Structural changes in cytochrome c oxidase induced by binding of sodium and calcium ions: an ATR-FTIR study.

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to investigate the binding of Na(+) and Ca(2+)cations to bovine cytochrome c oxidase in its fully oxidized and partially reduced, cyanide-ligated (a(2+)a3(3+)-CN) (mixed valence) forms. These ions induced distinctly different IR binding spectra, indicating that the induced structural changes are different. Despite this, their binding spectra were mutually exclusive, confirming their known competitive binding behavior. Dissociation constants for Na(+) and Ca(2+) with the oxidized enzyme were 1.2 mM and 11 μM, respectively and Na(+) binding appeared to involve cooperative binding of two Na(+). Ca(2+) binding induced a large IR spectrum, with prominent amide I/II polypeptide changes, bandshifts assigned to carboxylate and an arginine, and a number of bandshifts of heme a. The Na(+)-induced binding spectrum showed much weaker amide I/II and heme a changes but had similar shifts assignable to carboxylate and arginine residues. Yeast CcO also displayed a calcium-induced IR and UV/visible binding spectra, though of lower intensities. This was attributed to the difficulty in fully depleting Ca(2+) from its binding site, as has been found with bacterial CcOs. The implications of Ca(2+)/Na(+) ion binding are discussed in terms of structure and possible modulation of core catalytic function.