Experimental Evidence and Mechanistic Description of the Phenolic H-Transfer to the Cu2O2 Active Site of oxy-Tyrosinase.

Tyrosinase is a ubiquitous coupled binuclear copper enzyme that activates O2 toward the regioselective monooxygenation of monophenols to catechols via a mechanism that remains only partially defined. Here, we present new mechanistic insights into the initial steps of this monooxygenation reaction by employing a pre-steady-state, stopped-flow kinetics approach that allows for the direct measurement of the monooxygenation rates for a series of para-substituted monophenols by oxy-tyrosinase. The obtained biphasic Hammett plot and the associated solvent kinetic isotope effect values provide direct evidence for an initial H-transfer from the protonated phenolic substrate to the Cu2O2 core of oxy-tyrosinase. The correlation of these experimental results to quantum mechanics/molecular mechanics calculations provides a detailed mechanistic description of this H-transfer step. These new mechanistic insights revise and expand our fundamental understanding of Cu2O2 active sites in biology.

Transient binding sites at the surface of haloalkane dehalogenase LinB as locations for fine-tuning enzymatic activity.

The haloalkane dehalogenase LinB is a well-known enzyme that contains buried active site and is used for many modelling studies. Using classical molecular dynamics simulations of enzymes and substrates, we searched for transient binding sites on the surface of the LinB protein by calculating maps of enzyme-ligand interactions that were then transformed into sparse matrices. All residues considered as functionally important for enzyme performance (e.g., tunnel entrances) were excluded from the analysis to concentrate rather on non-obvious surface residues. From a set of 130 surface residues, twenty-six were proposed as a promising improvement of enzyme performance. Eventually, based on rational selection and filtering out the potentially unstable mutants, a small library of ten mutants was proposed to validate the possibility of fine-tuning the LinB protein. Nearly half of the predicted mutant structures showed improved activity towards the selected substrates, which demonstrates that the proposed approach could be applied to identify non-obvious yet beneficial mutations for enzyme performance especially when obvious locations have already been explored.

Elucidation of the tyrosinase/O2/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis.

Melanins are highly conjugated biopolymer pigments that provide photoprotection in a wide array of organisms, from bacteria to humans. The rate-limiting step in melanin biosynthesis, which is the ortho-hydroxylation of the amino acid L-tyrosine to L-DOPA, is catalyzed by the ubiquitous enzyme tyrosinase (Ty). Ty contains a coupled binuclear copper active site that binds O2 to form a µ:η2:η2-peroxide dicopper(II) intermediate (oxy-Ty), capable of performing the regioselective monooxygenation of para-substituted monophenols to catechols. The mechanism of this critical monooxygenation reaction remains poorly understood despite extensive efforts. In this study, we have employed a combination of spectroscopic, kinetic, and computational methods to trap and characterize the elusive catalytic ternary intermediate (Ty/O2/monophenol) under single-turnover conditions and obtain molecular-level mechanistic insights into its monooxygenation reactivity. Our experimental results, coupled with quantum-mechanics/molecular-mechanics calculations, reveal that the monophenol substrate docks in the active-site pocket of oxy-Ty fully protonated, without coordination to a copper or cleavage of the µ:η2:η2-peroxide O-O bond. Formation of this ternary intermediate involves the displacement of active-site water molecules by the substrate and replacement of their H bonds to the µ:η2:η2-peroxide by a single H bond from the substrate hydroxyl group. This H-bonding interaction in the ternary intermediate enables the unprecedented monooxygenation mechanism, where the µ-η2:η2-peroxide O-O bond is cleaved to accept the phenolic proton, followed by substrate phenolate coordination to a copper site concomitant with its aromatic ortho-hydroxylation by the nonprotonated µ-oxo. This study provides insights into O2 activation and reactivity by coupled binuclear copper active sites with fundamental implications in biocatalysis.

Comprehensive Theoretical View of the [Cu2 O2 ] Side-on-Peroxo-/Bis-µ-Oxo Equilibria.

Coupled binuclear copper (CBC) sites are employed by many metalloenzymes to catalyze a broad set of biochemical transformations. Typically, the CBC catalytic sites are activated by the O2 molecule to form various [Cu2 O2 ] reactive species. This has also inspired synthesis and development of various biomimetic inorganic complexes featuring the CBC core. From theoretical perspective, the [Cu2 O2 ] reactivity often hinges on the side-on-peroxo-dicopper(II) (P) vs. bis-µ-oxo-dicopper(III) (O) isomerism - an equilibrium that has become almost iconic in theoretical bioinorganic chemistry. Herein, we present a comprehensive calibration and evaluation of the performance of various composite computational protocols available in contemporary computational chemistry, involving coupled-cluster and multireference (relativistic) wave function methods, popular density functionals and solvation models. Starting with the well-studied reference [Cu2 O2 (NH3 )6 ]2+ system, we compared the performance of electronic structure methods and discussed the relativistic effects. This allowed us to select several 'calibrated' DFT functionals that can be conveniently employed to study ten experimentally well-characterized [Cu2 O2 ] inorganic systems. We mostly predicted the lowest-energy structures (P vs. O) of the studied systems correctly. In addition, we present calibration of the used electronic structure methods for prediction of the spectroscopic features of the [Cu2 O2 ] core, mostly provided by the resonance Raman (rR) spectroscopy.

Evidence for H-bonding interactions to the µ-η2:η2-peroxide of oxy-tyrosinase that activate its coupled binuclear copper site.

The factors that control the diverse reactivity of the µ-η2:η2-peroxo dicopper(II) oxy-intermediates in the coupled binuclear copper proteins remain elusive. Here, spectroscopic and computational methods reveal H-bonding interactions between active-site waters and the µ-η2:η2-peroxide of oxy-tyrosinase, and define their effects on the Cu(II)2O2 electronic structure and O2 activation.

Bioengineering of Cytochrome P450 OleTJE: How Does Substrate Positioning Affect the Product Distributions?

The cytochromes P450 are versatile enzymes found in all forms of life. Most P450s use dioxygen on a heme center to activate substrates, but one class of P450s utilizes hydrogen peroxide instead. Within the class of P450 peroxygenases, the P450 OleTJE isozyme binds fatty acid substrates and converts them into a range of products through the α-hydroxylation, ß-hydroxylation and decarboxylation of the substrate. The latter produces hydrocarbon products and hence can be used as biofuels. The origin of these product distributions is unclear, and, as such, we decided to investigate substrate positioning in the active site and find out what the effect is on the chemoselectivity of the reaction. In this work we present a detailed computational study on the wild-type and engineered structures of P450 OleTJE using a combination of density functional theory and quantum mechanics/molecular mechanics methods. We initially explore the wild-type structure with a variety of methods and models and show that various substrate activation transition states are close in energy and hence small perturbations as through the protein may affect product distributions. We then engineered the protein by generating an in silico model of the double mutant Asn242Arg/Arg245Asn that moves the position of an active site Arg residue in the substrate-binding pocket that is known to form a salt-bridge with the substrate. The substrate activation by the iron(IV)-oxo heme cation radical species (Compound I) was again studied using quantum mechanics/molecular mechanics (QM/MM) methods. Dramatic differences in reactivity patterns, barrier heights and structure are seen, which shows the importance of correct substrate positioning in the protein and the effect of the second-coordination sphere on the selectivity and activity of enzymes.

Can a Mononuclear Iron(III)-Superoxo Active Site Catalyze the Decarboxylation of Dodecanoic Acid in UndA to Produce Biofuels?

Decarboxylation of fatty acids is an important reaction in cell metabolism, but also has potential in biotechnology for the biosynthesis of hydrocarbons as biofuels. The recently discovered nonheme iron decarboxylase UndA is involved in the biosynthesis of 1-undecene from dodecanoic acid and using X-ray crystallography was assigned to be a mononuclear iron species. However, the work was contradicted by spectroscopic studies that suggested UndA to be more likely a dinuclear iron system. To resolve this controversy we decided to pursue a computational study on the reaction mechanism of fatty acid decarboxylation by UndA using iron(III)-superoxo and diiron(IV)-dioxo models. We tested several models with different protonation states of active site residues. Overall, however, the calculations imply that mononuclear iron(III)-superoxo is a sluggish oxidant of hydrogen atom abstraction reactions in UndA and will not be able to activate fatty acid residues by decarboxylation at room temperature. By contrast, a diiron-dioxo complex reacts with much lower hydrogen atom abstraction barriers and hence is a more likely oxidant in UndA.

AQUA-DUCT 1.0: structural and functional analysis of macromolecules from an intramolecular voids perspective.

MOTIVATION: Tunnels, pores, channels, pockets and cavities contribute to proteins architecture and performance. However, analysis and characteristics of transportation pathways and internal binding cavities are performed separately. We aimed to provide universal tool for analysis of proteins integral interior with access to detailed information on the ligands transportation phenomena and binding preferences. RESULTS: AQUA-DUCT version 1.0 is a comprehensive method for macromolecules analysis from the intramolecular voids perspective using small ligands as molecular probes. This version gives insight into several properties of macromolecules and facilitates protein engineering and drug design by the combination of the tracking and local mapping approach to small ligands. AVAILABILITY AND IMPLEMENTATION: http://www.aquaduct.pl. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Reaction mechanism between Cu(II)-enolate complex and O2 as a test case for methodology used in DFT computational studies.

The reaction mechanism of an intricate oxidation reaction of chlorodiketonate ligand of mononuclear Cu(II) complex was studied computationally employing five different models that differ in: a) basis set, b) the way that solvent corrections are included, and c) DFT functional. Qualitative and quantitative comparison of structures and enthalpy reaction profiles enabled us to assess how sensitive they are to the changes in computational methodology. Graphical abstract Comparison of enthalpy reaction profiles and molecular structures demonstrate how the qualitative picture on Cu(II)-catalyzed reaction changes upon variation of computational methodology.

Exploring Solanum tuberosum Epoxide Hydrolase Internal Architecture by Water Molecules Tracking.

Several different approaches are used to describe the role of protein compartments and residues in catalysis and to identify key residues suitable for the modification of the activity or selectivity of the desired enzyme. In our research, we applied a combination of molecular dynamics simulations and a water tracking approach to describe the water accessible volume of Solanum tuberosum epoxide hydrolase. Using water as a molecular probe, we were able to identify small cavities linked with the active site: (i) one made up of conserved amino acids and indispensable for the proper positioning of catalytic water and (ii) two others in which modification can potentially contribute to enzyme selectivity and activity. Additionally, we identified regions suitable for de novo tunnel design that could also modify the catalytic properties of the enzyme. The identified hot-spots extend the list of the previously targeted residues used for modification of the regioselectivity of the enzyme. Finally, we have provided an example of a simple and elegant process for the detailed description of the network of cavities and tunnels, which can be used in the planning of enzyme modifications and can be easily adapted to the study of any other protein.