Light induced EPR spectra of reaction centers from Rhodobacter sphaeroides at 80K: Evidence for reduction of Q(B) by B-branch electron transfer in native reaction centers

Abstract

Photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides capture solar energy by electron transfer from primary donor, D, to quinone acceptor, Q(B,) through the active A-branch of electron acceptors, but not the inactive B-branch. The light induced EPR spectrum from native RCs that had Fe(2+) replaced by Zn(2+) was investigated at cryogenic temperature (80K, 35 GHz). In addition to the light induced signal due to formation of D(+•)Q(A) (-•) observed previously, a small fraction (~5%) of the signal displayed very different characteristics: (1) The signal was absent in RCs in which the Q(B) was displaced by the inhibitor stigmatellin. (2) Its decay time (τ=6 s) was the same as observed for D(+•)Q(B) (-•) in mutant RCs lacking Q(A,) which is significantly slower than for D(+•)Q(A) (-•) (τ=30 ms). (3) Its EPR spectrum was identical to that of D(+•)Q(B) (-•). (4) The quantum efficiency for forming the major component of the signal was the same as that found for mutant RCs lacking Q(A) (Φ =0.2%) and was temperature independent. These results are explained by direct photochemical reduction of Q(B)via B-branch electron transfer in a small fraction of native RCs.

Figure 1

Structure of the cofactors in Rb. sphaeroides RCs (based on coordinates 1AIG and 1AIJ [17]. Electron transfer occurs with high efficiency along the A branch (solid arrow). Following light excitation, electron transfer occurs from the excited state of the donor, D, to the primary quinone electron acceptor, Q_A. This transfer occurs even at cryogenic temperature. Electron transfer from Q_A^−• to Q_B is temperature dependent and inhibited at cryogenic temperatures. Two positions for Q_B, distal and proximal, are indicated; their properties are discussed in the text. Possible electron transfer along the B branch (dotted arrow) with low quantum yield is the focus of this study.

Figure 2

EPR spectrum of RCs frozen in the dark (T= 80 K, ν = 35.03 GHz). The dark heavy line is the spectrum generated by illumination of the RCs in the EPR cavity. The lowest magnetic field peak is due to the ubisemiquinone Q^−•; the middle field peak to contributions from the oxidized donor, D^+• (predominant), and the reduced acceptor, Q^−• (lesser); and the high field peak a g-marker (g=1.99891). The baseline spectrum before illumination is shown (thinner grey line). The arrow indicates the magnetic field position used for the kinetic studies. (Conditions: 100μM RCs in TMK and D₂O, 80K, ν = 35 GHz, magnetic field modulation (f=270 Hz, ΔH=1.5 G), average 28 traces, time constant 20ms, scan time 30s.

Figure 3

Formation and decay kinetics of the light induced EPR signal of D^+•Q^−• at higher light intensity (A) (I=100%), and at lower light intensity (B) (I=2%). The sample is illuminated from t=−4 s to t=0 s. At high light intensity the decay of the signal consists of a fast phase (τ=30 ms, unresolved in this trace) that has been previously reported [15]. In addition a slow phase with decay time (τ = 6.0±1 s) that represents about 5% of the amplitude is also seen (arrow). The two kinetic phases are more apparent at lower light intensity (B) due the decrease in amplitude of the fast phase relative to that of the slow phase. In addition, a slow phase in the rise of the signal is also apparent. The amount of the slowly decaying component was equal to that of the slowly rising component. (Conditions: same as in Figure 2, magnetic field fixed at 12,493 G, field modulation ΔH= 10 G, average of 10–100 traces.)

Figure 4

Dependence of fast and slow phase amplitudes on illumination time. The illumination time was varied at low light intensity (2%). The amplitude of the slowly decaying component increases with illumination time while that of the fast phase is relatively constant. This indicates the quantum yield for the slow component is lower than for the fast component. The amplitude of the slower rising component is the same as that of the slower decaying component, showing that the slow rise and slow decay are properties of the same charge separated state. (Conditions: same as in Figure 3.)

Figure 5

Effect of Stigmatellin on the slow phase. Kinetic traces are shown for the light induced EPR signals of RCs to which either excess Q₁₀ (+ Q₁₀,) or stigmatellin (+ Stig,) was added (I=2%). The RCs containing stigmatellin lack the slowly rising and slowly decaying phases seen in RCs having added Q₁₀. Since stigmatellin is a potent inhibitor of Q_B function, these results show that the slow rise and decay kinetic phases are due to Q_B reduction and oxidation. The time scales of the two traces are slightly displaced for better viewing. (Conditions: same as Figure 3)

Figure 6

EPR spectra at high (100%) and low light (2%) intensities. A) The spectra of D^+• and Q^−• are normalized to the D^+• peak (middle). A small shift in the Q^−• spectrum (around the arrow) is observed. The scale is expanded in B) to better illustrate the observed shift in the spectrum of the Q^−• peak. The spectra at low light intensity can be identified by its lower signal-to-noise ratio. (Conditions: same as Figure 2, average of 28 traces for 100% transmission and 160 traces for 2% transmission.)

Figure 7

EPR difference spectrum in the semiquinone region associated with Q_X^−•. The Q_A^−• contribution to the observed spectrum (I=2%) was removed as described in the text. The spectra for Q_A^−• (grey line) and Q_B^−• (black line) are shown for comparison. The difference spectrum of Q_X^−• matches that of the Q_B^−•. This indicates that the component that gives rise to the slow phase is Q_B^−•. (Conditions: same as Figure 6.)

Figure 8

The amplitudes of the slow and fast components as a function of the light intensity (plotted as a % of maximal). The amplitudes were normalized to 1.0 at infinite light intensity. This resulted in an 18-fold increase in the relative amplitude of the slow component, which is smaller than that of the fast at all light intensities; it had a value near 5% at I=100%. The fast phase amplitude was fit with eqn. (7) to obtain the value of σ. Eqn. (9) was used to fit the slow phase data using a heterogeneous distribution of two RC fractions capable of B-branch transfer: 1) a large fraction (α₁ = 15%) with low quantum yield (Φ_B1= 0.2%) and 2) a smaller fraction (α₂ =1.5%) with high quantum yield (Φ_B2 = 100%).