THE SUBUNIT LOCATION OF MAGNESIUM IN CYTOCHROME-C-OXIDASE

The magnesium ion in bovine heart cytochrome c oxidase can be depleted up to 75% by heat treatment of the enzyme at 43-degrees-C followed by dialysis against EDTA buffer solution. The magnesium-depleted enzyme so obtained retains 40% of the activity of the native enzyme. This is the first attempt to deplete magnesium ion from bovine heart cytochrom e c oxidase without denaturation of the protein. Magnesium depletion e xposes at least one carboxyl group on subunit IV for labeling by ycloh exyl-N'-(4-dimethylaminonaphthyl)carbodiimide (NCD-4). The NCD-4 label ing of subunit IV of the magnesium-depleted enzyme is significantly en hanced relative to what is observed for the native and heat-treated ox idase, suggesting that the magnesium ion is located in subunit IV with at least one carboxyl ligand. By comparing the activity of the magnes ium-depleted enzyme with that of a control sample of heat-treated oxid ase, the influence of divalent magnesium on the activity of the enzyme is assessed.