Capturing the Binuclear Copper State of Peptidylglycine Monooxygenase Using a Peptidyl-Homocysteine Lure.

Peptidylglycine monooxygenase is a copper-dependent enzyme that catalyzes C-alpha hydroxylation of glycine extended pro-peptides, a critical post-translational step in peptide hormone processing. The canonical mechanism posits that dioxygen binds at the mononuclear M-center to generate a Cu(II)-superoxo species capable of H atom abstraction from the peptidyl substrate, followed by long-range electron tunneling from the CuH center. Recent crystallographic and biochemical data have challenged this mechanism, suggesting instead that an "open-to-closed" transition brings the copper centers closer, allowing reactivity within a binuclear intermediate. Here we present the first direct observation of an enzyme-bound binuclear copper species, captured by the use of an Ala-Ala-Phe-hCys inhibitor complex. This molecule reacts with the fully reduced enzyme to form a thiolate-bridged binuclear species characterized by EXAFS of the WT and its M314H variant and with the oxidized enzyme to form a novel mixed valence entity characterized by UV/vis and EPR. Mechanistic implications are discussed.

Pre-Steady-State Reactivity of Peptidylglycine Monooxygenase Implicates Ascorbate in Substrate Triggering of the Active Conformer.

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 O2 at catalytic rates in the absence of peptidyl substrate. Next, we investigated reactivity in the presence of the substrate dansyl-YVG. Surprisingly, when ascorbate-free di-Cu(I) PHM was shot against oxygenated buffer containing the dansyl-YVG substrate, <15% of the expected product was formed. Substoichiometric reactivity was confirmed by stopped-flow and RFQ EPR spectroscopy. Product generation reached a maximum of 70% by the addition of increasing amounts of the ascorbate cosubstrate in a process that was not the result of multiple turnovers. FTIR spectroscopy of the Cu(I)-CO reaction chemistry was then used to show that increasing ascorbate concentrations correlated with a substrate-induced Cu(I)M-CO species characteristic of an altered conformation. We conclude that ascorbate and peptidyl substrate work together to induce a transition from an inactive to an active conformation and suggest that the latter may represent the "closed" conformation (Cu-Cu of ∼4 Å) recently observed for both PHM and its sister enzyme DBM by crystallography.

Catalytic M Center of Copper Monooxygenases Probed by Rational Design. Effects of Selenomethionine and Histidine Substitution on Structure and Reactivity.

The M centers of the mononuclear monooxygenases peptidylglycine monooxygenase (PHM) and dopamine ß-monooxygenase bind and activate dioxygen en route to substrate hydroxylation. Recently, we reported the rational design of a protein-based model in which 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 His2Met ligand set, a fluxional Cu(I)-S(Met) bond, tight binding of exogenous ligands CO and N3-, 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 the catalytic activity despite the propensity of CuI-His3 centers to bind and activate dioxygen in other metalloenzyme systems. Here, we further develop the CusF-based model to explore methionine variants in which Met is replaced by selenomethionine (SeM) and histidine. We examine the effects on coordinate structure and exogenous ligand binding via X-ray absorption spectroscopy and electron paramagnetic resonance and probe the consequences of mutations on redox chemistry via studies of the reduction by ascorbate and oxidation via molecular oxygen. The M-site model is three-coordinate in the Cu(I) state and binds CO to form a four-coordinate carbonyl. In the oxidized forms, the coordination changes to tetragonal five-coordinate with a long axial Met ligand that 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 provide unique information about the nature of the Cu-methionine bond that is likewise weak and fluxional. Kinetic studies document the 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 a critical re-examination of the canonical reaction mechanisms of the mononuclear copper monooxygenases.

Rational Design of a Histidine-Methionine Site Modeling the M-Center of Copper Monooxygenases in a Small Metallochaperone Scaffold.

Mononuclear copper monooxygenases peptidylglycine monooxygenase (PHM) and dopamine ß-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 Å. 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.

New structures reveal flexible dynamics between the subdomains of peptidylglycine monooxygenase. Implications for an open to closed mechanism.

Peptidylglycine monooxygenase (PHM) is essential for the biosynthesis of many neuroendocrine peptides via a copper-dependent hydroxylation of a glycine-extended pro-peptide. The "canonical" mechanism requires the transfer of two electrons from one mononuclear copper (CuH, H-site) to a second mononuclear copper (CuM, M-site) which is the site of oxygen binding and catalysis. In most crystal structures the copper centers are separated by 11 Å of disordered solvent, but recent work has established that a PHM variant H108A forms a closed conformer in the presence of citrate with a reduced Cu-Cu site separation of ~4 Å. Here we report three new PHM structures where the H and M sites are separated by a longer distance of ~14 Å. Variation in Cu-Cu distance is the result of a rotation of the M subdomain about a hinge point centered on the pro199 -leu200 -ile201 triad which forms the linker between subdomains. The energetic cost of domain dynamics is likely small enough to allow free rotation of the subdomains relative to each other, adding credence to recent suggestions that an open-to-closed transition to form a binuclear oxygen binding intermediate is an essential element of catalysis. This inference would explain many experimental observations that are inconsistent with the current canonical mechanism including substrate-induced oxygen activation and isotope scrambling during the peroxide shunt.

Copper monooxygenase reactivity: Do consensus mechanisms accurately reflect experimental observations?

An important question is whether consensus mechanisms for copper monooxygenase enzymes such as peptidylglycine monooxygenase (PHM) and dopamine ß-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.