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Peptidylglycine monooxygenase (PHM) is essential for the posttranslational amidation of neuroendocrine peptides. An important aspect of the PHM mechanism is the complete coupling of oxygen reduction to substrate hydroxylation, which implies no oxygen reactivity of the fully reduced enzyme in the absence of peptidyl substrates. As part of studies aimed at investigating this feature of the PHM mechanism, we explored pre-steady-state kinetics using chemical quench (CQ) and rapid freeze-quench (RFQ) studies of the fully reduced ascorbate-free PHM enzyme. First, we confirmed the absence of Cu(I)-enzyme oxidation by O

The M-centers of the mononuclear monooxygenases peptidylglycine monooxygenase (PHM) and dopamine β-monooxygenase (DBM) bind and activate dioxygen on route to substrate hydroxylation. Recently we reported the rational design of a protein-based model wherein the CusF metallochaperone was repurposed via a His to Met mutation to act as a structural and spectroscopic biomimic. The PHM M-site exhibits a number of unusual attributes including a His(2)Met ligand set, a fluxional Cu(I)-S(Met) bond, tight binding of exogenous ligands CO and N(3)(−), and complete coupling of oxygen reduction to substrate hydroxylation even at extremely low turnover rates. In particular, mutation of the Met ligand to His completely eliminates catalytic activity despite the propensity of Cu(I)-His(3) centers to bind and activate dioxygen in other metalloenzyme systems. Here we further develop the CusF-based model to explore methionine variants where Met is replaced by selenomethionine (SeM) and histidine. We examine the effects on coordinate structure and exogenous ligand binding via XAS and EPR and probe the consequences of mutations on redox chemistry via studies on the reduction by ascorbate, and oxidation via molecular oxygen. The M-site model is 3-coordinate in the Cu(I) state and binds CO to form a 4-coordinate carbonyl. In the oxidized forms the coordination changes to tetragonal 5-coordinate with a long axial Met ligand which like the enzymes is undetectable at either the Cu or Se K edges. The EXAFS data at the Se K-edge of the SeM variant provides unique information on the nature of the Cu-methionine bond which is likewise weak and fluxional. Kinetic studies document sluggish reactivity of the Cu(I) complexes with molecular oxygen and rapid rates of reduction of the Cu(II) complexes by ascorbate, indicating a remarkable stability of the Cu(I) state in all three derivatives. The results show little difference between the Met ligand and its SeM and His congeners and suggest that the Met contributes to catalysis in ways that are more complex than simple perturbation of the redox chemistry. Overall the results stimulate critical re-examination of the canonical reaction mechanisms of the mononuclear copper monooxygenases.

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.