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 (Qp') and secondary (QB') ubiquinone electron acceptors in reaction centers (RCs) of the photosynthetic bacterium Rhodobacter sphaeroides. To reduce the EPR linewidth, the high-spin Fe e 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 Qp' and QR', which are attributed to different environments of the quinones in the RC. The differences exhibited themselves in: (i) the g-tensors, (ii) the "O and 13 C hyperfine coupling (hfc) constants of the quinones labeled at the carbonyl group, (iii) the 'H-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 Qp' and QB' have at least two hydrogen bonds of different strengths to the carbonyl oxygens. The hydrogen bonds for Qp' are stronger and more asymmetric than for Q. For Q the stronger bond (to 04) was assigned to His(M219) and the weaker (to 0,) to Ala(M260). For Q the stronger bond (to 0 4) was assigned to His(L190), with several weaker bonds (to 0,) to Ser(L223), lle(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 they were assigned to N 8 of His(M219) and to the peptide backbone nitrogen of Ala(M260) and for QB' to N d 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 maghetic resonance results, the function of the quinone acceptors in the reaction center is discussed.

Trapped Conformational States of Semiquinone (D+•QB-•) Formed by B-Branch Electron Transfer at Low Temperature in Rhodobacter sphaeroides Reaction Centers

“…In contrast, Q B −• , the reduced anionic semiquinone, has always been found to be bound in the proximal site (27,28) consistent with the stabilization of the negative charge by H-bonds. This binding position is supported by EPR and ENDOR (31,32) and FTIR (33,34) spectroscopic studies.…”

“…The Quintuple mutant RC sample frozen in the dark had essentially no (< 1% of maximum) EPR signal (Frozen Dark trace, 2.0053 (32). The uncoupled semiquinone would be expected to result in an observable shift in the g-value (2.0036) and introduce an asymmetry in the lineshape (1.5:1 ratio in relative amplitude of the peak and trough) (see e.g.…”

Determination of the Proton Environment of High Stability Menasemiquinone Intermediate in Escherichia coli Nitrate Reductase A by Pulsed EPR

“…In particular, the spin density on the C␣ at the 2-position, which is sensed by the methyl protons, is reduced by ϳ30% in MSQ D as compared with the symmetrically hydrogen bonded MSQ-4 prepared in 2-propanol (39). This decrease can be explained by a strong asymmetry of hydrogen bonding to the carbonyl oxygens of MSQ D in NarGHI, which leads to a redistribution of both the spin density and charges within the quinone ring (2,43,44). Indeed, a stronger hydrogen bond to the carbonyl oxygen O1, as compared with oxygen O4, is expected to lead to an increase of spin density on carbon 3 but a decrease of the spin density on carbon 2 as observed here for MSQ D .…”

“…They are characterized by an almost axial hyperfine tensor, a predominant isotropic hyperfine coupling value in the range of 6.8 -12.3 MHz, and a characteristic relative hyperfine anisotropy (A ʈ ϪA Ќ )/A iso in the range of 0.26 -0.45 with A ʈ Ͼ A Ќ Ͼ 0. The latter value increases up to 0.76 for the methyl protons of the asymmetrically bound ubisemiquinone Q A ⅐ Ϫ in the RC from R. sphaeroides (2). Based on these results, we assign H1 to the methyl protons of MSQ D, with A iso ϭ ϩ5.53 MHz and T ϭ ϩ1.25 MHz leading to a (A ʈ Ϫ A Ќ )/A iso value of ϳ0.68 (Fig.…”

Elucidating mechanisms in haemcopperoxidases: The high-affinity QHbinding site in quinol oxidase as studied by DONUT-HYSCOREspectroscopy and density functional theory

“…This has previously discussed in detail in bacterial reaction centres (e.g. 26 ), where asymmetric binding of the quinone radical is suggested to be a prerequisite for efficient sequential electron transfer. In this work we use variants that perturb the hydrogen-bonding environment between the bound quinone and potential ligands and characterise them by the two-dimensional pulsed EPR techniques HYSCORE (HYperfine Sublevel CORElation) and DONUT-HYSCORE (DOuble NUclear coherence Transfer HYSCORE).…”