The state of the copper in cytochrome c oxidase.

Nutritional studies have long supported the view that copper may be a part of cytochrome c oxidase (l-3). However, direct evidence did not become available until Eichel et al. (4) found that there was a good correlation between the copper content and the a-absorption by the reduced enzyme in fractions of heart mitochondria prepared with deoxycholate. More recently, Green et al. (5) and Okunuki et al. (6) have presented analyses of their cytochrome c oxidase preparations and have invariably found a high copper content. In the most recent publication from our laboratory, Wainio et al. (7) have established a good correlation between the copper content, the heme content, and the activity of cytochrome c oxidase. In this study we present evidence to show that the copper of cytochrome c oxidase is firmly bound to the enzyme (probably to-the protein) and that it is mostly in the cuprous state in the oxidized enzyme. The cytochrome c oxidase was a soluble Preparation 2.05-2.0 (4). Copper was determined by a modification of the method of Peterson and Bollier (8) which uses cuprizone (biscyclohexanone-oxalyldihydrazone) to develop the color. Iron was also determined calorimetrically (9). The care of the glassware is described in a previous publication (7). Cytochrome c oxidase activity was determined spectrophotometrically (7). Dialysis of the soluble enzyme for 12 hours against different reagents, followed by dialysis for 12 hours against water, led to the results presented in Table I (cf. Okunuki et al. (6)). Control samples were dialyzed against water for 24 hours. The activity in each instance is approximately proportional to the copper content. Iron was determined in the samples that were dialyzed against cyanide; as can be seen, the relative iron content rose to a constant value in each of the 3 samples. Attempts to regenerate the soluble enzyme with cupric ion after dialysis against 1 X 10-l M diethyldithiocarbamate for 12 hours and against water for 12 hours were unsuccessful. Although the relative copper content was increased to 182% after further dialysis against 1 X 10V6 M CuSOd and water for 12 hours each, the activity remained zero. Application of the cuprous ion was more successful. The copper of the enzyme was removed with 1 X 10-l M diethyldithiocarbamate, and after dialysis against 1 X 10db M CUSO~ + ascorbic acid and then water, the relative activity in one experiment was 320 ‘% and the