Mechanisms of Copper Selectivity and Release by the Metallochaperone CusF: Insights from CO-Binding, Rapid-Freeze-Quench EXAFS, and Unnatural Amino Acid Substitution.

Metallochaperones are small proteins that shuttle essential metal ions such as Cu selectively to their cellular targets. CusF has unusual Cu(I) coordination, bound by two methionines, one histidine and a capping tryptophan residue, W44. Here we compare the CO binding reactivity of the wild type (WT) protein and its W44A, F, and M variants.

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.

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.

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.

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 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.

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 β-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 HisMet ligand set, a fluxional Cu(I)-S(Met) bond, tight binding of exogenous ligands CO and N, and complete coupling of oxygen reduction to substrate hydroxylation even at extremely low turnover rates.

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 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 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.