Ca²⁺ ions inhibit cytochrome <i>c-</i> catalyzed oxidation of ferrocyanide by cytochrome oxidase.

<p>(<b>A</b>) 0.3 µM bovine COX in the basic medium (100 µM EGTA present) with 40 nM cytochrome <i>c</i>. Where indicated, 1 mM ferrocyanide (ferro) was added, and its oxidation to ferricyanide was followed spectrophotometrically at 420 nm vs the 500 nm reference. Trace <i>1</i>, control recording; trace <i>2</i>, +200 µM CaCl₂ (100 µM excess over EGTA); trace <i>3</i>, +0.4 mM Mg²⁺ (300 µM excess over EGTA). (<b>B</b>) Conditions, as in panel A, trace <i>2</i> (100 µM excess of CaCl₂ over EGTA). In trace <i>2</i>, 300 µM EGTA has been added. (<b>C</b>) 0.2 µM wild-type COX from <i>R.sphaeroides</i>. Trace <i>1</i>, control (100 µM EGTA). Trace <i>2</i>, with 200 µM CaCl₂. (<b>D</b>) Concentration dependence of the inhibition. Initial rate of ferrocyanide oxidation by COX (0.3 µM) is plotted vs concentration of free Ca²⁺ buffered with 1 mM NTA (squares) or 1 mM BAPTA (circles). The curve corresponds to K_i of 0.9 µM and maximal inhibition of 60%.