EPR and solid-state NMR studies on the mechanism of cytochrome BO3 ubiquinol oxidase from Escherichia coli

Cytochrome bo3 ubiquinol oxidase from E. coli is a member of heme-copper oxidase superfamily. This trans-membrane enzyme complex catalyzes two-electron oxidation of ubiquinol and reduction of molecular oxygen to water. During the process, the protons from ubiquinol are released to the periplasmic side of the membrane, whereas the protons used in the reduction of O2 are taken from the cytoplasmic side. In addition to the protons involved in the redox reaction, the enzyme also translocates four protons from the cytoplasmic side to the periplasmic side for each molecule of oxygen reduced. Thus, cytochrome bo 3 contributes to the electrochemical potential difference across the membrane. During turnover, electrons from the ubiquinol pool are first transferred to a ubiquinone cofactor bound at the high affinity binding site known as the QH-site, from which electrons are moved one at a time sequentially to the low-spin heme b and the CuB-heme o3 catalytic site. In this study, E. coli C43(DE3) auxotroph strains were constructed with highly efficient lambda-Red recombination system. Wild-type and D75H mutant cytochrome bo 3 samples with selectively isotope labeled Arg, Gln or His were prepared from these C43(DE3) auxotroph strains, and the semiquinone radicals at the QH-site of these samples were analyzed by pulsed EPR spectroscopy. Selective 15N labeling has revealed the N&egr; of R71 in the wild type cytochrome bo3 and the N &egr; of H75 in the D75H mutant cytochrome bo 3 as the nitrogen atoms that are strongly coupled with the carbonyl oxygen-1 of the semiquinone and produce 14N ESEEM features observed in previous studies. Pulsed EPR experiments performed on these selectively 15N labeled samples also enabled the investigation of nitrogen nuclei weakly coupled to the semiquinone. In addition, three amino-acid pair-wise isotope labeled cytochrome bo3 samples were prepared from C43(DE3) auxotrophs for solid-state NMR (SSNMR) experiments. The initial SSNMR spectra proved that clean isotope labeling at the selected amino acid types was achieved through the use of auxotroph strains. This selective labeling approach dramatically simplifies the SSNMR signals and presents a great possibility to accomplish the chemical shift assignments of critical residues at the active site of such a large membrane protein complex. Furthermore, several cytochrome bo3 samples with selectively isotope labeled ubiquinone cofactors were successfully assembled. The procedure described in this study establishes a major step towards structural and mechanistic studies on the ubiquinone binding site of cytochrome bo 3 by EPR and SSNMR techniques.