The catalytic role of the copper ligand H172 of peptidylglycine α-hydroxylating monooxygenase: A kinetic study of the H172A mutant

An essential histidine ligand to the electron transfer copper (Cu _H) of peptidylglycine α-hydroxylating monooxygenase (PHMcc) was mutated to an alanine and found to retain copper binding and hydroxylase activity [Jaron, S., et al. (2002) Biochemistry 41, 13274-13282]. An extensive kinetic and deuterium isotope effect study finds this mutant to maintain full coupling of O₂ consumed to product formed despite a 3 order-of-magnitude decrease in k_cat and a 300-fold decrease in k _cat/K_m(O₂). Unexpectedly, electron transfer is not rate-limiting in H172A. Rather, the increased kinetic isotope effect (KIE) on k_cat of 3.27 ± 0.39 suggests that C-H bond cleavage has become more rate-limiting, implicating a role for His¹⁷² that goes beyond that of a simple ligand to CU_H. The mechanistic implications are discussed.