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 O 2 consumed to product formed despite a 3 order-of-magnitude decrease in k cat and a 300-fold decrease in k cat / K m (O 2 ). 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 His 172 that goes beyond that of a simple ligand to Cu H . The mechanistic implications are discussed.