Structural and chemical changes of the P(M) intermediate of paracoccus denitrificans cytochrome c oxidase revealed by IR spectroscopy with labeled tyrosines and histidine.

Structural and chemical changes in the P(M) intermediate of Paracoccus denitrificans cytochrome c oxidase have been investigated by attenuated total reflection-Fourier transform infrared spectroscopy. Prior studies of P(M) minus oxidized (O) IR difference spectra of unlabeled, universally (15)N-labeled and ring-d(4)-tyrosine-labeled proteins (Iwaki, M., Puustinen, A., Wikström, M., and Rich, P. R. (2004) Biochemistry 43, 14370-14378). provided a basis for band assignments to changes in metal centers and the covalently linked His-Tyr ligand of Cu(B) and highlighted a structural alteration of the protonated Glu278 in the P(M) intermediate. This work has been extended to equivalent measurements on enzymes with (13)C(9)(15)N-labeled and ring-(13)C(6)-labeled tyrosine and with (13)C(6)(15)N(3)-labeled histidine. Histidine labeling allows the assignment of troughs at 1104 and 973 cm(-1) in reduced minus O spectra to histidine changes, whereas tyrosine labeling moves otherwise obscured tyrosine bandshifts to 1454-1437 and 1287-1284 cm(-1). P(M) minus O spectra reveal bands at 1506, 1311, and 1094 cm(-1) in the oxidized state that are replaced by a band at 1519 cm(-1) in P(M). These bands shift with both tyrosine- and histidine-labeling, providing evidence for their assignment to the covalent His-Tyr and for its chemical change in P(M). Comparisons of isotope effects on the amide I regions in P(M) minus O spectra demonstrate that amide carbonyl bonds of tyrosine and histidine are major contributors. This suggests a structural alteration in P(M) that is centered on the His276-Pro277-Glu278-Val279-Tyr280 pentapeptide formed by the His-Tyr covalent linkage. This structural change is proposed to mediate the perturbation of the IR band of the protonated Glu278 headgroup.