Low-temperature pulsed EPR study at 34 GHz of the triplet states of the primary electron Donor P865 and the carotenoid in native and mutant bacterial reaction centers of Rhodobacter sphaeroides

Abstract

The photosynthetic charge separation in bacterial reaction centers occurs predominantly along one of two nearly symmetric branches of cofactors. Low-temperature EPR spectra of the triplet states of the chlorophyll and carotenoid pigments in the reaction center of Rhodobacter sphaeroides R-26.1, 2.4.1 and two double-mutants GD(M203)/AW(M260) and LH(M214)/AW(M260) have been recorded at 34 GHz to investigate the relative activities of the "A" and "B" branches. The triplet states are found to derive from radical pair and intersystem crossing mechanisms, and the rates of formation are anisotropic. The former mechanism is operative for Rb. sphaeroides R-26.1, 2.4.1, and mutant GD(M203)/AW(M260) and indicates that A-branch charge separation proceeds at temperatures down to 10 K. The latter mechanism, derived from the spin polarization and operative for mutant LH(M214)/AW(M260), indicates that no long-lived radical pairs are formed upon direct excitation of the primary donor and that virtually no charge separation at the B-branch occurs at low temperatures. When the temperature is raised above 30 K, B-branch charge separation is observed, which is at most 1% of A-branch charge separation. B-branch radical pair formation can be induced at 10 K with low yield by direct excitation of the bacteriopheophytin of the B-branch at 590 nm. The formation of a carotenoid triplet state is observed. The rate of formation depends on the orientation of the reaction center in the magnetic field and is caused by a magnetic field dependence of the oscillation frequency by which the singlet and triplet radical pair precursor states interchange. Combination of these findings with literature data provides strong evidence that the thermally activated transfer step on the B-branch occurs between the primary donor, P865, and the accessory bacteriochlorophyll, whereas this step is barrierless down to 10 K along the A-branch.

Figure 1

Cofactor arrangement in the bacterial reaction centers of (a) Rb. sphaeroides R-26.1 (PDB code: 1pcr), (b) Rb. sphaeroides 2.4.1 (PDB code: 4rcr), (c) Rb. sphaeroides double mutant GD(M203)/AW(M260) and (d) Rb. sphaeroides double mutant LH(M214)/AW(M260). The mutants are modeled after the structure of a quintuple mutant that contains all these mutations (PDB code: 1yf6) (15).

Figure 2

Q-band transient EPR spectrum of the bRC of Rb. sphaeroides R-26.1 at T = 10 K.

Figure 3

Q-band ESE-detected triplet EPR spectra in bRCs in Rb. sphaeroides at T = 10 K (left) and T = 50 K (right). (a) Rb. sphaeroides R-26.1, (b) Rb. sphaeroides 2.4.1, (c) Rb. sphaeroides GD(M203)/AW(M260), (d) Rb. sphaeroides LH(M214)/AW(M260). (e) ³BChla in pyridine. The signal marked with (*) is assigned to a radical signal. The microwave frequency was typically 33.9 GHz with a variation of 0.2 GHz depending on the sample.

Figure 4

(a) ESE-detected EPR spectra of the ³P₈₆₅ and ³Car triplet signals in Rb. sphaeroides 2.4.1 at 50K recorded at different times after the laser flash. Dotted lines indicate the canonical orientations of ³P₈₆₅, dashed lines those of ³Car; (b) Time traces of the EPR signal recorded at B ‖ X_II and B ‖ Z_I and simulations (dashed lines) using the model described in the supporting information; (c,d) Temperature dependence of time constants for ³Car formation and decay in Rb. sphaeroides 2.4.1; (e,f) Temperature dependence of time constants for ³Car formation and decay in Rb. sphaeroides GD(M203)/AW(M260).

Figure 5

(a) Schematic energy level diagram for a nuclear spin I = 1 and electron spin S = 1 in a magnetic field, including nuclear Zeeman, hyperfine (hfi) and nuclear quadrupole (nqi) interactions. The three frequencies ν_sq(1), ν_sq(2) and ν_dq of the M_S = 0 manifold dominate the ESEEM spectrum. (b) Two-pulse ESEEM spectra and modulation patterns (inset) of ³P₈₆₅ recorded for Rb. sphaeroides GD(M203)/AW(M260) at the Z_I (low field) and Z_II (high field) canonical orientations (see figure 2a). The modulation depth at Z_I is about four times larger than at Z_II. The temperature is 10 K and no contribution of carotenoid was detected in the EPR spectrum.

Figure 6

(top) UV-VIS spectrum and ESE-detected EPR spectra of ³P₈₆₅ in Rb. sphaeroides LH(M214)/AW(M260) at T = 10 K. (bottom) EPR spectra recorded with laser excitation of wavelength (a) 537 nm, (b) 590 nm and (c) 865 nm. The labels in the UV-VIS spectrum indicate absorbing cofactors. The arrows at the spectrum with (a) λ_exc = 537 nm and T = 10 K indicate additional signals of low intensity which correspond to the RP triplet state of ³P₈₆₅.

Figure 7

EPR spectra (upper trace) and simulations (lower traces) of the ³P₈₆₅ and ³Car triplet states in bacterial reaction centers of Rb. sphaeroides. (a) transient EPR, ³P₈₆₅ in Rb sphaeroides R-26.1, T = 10 K; (b) ESE-detected EPR, ³P₈₆₅ in Rb. sphaeroides LH(M214)/AW(M260), T = 10 K, t_DAF = 500 ns; (c) ESE-detected EPR, ³Car in Rb. sphaeroides GD(M203)/AW(M260), T=70K, t_DAF=500 ns; (d) ESE-detected EPR, ³Car in Rb. sphaeroides GD(M203)/AW(M260), T=70K, t_DAF=25 μs; (e) ESE-detected EPR, ³Car in Rb. sphaeroides LH(M214)/AW(M260), T = 50K, t_DAF = 500 ns. The signals marked with * belong to a small contribution of ³P₈₆₅, which has not yet fully decayed. The middle trace in (e) is a simulation assuming a pure ISC mechanism, in the lower trace 50% ISC and 50% RP mechanism efficiency was used.

Figure 8

(a) Triplet formation in bacterial reaction centers of Rb. sphaeroides R-26.1, 2.4.1 and GD(M203)/AW(M260) by the radical pair mechanism. Energies are in [cm⁻¹] and reproduced from references (5;13). Also indicated is the splitting for the triplet sublevels for the magnetic field direction parallel to the X, Y or Z principal axis of the ZFS tensor. All population is located in the M_S = 0 sublevel. The resulting polarization pattern of the ESE-detected EPR spectrum; (b) Triplet formation in the bacterial reaction center of Rb. sphaeroides LH(M214)/AW(M260) by the intersystem crossing mechanism. The splitting of the triplet sublevels is identical to (a), but the population is distributed as described by equation (1), resulting in a different polarization pattern. P: special pair (bacteriochlorophyll dimer), B_A: accessory bacteriochlorophyll, H_A: bacteriopheophytin (both in the A-branch).

Figure 9

(a) Formation of ³P₈₆₅ at T = 10 K in Rb. sphaeroides LH(M214)/AW(M260) by the intersystem crossing mechanism at excitation of 865 nm; (b) possible HOMO-based formation of ³P₈₆₅ by the radical pair mechanism at excitation of 537 nm.

Scheme 1