Protein−Cofactor Interactions and EPR Parameters for the Q_H Quinone Binding Site of Quinol Oxidase. A Density Functional Study

Abstract

Recent multifrequency EPR studies of the "high-affinity" quinone binding site of quinol oxidase (Q(H) site) have suggested a very asymmetric hydrogen-bonding environment for the semiquinone radical anion state. Single-sided hydrogen bonding to the O1 carbonyl position was one of the proposals, which contrasts with some previous experimental indications. Here density functional calculations of the EPR parameters (g-tensors, 13C, 1H, and 17O hyperfine tensors) for a wide variety of supermolecular model complexes have been used to provide insight into the detailed relations among structure, environment, and EPR parameters of ubisemiquinone radical anions. A single-sided binding model is not able to account for the experimentally observed low g(x) component of the g-tensor or for the observed magnitude of the asymmetry of the 13C carbonyl HFC tensors. Based on the detailed comparison between computation and experiment, a model with two hydrogen bonds to O1 and one hydrogen bond to O4 is suggested for the Q(H) site, but a model with one more hydrogen bond on each side cannot be excluded. Several general conclusions on the interrelations between EPR parameters and hydrogen bond patterns of ubisemiquinones in proteins are provided.

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

“…As these reference systems feature extensive (double-sided) hydrogen bonding and a low Dg XX is representative of strong hydrogen bonding, we must conclude that the low Dg XX can only be explained by strong hydrogen bonding. 25 Similar to our earlier observation 25 the computed Dg XX values for the models are significantly too large for most of the structures (even after scaling by 0.88 to account for systematic deficiencies of the DFT methods used) except for ones with two hydrogen bonds to the carbonyl and either none or one to the hydroxyl oxygen. However, the latter structures contradict the binding situation suggested by recent work of Yap et al 18,24 who proposed a hydrogen bonding network with two hydrogen bond donors to the hydroxyl oxygen (O 1 ) and no additional hydrogen bond to the carbonyl oxygen (O 4 ).…”

“…8b). Unlike our previous studies on models involving semiquinone radical anion, 25 where dihedral angles g were typically below 30 -40 , significantly different conformations of In this experiment the time between the first two (both p/2) microwave pulses was s ¼ 128 ns and the times t 1 (between the second (p/2) and third (p) microwave pulses) and t 2 (between the third (p) and fourth (p/2) microwave pulses) were incremented in steps of 32 ns from their initial values and 256 points were recorded in each dimension. Experimental conditions: p/2 and p pulse lengths: 16 ns and 12 ns (optimised for maximal echo inversion) respectively, temperature: 20 K, microwave frequency: 9.764 GHz, shot repetition time: 20 ms. (C) Mims 1 H-ENDOR 79 spectra of Q _ H in 15 N-labelled QOX under reducing conditions.…”

“…We have also shown that single-sided hydrogen bonding in an anionic model would cause even larger differences between the two carbonyl sites than observed experimentally and in particular an even lower A ZZ for the C 4 position. 25 In contrast, unsymmetrical double-sided anionic models, especially 2/1 models, provide much better agreement with the measured data for the Q H site (see Table 4). Recently it has been suggested that exact measurements of the complete 13 C hyperfine tensors for C 1 and C 4 could unambiguously resolve between an anionic and a neutral form of semiquinone radicals.…”

“…2/0-b 1HO-1HO) enforce hydrogen bonds even shorter than those found for semiquinone radical anion models. 25 A second water molecule, introduced as a potential hydrogen-bond donor on either O 1 or O 4 sides, always imposed a strong out-of-plane conformation on one of them with a simultaneous relocation of a second one to bridge between the first water molecule and a methoxy oxygen. This effect is due to the steric hindrance of the substituents of ubisemiquinone.…”

“…In ref. 25 we have shown that the differences between the 13 C hyperfine couplings of the carbonyl groups reflect specifically the asymmetry of the hydrogen-bonding framework, if one keeps in mind that one may not straightforwardly transfer HFC values from symmetrical to asymmetrical environments. We have also shown that single-sided hydrogen bonding in an anionic model would cause even larger differences between the two carbonyl sites than observed experimentally and in particular an even lower A ZZ for the C 4 position.…”