J. Phys. Chem. 1993,97, 9831-9836
9831
What Can Be Learned from the Singlet-Triplet Splitting of the Radical Pair P+H- in the Photosynthetic Reaction Center? Conclusions from Electric Field Effects on the P+HRecombination Dynamics Martin Volk,'lt Tilmann Haberle,? Reiner Feick,$ Alexander Ogrodnik,? and Maria-Elisabeth Michel-Beyerlet Institut fur Physikalische und Theoretische Chemie, Technische Universitiit Miinchen, 85747 Garching, Germany, and Max- Planck- Institut f i r Biochemie, 82152 Martinsried, Germany Received: March 31, 1993; In Final Form: June 29, 1993"
The singlet-triplet splitting J of the radical pair P+H- in the photosynthetic reaction center critically depends on the energy separation between P+H- and the triplet state 3P*. Due to its large dipole moment, the energy of P+H-can be considerably shifted by the application of an external electric field. Following a simple perturbation theoretical approach, such a shift should enlarge J to values which do not allow singlet-triplet-mixing in P H - , thereby inhibiting the triplet recombination channel P+H--c 3P*. In contrast to this prediction, the recombination dynamics of P+H- and the yield of 3P* in reaction centers of Rb. sphaeroides were found to change only slightly in an external electric field. This insensitivity of the recombination dynamics to the energy of P+H- is predicted in an extended theoretical treatment of J taking into account nuclear oscillations. Following the extended treatment, J is much less sensitive to the energy of P+H-. This has important consequences for the discussion of J in the context of primary charge separation and also for the values of J to be expected in reaction centers in which the energetics have been altered by chemical or genetic modifications.
I. Introduction After the excitation of photosynthetic reaction centers (RCs) of Rb. sphaeroides, an electron is transferred from the primary donor P, a bacteriochlorophyll (BChl) dimer, to a bacteriopheophytin (H) within 3-4 p ~ . l - The ~ detailed mechanism of this primary charge separation, especially the role of the accessory BChl monomer (B), is still discussed controversially.5" Under debate are models, in which electron transfer either occurs in two steps via B or directly with B enhancing the electronic interaction between P and H via the superexchange mechanism. Besides picosecond time resolved spectroscopyz" and hole burn experiments? another fingerprint feature of the RC has been used to draw conclusions on the mechanism of primary charge separation. This is the exchange interaction J o f the radical pair P+H-,10-15 i.e., the splitting between singlet and triplet phased radical pair, which can be determined from the magnetic field dependent recombination dynamics of P+H-."5J7 Even before the X-ray crystal structure of the RC revealed the location of B between P and H,18-20the observed small value of J led to the proposal of two-step charge separation.1° This was necessary to reconcile the small value of J with the strong electronicinteraction concluded from fast electron transfer. Alternatively, (i) a compensation of the singlet and triplet contribution to J or (ii) relaxational effects were discussed to resolve this discrepancy.13J4 In a perturbation theoretical approach, J is determined by the electronic interaction between P+H- and the singlet and triplet states IP*and 3P*,respectively, and by the energy denominators 6$ and 6,$ defining the vertical energy separation between P+H- and IP*and P+H- and 3P*, respectively, at the equilibrium nuclear configuration of P+H-. The vertical energy separations 6E0 can be calculated from the sum of the free energy difference AG and the reorganization energy A, as depicted in Figure 1. The activationless rate kT for P+H- 3P*21implies a small value of and thus similar absolute values of ACT = AG(P+H--3P*) and XT = X(P+H--3P*). Therefore, even small changes of the energy of P+H- should cause large relative changes of 6,$ and
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Technische UniversitHt Mtinchen. :Max-Planck-Institut ftir Biochemie. .Abstract published in Aduance ACS Abstracrs, September 1, 1993.
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NUCLEAR COORDINATE q
Figure 1. One-dimensional potential energy surfaces of IP*,3P*,and P+H-showingthe freeenergydifferencesAGand A&, the reorganization energies Aq. and AT and the vertical energy separations 6l$ and 6$ at the equilibrium coordinate of P+H- (q = 0).
considerably affect the triplet contribution to J , while hardly influencing the singlet contribution. This different sensitivity of the singlet and the triplet contribution to small changes of the energy of P+H- should allow one to identifycompensational effects (i) and, thus, allow conclusions on the mechanism of primary charge separation. Furthermore, changing the energy of P+Hmight lead to a more precise determination of SP,, which in turn would allow the precisedetermination of the reorganizationenergy AT for electron transfer from P+H- to 3P*since the corresponding free energy gap ACT is A powerful tool for experimentally altering the energies of charge-separated states with large dipole moments is the appliThis technique does not cation of external electric suffer from the usual uncertainty of unpredictable structural changes in chemically modified or genetically altered RCs. In this paper we report measurements of the electric field dependence
0022-365419312097-9831$04.00/0 0 1993 American Chemical Society
9832 The Journal of Physical Chemistry, Vol. 97, No. 38, 1993
Volk et al.
oftherecombinationdynamicsofP+H-inRCsofRb.sphaeroides. It will be shown that, on the basis of these measurements, a compensation of large singlet and triplet contributions to J (i) can be exclused. Furthermore, the experimental results forced us toextend theconventional theoretical treatment of J. A precise determination of and AT from these results, however, is not possible.
6e
11. Recombination Dynamics of P+HIn quinone-depleted RCs of Rb.sphaeroides the radical pair P+H- recombines on the nanosecond time scale.16~2"30Recombination proceeds from the singlet-phased radical pair to the ground state P with the rate ks or after hyperfine induced singlettriplet mixing (S-T mixing) from the triplet-phased radical pair to 3P* with the (faster) rate kT. At low magnetic fields, S-T mixing involving all three triplet levels is possible. A magnetic field of several 100 G removes the degeneracy of two of the three triplet levels '(P+H-) with the singlet level l(P+H-). This leads to a reduction of the efficiency of the isoenergetic S-T mixing, thus reducing the yield @T of 3P* and, concomitantly, increasing the radical pair lifetime TRP. Therefore, on increasing the magnetic field a steady decrease of @T is observed, saturating above 500 G (magnetic field dependence of the reaction yield, MARY). The half-width Hl12 of the MARY curve is defined as the magnetic field at which half of the maximum magnetic field effect on @T is found. Its value is mainly determined by the energetic spread of the singlet and triplet radical pair levels, respectively, which arise from the hyperfine interaction and especially the lifetime broadening. The latter is due to the recombination rates ks and kT, respectively. Since kT >> ks,H1/2 is a measure for k ~ . The ~ l singlet recombination rate ks can be directly obtained from TW and @ ~ . 2 2 , 3 1 For RCs of Rb. sphaeroides a value of approximately 5 X lo* s-1 was found for kT, independent of the t e m p e r a t ~ r e , 2 ~while ~ ~ ~ks 8 decreases ~~ from 5 X lo7s-' at room temperature to 1.5 X lo7s-l at 90 K.22,34The exchange interaction J, which also removes the degeneracy of cm-I) singlet and triplet levels, is found to be small (1 X compared to Hlp ( 5 5 G N 5 X 10-3 cm-l).21,32
III. Electric Field Dependent Measurements Materials and Methods. The preparation of RCs of Rb. sphaeroides R26 depleted of both quinones has been described.22 The RC concentration was adjusted to 160 pM in Tris buffer (20 mM, pH 8.0) with 0.1% Nonidet P40. RCs were embedded in poly(viny1 alcohol) (PVA), yielding a film of 80-pm thickness after drying for 48 h at 4 "C in a N2 gas flow. Nanosecond time resolved absorbance measurements were performed as described p r e v i ~ u s l y .RCs ~ ~ were excited at 600 nm with a Nd:YAG laser pumped dye laser (pulse width 1.8 ns) with a maximum intensity of 0.5 mJ/cm2 which corresponds to a yield of approximately 20% excited RCs. Absorbance changes were probed in the Quband of P around 870 nm with a N2 laser pumped dye laser (pulse width 1.5 ns). The delay time between excitation and probing pulse was adjusted electronically between 0 ns and 10 ms in steps of 1 ns. PVA films were sandwiched between glass slides coated with a transparent Sn02 layer to which square pulses of up to 5 kV with 20-ms duration were applied. The Stark effect on the absorbance of the sample was measured in situ by measuring the difference of the absorbance at the probing wavelength with and without the electric field in the absence of actinic light. The calibration of the electric field was achieved via the Stark e f f e ~ t . ' ~At . ~ 90 ~ and 210 K an electric field induced reduction of the P absorbance around 870 nm by 0.7% was observed when applying 5 kV, implying an electric field of approximately 6 X lo5 V/cm, which is in good agreement with the value estimated from the applied voltage and the thickness of the PVA film.
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