The primary and secondary acceptors in bacterial photosynthesis III. Characterization of the quinone radicals Q A − ⋅ and Q B − ⋅ by EPR and ENDOR

Abstract

Electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) techniques were used to investigate the electronic structure of the primary (Q A −⋅ ) and secondary (Q B −⋅ ) ubiquinone electron acceptors in reaction centers (RCs) of the photosynthetic bacteriumRhodobacter sphaeroides. To reduce the EPR linewidth, the high-spin Fe2+ present in native RCs was replaced by diamagnetic Zn2+. Experiments were performed both on frozen solutions and single crystals at microwave frequencies of 9, 35 and 94 GHz. Differences in the EPR/ENDOR spectra were observed for Q A −⋅ and Q B −⋅ , which are attributed to different environments of the quinones in the RC. The differences exhibited themselves in: (i) the g-tensors, (ii) the17O and13C hyperfine coupling (hfc) constants of the quinones labeled at the carbonyl group, (iii) the1H-hfcs of the quinone ring and (iv) the exchangeable protons hydrogen bonded to the carbonyl oxygens. From these results and from H/D exchange experiments, the following conclusions were drawn: both Q A −⋅ and Q B −⋅ have at least two hydrogen bonds of different strengths to the carbonyl oxygens. The hydrogen bonds for Q A −⋅ are stronger and more asymmetric than for Q B −⋅ . For Q A −⋅ the stronger bond (to O4) was assigned to His(M219) and the weaker (to O1) to Ala(M260). For Q B −⋅ the stronger bond (to O4) was assigned to His(L190), with several weaker bonds (to O1) to Ser(L223), Ile(224) and Gly(L225). From the temperature dependence of the hfcs of the exchangeable protons some dynamic properties of the RC were deduced. Hfcs with more distant nitrogens were observed by electron spin echo envelope modulation (ESEEM). For Q A −⋅ they were assigned to Nδ of His(M219) and to the peptide backbone nitrogen of Ala(M260) and for Q B −⋅ to Nδ of His(L190). These interactions indicate the extent of the electron wave function, which is important for the understanding of the electron transfer mechanism. Based on the magnetic resonance results, the function of the quinone acceptors in the reaction center is discussed.