Isotropic ¹³C Hyperfine Coupling Constants Distinguish Neutral from Anionic Ubiquinone‐Derived Radicals

Abstract

Ubiquinol oxidation to ubiquinone and/or the reverse reaction, ubiquinone reduction to ubiquinol (Scheme 1), is critical in the actions of several enzymes, including cytochrome (cyt) bc 1 [1][2][3] and cyt bo 3 ubiquinol oxidase, [4] as well as photosynthesis in bacterial photosynthetic reaction centers.[5] For example, cyt bo 3 from E. coli reportedly contains two ubiquinol/ubiquinone binding sites, a low-affinity site (QL), where ubiquinol is oxidized and a high-affinity site (QH) where a bound ubiquinone species acts as a conduit for electrons. The QH site binds a quinone tightly and stabilizes a transient intermediate, the one-electron reduced quinone (or semiquinone), through hydrogen bonding by the protein. The best available evidence provides conflicting reports about the exact identity of the intermediate. Multi-frequency EPR experiments with 13 C-labeled ubiquinone-2 [6] implies binding of an anionic semiquinone radical to the protein via a one-sided hydrogen bond or a strongly asymmetric hydrogen-bonding network. In contrast, two-dimensional electron spin-echo envelope modulation experiments with the exchangeable protons implies "significant covalent OÀH binding of carbonyl oxygen O1 that is characteristic of a neutral radical". [7,8] Computed isotropic hyperfine coupling constants (hfccs) for a model of the ubiquinone-derived radicals presented herein show that 13 C hfccs for the carbonyl carbon atoms can be more than twenty times larger for the protonated, neutral radical (UQHC, Scheme 2) than for the anionic semiquinone radical (UQC À ) and confirm that the signs of 13

Interactions of Intermediate Semiquinone with Surrounding Protein Residues at the QH Site of Wild-Type and D75H Mutant Cytochrome bo3 from Escherichia coli

“…6. Previous DFT studies 33–35 using SQ anion models have been unable to match the experimental value for this position and this can be mainly attributed to the use of water or amide groups as the hydrogen bonding groups in these modeling studies. Because the elevated 5-CH 3 hyperfine coupling was similar to that found for neutral semiquinone free radicals, it has been proposed that the Q H site SQ is a neutral radical.…”

“…33–35 In our work, based on the X-ray structure, Fig. 1, and the EPR and mutational data for the WT enzyme, the SQ H was modeled to have hydrogen bonds to the OH of the carboxylic acid group of D75, the N ε H of the imidazole group of H98 and the N ε H group of the guanidium group of R71.…”

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

“…In a simultaneous recent study Boesch et al 27 also briefly addressed this problem. Here we provide a comprehensive discussion of various EPR parameters with the aim to establish the exact nature of the bound semiquinone.…”

“…On the quantum-chemical side we extend our set of models to those having a neutral semiquinone radical as a stabilised cofactor in the Q H site. In a simultaneous recent study Boesch et al 27 also briefly addressed this problem. Here we provide a comprehensive discussion of various EPR parameters with the aim to establish the exact nature of the bound semiquinone.…”

Exploring by Pulsed EPR the Electronic Structure of Ubisemiquinone Bound at the QH Site of Cytochrome bo3 from Escherichia coli with in Vivo 13C-Labeled Methyl and Methoxy Substituents

“…These data indicate the substantial shift of the unpaired spin density toward the O1 side of the Q H SQ. Unfortunately, very recent calculations devoted to the comparison of the unpaired spin density distributions in neutral and anionic SQ species of UQ (54,55) do not report the values of the 13 C methyl couplings, precluding any comparison of the values shown in Table 2 with theoretical values.…”