Two Fe2+ atoms are present in activated prolyl 4-hydroxylase.

A linear relationship was found between the activity of prolyl 4-hydroxylase (EC 1.14.11.2) and the amount of Fe2+ bound per mol enzyme. At maximal activity (2.1 mumol X min-1 per mg protein) the enzyme contains 2.1 mol Fe2+ specifically bound per mol enzyme tetramer, indicating two Fe2+-binding sites on prolyl 4-hydroxylase. The half-maximal concentration of added Fe2+ for enzyme activity depends on the nature of sulphydryl compounds present in the reaction medium.