The protein collagen is unusual in its widespread modification of proline to 4-hydroxyproline (also called hydroxyproline). The OH groups of hydroxyproline participate in stabilizing the structure. Hydroxylation of lysine residues in collagen also occurs but is much less frequent. It plays a different role, serving to form attachment sites for polysaccharides.

Hydroxyproline residues are generated by posttranslational modification, following completion of the polypeptide chain. The nonhydroxylated collagen precursor is called procollagen (Figure 6.14, Figure 21.4). In this polypeptide, a proline residue two positions to the carboxyl side of a glycine residue is the preferred substrate for the action of procollagen proline hydroxylase (Figure 21.4). This unusual enzyme requires ferrous iron, ascorbic acid, molecular oxygen, and -ketoglutarate. The last substance is oxidized during the reaction to succinate and CO2. One of the atoms from O2 is incorporated into succinate, and the other ends up in the hydroxyproline hydroxyl group.