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Mononuclear copper monooxygenases peptidylglycine monooxygenase (PHM) and dopamine β-monooxygenase (DBM) catalyze the hydroxylation of high energy C-H bonds utilizing a pair of chemically distinct copper sites (CuH and CuM) separated by 11 A. In earlier work, we constructed single-site PHM variants that were designed to allow the study of the M- and H-centers independently in order to place their reactivity sequentially along the catalytic pathway. More recent crystallographic studies suggest that these single-site variants may not be truly representative of the individual active sites. In this work, we describe an alternative approach that uses a rational design to construct an artificial PHM model in a small metallochaperone scaffold. Using site-directed mutagenesis, we constructed variants that provide a His2Met copper-binding ligand set that mimics the M-center of PHM. The results show that the model accurately reproduces the chemical and spectroscopic properties of the M-center, including details of the methionine coordination, and the properties of Cu(I) and Cu(II) states in the presence of endogenous ligands such as CO and azide. The rate of reduction of the Cu(II) form of the model by the chromophoric reductant N,N'-dimethyl phenylenediamine (DMPD) has been compared with that of the PHM M-center, and the reaction chemistry of the Cu(I) forms with molecular oxygen has also been explored, revealing an unusually low reactivity toward molecular oxygen. This latter finding emphasizes the importance of substrate triggering of oxygen reactivity and implies that the His2Met ligand set, while necessary, is insufficient on its own to activate oxygen in these enzyme systems.

An important question is whether consensus mechanisms for copper monooxygenase enzymes such as peptidylglycine monooxygenase (PHM) and dopamine β-monooxygenase (DBM) generated via computational and spectroscopic approaches account for important experimental observations. We examine this question in the light of recent crystallographic and QMMM reports which suggest that alternative mechanisms involving an open to closed conformational cycle may be more representative of a number of experimental findings that remain unaccounted for in the canonical mononuclear mechanisms. These include (i) the almost negligible reactivity of the catalytic copper site (CuM) with oxygen in the absence of substrate, (ii) the carbonyl chemistry and in particular the substrate-induced activation exemplified by the lowered CO stretching frequency, (iii) the peroxide shunt chemistry which demands an intermediate that facilitates equilibrium between a Cu(II)-peroxo state and a Cu(I)-dioxygen state, and (iv) clear evidence for both closed and open conformational states in both PHM and DBM. An alternative mechanism involving a dinuclear copper intermediate formed via an open to closed conformational transition appears better able to accommodate these experimental observations, as well as being shown by QMMM methodologies to be energetically feasible. This suggests that future experiments should be designed to distinguish between these competing mechanisms and the factors that govern the oxygen reactivity of the copper centers. In particular, determining how oxygen reactivity is activated by binding of substrate, should be considered an important new challenge.