Selective 15N isotope labeling of the cytochrome bo3 ubiquinol oxidase from E. coli with auxotrophs was used to characterize the hyperfine couplings with the side-chain nitrogens from R71, H98, and Q101 residues and peptide nitrogens from R71 and H98 residues around the semiquinone (SQ) at the high-affinity QH site. The 2D ESEEM (HYSCORE) data have directly identified the Nε of R71 as an H-bond donor carrying the largest amount of the unpaired spin density. In addition, weaker hyperfine couplings with the side-chain nitrogens from all residues around the SQ were determined. These hyperfine couplings reflect a distribution of the unpaired spin density over the protein in the SQ state of the QH site and strength of interaction with different residues. The approach was extended to the virtually inactive D75H mutant, where the intermediate SQ is also stabilized. We found that the Nε from a histidine residue, presumably H75, carries most of the unpaired spin density instead of the Nε of R71, as in the wild-type bo3. However, the detailed characterization of the weakly coupled 15Ns from selective labeling of R71 and Q101 in D75H was precluded by overlap of the 15N lines with the much stronger ~1.6 MHz line from quadrupole triplet of the strongly coupled 14Nε from H75. Therefore, a reverse labeling approach, in which the enzyme was uniformly labeled except for selected amino acid types, was applied in order to probe the contribution of R71 and Q101 to the 15N signals. Such labeling has shown only weak coupling with all nitrogens of R71 and Q101. We utilize density functional theory based calculations to model the available information about 1H, 15N and 13C hyperfine couplings for the QH site and to describe the protein-substrate interactions in both enzymes. In particular, we identify the factors responsible for the asymmetric distribution of the unpaired spin density and ponder the significance of this asymmetry to the quinone’s electron transfer function.
QM/MM calculations have been used to monitor the oxidation of the D2-Tyr160, Tyr(D), residue involved in redox reactions in Photosystem II. The results indicate that in the reduced form the residue is involved in hydrogen bond donation via its phenolic head group to the tau-nitrogen of the neighboring D2-His189 residue. Oxidation to form the radical is accompanied by spontaneous transfer of the phenolic hydrogen to the tau-nitrogen of D2-His189 leading to the formation of a tyrosyl-imidazolium ion complex. Deprotonation of the imidazolium ion leads to the formation of a tyrosyl-imidazole neutral hydrogen-bonded complex. Comparison of calculated and experimental hyperfine coupling tensors and g-tensors suggests that the neutral imidazole complex is formed at physiological temperatures while the imidazolium complex may be stabilized at cryogenic temperatures.
The synthesis and characterization of three new complexes, BiCl 3 (mipit) 2 , BiCl 3 (emit) 2 and BiCl 3 (mnpit) 2 are reported where emit ¼ 1-ethyl-3-methyl-2(3H)-imidazolethione; mnpit ¼ 1-methyl-3-(1-propyl)-2(3H)-imidazolethione;mipit ¼ 1-methyl-3-(2-propyl)-2(3H)-imidazolethione. X-ray crystallographic results are reported for the mnpit and mipit complexes whereas the structure of the emit complex is under reinvestigation because of disorder. BiCl 3 (mnpit) 2 crystallizes in a triclinic space group (P1) with lattice parameters: a ¼ 9.4223(6) Å , b ¼ 10.6275(6) Å , c ¼ 12.8860(8) Å , a ¼ 108.329(1)8, b ¼ 90.388(1)8, g ¼ 115.200(1)8, V ¼ 1093(1) Å 3 , Z ¼ 2. BiCl 3 (mipit) 2 crystallizes in a triclinic space group (P1) with lattice parameters: a ¼ 10.4347(8) Å , b ¼ 11.0018(9) Å , c ¼ 11.2075(9) Å , a ¼ 95.827(1)8, b ¼ 104.890(1)8, g ¼ 117.064(1)8, V ¼ 1071(1) Å 3 , Z ¼ 2. Both complexes show the same pattern with two BiL 2 Cl 4 (L ¼ ligand) octahedra sharing a common edge through bridging chlorine atoms and the ligands occupying trans positions perpendicular to the plane made by the two bismuth atoms and six chlorine atoms. No strong evidence for a hemidirected lone pair is observed in either of the two complexes.
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