Redox-induced protein structural changes in cytochrome bo revealed by Fourier transform infrared spectroscopy and [13C]Tyr labeling

Cytochrome bo is a heme-copper terminal ubiquinol oxidase of Escherichia coli under highly aerated growth conditions. Tyr-288 present at the end of the K- channel forms a C-epsilon-N-epsilon covalent bond with one of the Cu-B ligand histidines and has been proposed to be an acid-base catalyst essential for the O-O bond cleavage at the Oxy-to-P transition of the dioxygen reduction cycle ( Uchida, T., Mogi, T., and Kitagawa, T. (2000) Biochemistry 39, 6669 - 6678). To probe structural changes at tyrosine residues, we examined redox difference Fourier transform infrared difference spectra of the wild-type enzyme in which either L-[1-C-13] Tyr or L-[4-C-13] Tyr has been biosynthetically incorporated in the tyrosine auxotroph. Spectral comparison between [1-C-13] Tyr-labeled and unlabeled proteins indicated that substitution of the main chain carbonyl of a Tyr residue(s) significantly affected changes in the amide-I (similar to 1620 - 1680 cm(-1)) and -II (similar to 1540 - 1560 cm(-1)) regions. In contrast, spectral comparison between [4-C-13] Tyr-labeled and unlabeled proteins showed only negligible changes, which was the case for both the pulsed and the resting forms. Thus, protonation of an OH group of tyrosines including Tyr- 288 in the vicinity of the heme o-Cu-B binuclear center was not detected at pH 7.4 upon full reduction of cytochrome bo. Redox-induced main chain changes at a Tyr residue(s) are associated with structural changes at Glu-286 near the binuclear metal centers and may be related to switching of the K-channel operative at the reductive phase to D-channel at the oxidative phase of the dioxygen reduction cycle via conformational changes in the middle of helix VI.