Cytochrome&IT c&IT oxidase inhibition by calcium at physiological ionic composition of the medium: Implications for physiological significance of the effect

Cytochrome c oxidase (CcO) from mammalian mitochondria binds Ca2+ and Na+ in a special cation binding site. Binding of Ca2+ brings about partial inhibition of the enzyme while Na(+ )competes with Ca2+ for the binding site and protects the enzyme from the inhibition [Vygodina, T., Kirichenko, A. and Konstantinov, A.A. (2013). Direct Regulation of Cytochrome c oxidase by Calcium Ions. PLoS One 8(9): e74436]. In the original studies, the inhibition was found to depend significantly on the ionic composition of the buffer. Here we describe inhibition of CcO by Ca2+ in media containing the main ionic components of cytoplasm (150 mM KCl, 12 mM NaCl and 1 mM MgCl2). Under these conditions, Ca2+ inhibits CcO with effective K-i of 20-26 mu M, that is an order of magnitude higher than determined earlier in the absence of Na+. At physiological value of ionic strength, the inhibition can be observed at any turnover number of CcO, rather than only at low TN (< 10 s(-1) ) as found previously. The inhibition requires partially oxidized state of cytochrome c and is favored by high ionic strength with a sharp transition at 0.1-0.2 M. The high K-i = 20-26 mu M found for CcO inhibition by calcium matches closely the known value of "K-m" for Ca2+-induced activation of the mitochondrial calcium uniporter. The inhibition of CcO by Ca2+ is proposed to modulate mitochondrial Ca2+-uptake via the mitochondrial calcium uniporter, promote permeability transition pore opening and induce reduction of Mia40 in the mitochondrial intermembrane space.