Vibrational modes of ubiquinone in cytochrome bo₃ from Escherichia coli identified by Fourier transform infrared difference spectroscopy and specific ¹³C labeling

In this study we present the infrared spectroscopic characterization of the bound ubiquinone in cytochrome bo₃ from Escherichia coli. Electrochemically induced Fourier transform infrared (FTIR) difference spectra of ΔUbiA (an oxidase devoid of bound ubiquinone) and ΔUbiA reconstituted with ubiquinone 2 and with isotopically labeled ubiquinone 2, where ¹³C was introduced either at the 1- or at the 4-position of the ring (C=O groups), have been obtained. The vibrational modes of the quinone bound to the discussed high-affinity binding site (Q(H)) are compared to those from the synthetic quinones in solution, leading to the assignment of the C=O modes to a split signal at 1658/1668 cm^-1, with both carbonyls similarly contributing. The FTIR spectra of ΔUbiA reconstituted with the labeled quinones indicate an essentially symmetrical and weak hydrogen bonding of the two C=O groups from the neutral quinone with the protein and distinct conformations of the 2- and 3-methoxy groups. Perturbations of the vibrational modes of the 5-methyl side groups are discussed for a signal at 1452 cm^-1. Only negligible shifts of the aromatic ring modes can be reported for the reduced and the protonated form of the quinone. Alterations of the protein upon quinone binding are reflected in the electrochemically induced FTIR difference spectra. In particular, difference signals at 1640- 1633 cm^-1 and 1700-1670 cm^-1 indicate variations of β-sheet secondary structure elements and loops, bands at 1706 and 1678 cm^-1 are tentatively attributed to individual amino acids, and a difference signal a 1540 cm^-1 is discussed to reflect an influence on C=C modes of the porphyrin ring or on deprotonated propionate groups of the hemes. Further tentative assignments are presented and discussed. The ¹³C labeling experiments allow the assignment of the vibrational modes of a bound ubiquinone 8 in the electrochemically induced FTIR difference spectra of wild-type bo₃.