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.

Hydrogen Bonding between the QBSite Ubisemiquinone and Ser-L223 in the Bacterial Reaction Center: A Combined Spectroscopic and Computational Perspective

“…The results of this work, showing no strong hydrogen bonding between Ser-L223 and Q B − in the Q A Q B − state, are in apparent contrast with the CW-ENDOR study by Paddock et al (27) The Paddock et al conclusions were based purely on the observation of 1 H ENDOR intensity changes, on mutation, which were attributed directly to hydrogen bonded proton hyperfine couplings from the L223-Ser hydroxyl group in the WT sample. As mentioned above, we do observe some intensity changes in this hydrogen bonding region on mutation but not as extensively as reported in the other study.…”

The impact of short‐term UV irradiation on grains of sensitive and tolerant cereal genotypes studied by EPR

“…However, the shape of radical spectra observed by us was similar to that found for barley grains irradiated with gamma rays, our interpretation of EPR signals was different. Signal I, present in the spectra of whole grains and seed coats, recorded at 293 K, exhibited parameters similar to that of semiquinone radical , , . Its contribution to the spectra was the highest in seed coat, containing high amounts of phenolic‐type compounds.…”

“…Signal I, present in the spectra of whole grains and seed coats, recorded at 293 K, exhibited parameters similar to that of semiquinone radical. 24,26,27 Its contribution to the spectra was the highest in seed coat, containing high amounts of phenolic-type compounds.…”

Pulse Q-Band EPR and ENDOR Spectroscopies of the Photochemically Generated Monoprotonated Benzosemiquinone Radical in Frozen Alcoholic Solution

“…Of particular interest is the protonated rhodosemiquinone radical (RQ B H • ) in the reaction center of Rhodobacter sphaeroides . This species has been recently observed by EPR spectroscopy and shown to be suitable for studying the magnetic properties of the crucial high energy intermediate Q B H • state involved in the reduction of Q B to Q B H 2 . , Another system in which the protonated semiquinone has been proposed is the cytochrome bo 3 ubiquinol oxidase from Escherichia coli . However, this assignment has been questioned by MacMillan et al who argued that the isotropic coupling calculated for the covalently bound proton was found to be larger than is observed experimentally .…”