane attenuator allowed the microwave pulse power to be varied. The pR yield is usually monitored at 870 nm, where the change in absorbance upon pR formation is largest, although similar field effects were found at other wave-
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Abbreviations: RYDMAR spectroscopy; reaction yield detected magnetic resonance spectroscopy; Q, ubiquinone; LDAO, lauryldimethylamine N-oxide.
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Proc. Natl. Acad. Sci. USA 78 (1981) pF
lengths throughout the visible spectrum. The delay of about 5 ttsec in sampling the spectrum after the laser pulse intentionally prevented observation of the pF spectrum so that only changes in the yield of PR and ground state P were seen. RESULTS AND DISCUSSION The relative yield of pR as a function of magnetic field has been determined for quinone-free reaction centers and for reaction centers with the quinone singly reduced. The PR yield in both samples is reduced 44% upon application of a strong magnetic field (24 kG) (1 G = 10-4 T). However, the field at which the reduction in pR is only half complete (22%) is quite different in the two samples, being about 64 G in the quinone-free reaction centers and about 210 G in the singly reduced reaction centers. 1 This difference may be due to changes in electron spin interactions between pF and the Fe-Q complex or to changes in electrostatic interactions. When microwaves are applied to the sample while pF is present, the pR yield in both samples exhibits an increase at about 3290 G as shown in Fig. 1. At the highest available microwave power of 2 kW, this resonant microwave effect amounts to about 3% of the total pR yield at zero field. Even at 2 kW, the signal amplitude is proportional to the microwave magnetic field. The full width at half height is 30 G for the quinone-free sample and 135 G for the reduced sample. The center of the resonances correspond to a g value of 2.00 ± 0.01. An energy level diagram is shown for the four spin states of pF at high magnetic field in Fig. 2. The highest and lowest levels are relatively pure high-field triplet eigenstates and are labeled with their angular momentum quantum numbers as T,1 and T_1. The two-center levels labeled fl and 42 are nearly degenerate and are strongly perturbed by electron nuclear hyperfine and by electron Zeeman energy differences, with the result that the eigenfunctions Xl and 42 are both a mixture of singlet and triplet electron spins. Since singlet and triplet are not good "quantum numbers" for 4l and 42, selection rules
A
Cd
g =2.00 +0.01
(D
field Magnetic FIG. 1. Magnetic resonance intensity vs. magnetic field. Magnetic resonance intensity is monitored experimentally as relative triplet yield of pR. (A) Room-temperature RYDMAR spectrum of bacteriochlorophyll2t bacteriopheophytin * (PF) of bacterial reaction centers depleted of ubiquinone. (B) Room-temperature RYDMAR spectrum of bacteriochlorophyll2t bacteriopheophytin * Q * Fe ofbacterial reaction centers with chemically reduced ubiquinone, Q-- Fe. The cen...
3P3*870
or pR
1P870 FIG. 2. Energy level diagram for the formation of P" and PR. Bold arrows represent microwave transitions. Other arrows represent various decay routes after excitation by a light quantum hv. Dashed arrows are decay routes enhanced by microwave transitions. Energy gaps within the sublevels of PR and pR are greatly exaggerated compared to the energy separations of 'P870, lp870, pF, and PR.
based on singlet and triplet are not valid for 41 and 42, although they are valid for T+1 and T-1. One consequence of this is that microwaves can induce the four transitions shown connecting 01 or 02 with T+1 or T_1. A second consequence is that a triplet state such as pR can be produced from any of the eigenstates of pF with triplet character-i.e., T+1, T_1, 41, or 02-whereas a singlet reaction product such as ground state singlet P can be produced only from Xl and 42 that have singlet character. A similar set ofrules describes the population ofthe pF eigenstates from its precursor 1P* The observation of an increase in triplet pR yield when resonant microwaves induce transitions between Xl and 42 and T+I or T-1 implies that Xl and 42 are initially populated from 'P*, while T,1 and T_1 are not. This means that the primary events of bacterial photosynthesis involve excited singlet state chemistry and not triplet state chemistry. The peak in the RYDMAR spectrum OfpF in the quinone-free reaction centers has a g value consistent with the spectrum of a pair composed of organic radicals such as P+ and F. The width of the RYDMAR spectrum of pF arises from four interactions: electron-nuclear hyperfine, electron-electron dipolar, electron exchange, and lifetime uncertainty broadening. Were hyperfine the only interaction present, the linewidth would be only 11 G rather than 30 G. If the width of the spectrum were due entirely to lifetime broadening, the lifetime of the triplet radical pair levels would have to be about 2 nsec. On the other hand, if the width is due to dipolar broadening, then P+ and F must be about 7 A apart (assuming a point-dipole interaction).tt Similarly, it can be said that the electron-exchange interaction between P+ and F in pF must be less than 30 G. The much larger width in the quinone reduced samples evidently reflects the additional interactions with the reduced Fe-Q complex. Because resonant microwaves are able to influence the recombination pathway of the pF radical pair in photosynthetic bacteria, the relative yields of the various recombination prod-
...
ters of the fields are not calibrated.
a1Because actually
an
iron
core
be swept
magnet is used, the field
through
zero.
at
the
sample might
not
tt A
delocalized dipole model is more realistic. Consideration of the delocalized nature of the pigment macrocycles would place the macrocycle-to-macrocycle, center-to-center distance of separation at greater than 4 A with the additional geometrical constraint that no edges of the macrocycle are closer than 1 A.
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Bowman et al.
ucts has proven to be a convenient and sensitive method ofdetecting the heretofore unobservable magnetic resonance spectrum of this short-lived radical pair. The RYDMAR or reaction yield. detected magnetic resonance spectra observed so far show that photosynthesis involves only singlet states, places limits on the distances and interactions involved in the pF radical pair state, and shows a sensitivity to the reduced Fe-Q complex. This form of RYDMAR should be a powerful technique for investigatingnot only bacterial photosynthesis but also green plant photosynthesis and model photosynthetic systems. We are particularly grateful to Dr. M. C. Thurnauer for many collaborations, discussions, and interactions. M.K.B. is grateful to Prof. E. L. Frankevitch of the Institute of Physical Chemistry, Moscow, U. S. S. R. for helpful discussions on RYDMAR. J. R. N. and M. K. B. also are indebted to Prof. Michel-Beyerle of the Technische Hochschule Munchen for discussions on the field dependence of the quantum yield of pR. This work was supported by the Office of Chemical Sciences, Division of Basic Energy Sciences, U.S. Department of Energy, under Contract W-31-109-Eng-38. D.E.B. acknowledges the support provided by Training Grant GM-01783 from the National Institutes of Health and G. L.C. is grateful for support by the National Science Foundation (CHE 7821789). C.A.W. acknowledges support from the National Science Foundation (PCM 8012032). 1. Fajer, J., Brune, D. C., Davis, M. S., Forman, A. & Spaulding, L. D. (1975) Proc. Natl. Acad. Sci. USA 72, 4956-4960. 2. Netzle, T. L., Rentzepis, P. M., Tiede, D. M., Prince, R. C. & Dutton, P. L. (1977) Biochim. Biophys. Acta 460, 467479. 3. Rockley, M. G., Windsor, M. W., Cogdell, R. J. & Parson, W. W. (1975) Proc. Natl. Acad. Sci. USA 72, 2251-2255. 4. Kaufmann, K. J., Dutton, P. L., Natzel, T. L., Leigh, J. S. & Rentzepis, P. M. (1975) Science 188, 1301-1304. 5. Netzel, T. L., Leigh, J. S. & Rentzepis, P. M. (1973) Science 182,
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