Probing heart cytochrome c oxidase structure and function by infrared spectroscopy.

IR spectra directly probe specific vibrators in bovine heart cytochrome c oxidase, yielding quantitative as well as qualitative information on structures and reactions at these vibrators. C-O IR spectra reveal that CO binds to Fe2+ a3 as two conformers each in isolated immobile environments sensitive to Fea and/or CuA oxidation state but remarkably insensitive to pH, medium, anesthetics, and other factors that affect activity. C-N IR spectra reveal that the one CN- that binds to fully and partially oxidized enzyme can be in three different structures. These structures vary in relative amounts with redox level, thereby reflecting dynamic electron exchange among Fea, CuA, and CuB with associated changes in protein conformation of likely significance in O2 reduction and H(+)-pumping. Azide IR spectra also reflect redox-dependent long-range effects. The amide I IR bands, due to C-O vibrators of peptide linkages and composed of multiple bands derived from different secondary structures, reveal high levels of alpha-helix (approximately 60%) and subtle changes with redox level and exposure to anesthetics. N2O IR spectra reveal that these anesthetic molecules at clinically relevant levels occupy three sites of different polarity within the enzyme as the enzyme is reversibly, but only partially, inhibited.