Interactions of intermediate semiquinone with surrounding protein residues at the QH site of wild-type and D75H mutant cytochrome bo 3 from Escherichia coli

Myat T. Lin, Amgalanbaatar Baldansuren, Richard Hart, Rimma I. Samoilova, Kuppala V. Narasimhulu, Lai Lai Yap, Sylvia K. Choi, Patrick J. O'Malley, Robert B. Gennis, Sergei A. Dikanov

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Selective 15N isotope labeling of the cytochrome bo3 ubiquinol oxidase from Escherichia coli with auxotrophs was used to characterize the hyperfine couplings with the side-chain nitrogens from residues R71, H98, and Q101 and peptide nitrogens from residues R71 and H98 around the semiquinone (SQ) at the high-affinity QH site. The two-dimensional ESEEM (HYSCORE) data have directly identified Nε of R71 as an H-bond donor carrying the largest amount of 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 the 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 Nε of a histidine residue, presumably H75, carries most of the unpaired spin density instead of N ε of R71, as in wild-type bo3. However, the detailed characterization of the weakly coupled 15N atoms 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 the quadrupole triplet of the strongly coupled 14Nε atom of H75. Therefore, a reverse labeling approach, in which the enzyme was uniformly labeled except for selected amino acid types, was applied 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.

Original languageEnglish (US)
Pages (from-to)3827-3838
Number of pages12
Issue number18
StatePublished - May 8 2012


ASJC Scopus subject areas

  • Biochemistry

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