EPR and ENDOR Investigation of Rhodosemiquinone in Bacterial Reaction Centers Formed by B-Branch Electron Transfer

Abstract

In photosynthetic bacteria, light-induced electron transfer takes place in a protein called the reaction center (RC) leading to the reduction of a bound ubiquinone molecule, Q(B), coupled with proton binding from solution. We used electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) to study the magnetic properties of the protonated semiquinone, an intermediate proposed to play a role in proton coupled electron transfer to Q(B). To stabilize the protonated semiquinone state, we used a ubiquinone derivative, rhodoquinone, which as a semiquinone is more easily protonated than ubisemiquinone. To reduce this low-potential quinone we used mutant RCs modified to directly reduce the quinone in the Q(B) site via B-branch electron transfer (Paddock et al. in Biochemistry 44:6920-6928, 2005). EPR and ENDOR signals were observed upon illumination of mutant RCs in the presence of rhodoquinone. The EPR signals had g values characteristic of rhodosemiquinone (g(x) = 2.0057, g(y) = 2.0048, g(z) ∼ 2.0018) at pH 9.5 and were changed at pH 4.5. The ENDOR spectrum showed couplings due to solvent exchangeable protons typical of hydrogen bonds similar to, but different from, those found for ubisemiquinone. This approach should be useful in future magnetic resonance studies of the protonated semiquinone.

Fig. 1

Structure of the quintuple RC. The cofactors and mutation sites are shown. The mutated residue Trp M260 is in the Q_A site and prevents Q_A binding. The other mutations enhance electron transfer directly to Q_B via the B-branch. Modified from Ref. [15] with permission. Copyright 2006 American Chemical Society

Fig. 2

Structures of a rhodoquinone and b ubiquinone. In rhodoquinone, an amino group replaces a methoxy group on the ubiquinone ring. The rhodoquinone compound used in this study has n = 3

Fig. 3

EPR powder spectra of rhodosemiquinone and ubisemiquinone at 80 K. a RQ^•− in nonprotic organic solvents, b ${\text{RQ}}_{\text{B}}^{•-}$ in quintuple mutant RCs at pH 9.5, c ${\text{RQ}}_{\text{B}}^{•-}$ (or RQ_BH^•) in quintuple mutant RCs at pH 4.5 and d ${\text{UQ}}_{\text{B}}^{•-}$ in quintuple mutant RCs at pH 7.1. Both g_x and g_y differ between the ${\text{RQ}}_{\text{B}}^{•-}$ and ${\text{UQ}}_{\text{B}}^{•-}$ samples and show slight shifts between pH 9.5 and 4.5. Experimental conditions: a MW frequency = 33.87 GHz, MW power = 3.2 × 10⁻⁴ W, spectrum obtained by pseudo-modulating the field-swept FID-detected EPR spectrum with 0.15 mT (see Sect. 2.2), average of 2 scans, 40 s per scan. The others: MW frequency = 35.03 GHz, MW power = 1 × 10⁻⁷ W, field modulation = 0.15 mT peak-to-peak at 270 Hz. Number of scans: b 7, c 29 and d 100. Scan time: 20 s

Fig. 4

ENDOR powder spectra of ${\text{RQ}}_{\text{B}}^{•-}$ and ${\text{UQ}}_{\text{B}}^{•-}$ in quintuple mutant RCs. Arrows indicate the couplings between exchangeable protein protons that form H-bonds with ${\text{UQ}}_{\text{B}}^{•-}$ (upper trace) and proposed to form H-bonds with ${\text{RQ}}_{\text{B}}^{•-}$ (middle trace). Assignment of these latter peaks to H-bonds is supported by their absence upon exchange into D₂O (lower trace). Spectra recorded at the magnetic field position corresponding to g_y (see Fig. 3). Experimental conditions: T = 80 K, MW frequency = 35.03 GHz, MW power = 3 × 10⁻⁶ W, frequency modulation (FM) = ±140 kHz at a rate of 947 Hz. Number of scans: 18,000 (top), 500 (middle) and 2,600 (bottom). Scan time: 4 s