Lysyl hydroxylase catalyzes the formation of hydroxylysine in collagens by a reaction that involves oxidative decarboxylation of 2-oxoglutarate. Its binding site can be divided into two main subsites: subsite I consists of a positively charged side-chain which binds the C-5 carboxyl group, while subsite II consists of two coordination sites of the enzyme-bound Fe2+ and is chelated by the C-1-C-2 moiety. In order to identify subsite I, we converted Arg-697, Arg-700 and Ser-705 individually to alanine and Arg-700 also to lysine, and expressed the mutant enzymes in insect cells. Arg-700-Ala inactivated lysyl hydroxylase completely, whereas Arg-697-Ala and Ser-723-Ala had only a relatively minor effect. Arg-700-Lys produced 93% inactivation under standard assay conditions, the main effect being a 10-fold increase in the Km for 2-oxoglutarate, whereas the Vmax was unchanged. Arg-700 thus provides the positively charged residue that binds the C-5 carboxyl group of 2-oxoglutarate, whereas Ser-705 appears to be of no functional significance in this binding.