The broad class of O2-activating coupled-binuclear copper (CBC) metalloenzymes contain a unique [Cu2O2] catalytic core. This core is responsible for catalyzing challenging biochemical transformations, particularly the regioselective monooxygenations/oxidations of substituted phenols. Despite almost four decades of intense experimental and theoretical research, the factors governing the diverse reactivity of CBC enzymes had remained only partially understood. In this review, we highlight the recent synergy between spectroscopy, kinetic experiments, and state-of-the-art computations (including hybrid quantum and molecular mechanical, QM/MM, and advanced wave function theory, WFT, methods) that provided a conclusive mechanistic picture of the initial stages of the ortho-hydroxylation of phenolic substrates catalyzed by the CBC enzyme tyrosinase (Ty). We emphasize the power of calibrated theoretical calculations, supported by experimental spectroscopic and kinetic data on intermediates, in providing definitive insight into the catalytic reaction coordinate. We provide a critical review of previous efforts towards elucidating structure-function correlations over the four CBC protein classes (hemocyanins, catechol oxidases, tyrosinases, o-aminophenol oxygenases). We outline how a systematic mechanistic understanding across the different CBC enzyme classes could uncover their elusive structure-function correlations, opening new possibilities for utilizing the [Cu2O2] catalytic core outside its native biological context for applications in materials and biocatalysis.

• Klíčová slova
• QM/MM modeling, Structure/Function Correlations, WFT & DFT, [Cu2O2] active sites, coupled binuclear Cu(CBC), kinetics, spectroscopy,
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• fenoly chemie   MeSH
• katalytická doména MeSH
• katecholy chemie   MeSH
• kinetika MeSH
• měď * chemie   MeSH
• tyrosinasa * chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• fenoly MeSH
• katecholy MeSH
• měď * MeSH
• tyrosinasa * MeSH

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.

• Klíčová slova
• binuclear copper, melanin biosynthesis, monooxygenase, oxygen activation, tyrosinase,
• MeSH
• bakteriální proteiny * MeSH
• fenoly * chemie   MeSH
• katalýza MeSH
• kyslík * metabolismus   MeSH
• měď chemie   MeSH
• melaniny * biosyntéza   MeSH
• peroxidy chemie   MeSH
• Streptomyces * enzymologie   MeSH
• tyrosinasa * chemie   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• bakteriální proteiny * MeSH
• fenoly * MeSH
• kyslík * MeSH
• měď MeSH
• melaniny * MeSH
• peroxidy MeSH
• tyrosinasa * MeSH

Activation of dioxygen attracts enormous attention due to its potential for utilization of methane and applications in other selective oxidation reactions. We report a cleavage of dioxygen at room temperature over distant binuclear Fe(II) species stabilized in an aluminosilicate matrix. A pair of formed distant α-oxygen species [i.e., (Fe(IV)═O)2+] exhibits unique oxidation properties reflected in an outstanding activity in the oxidation of methane to methanol at room temperature. Designing a man-made system that mimicks the enzyme functionality in the dioxygen activation using both a different mechanism and structure of the active site represents a breakthrough in catalysis. Our system has an enormous practical importance as a potential industrial catalyst for methane utilization because (i) the Fe(II)/Fe(IV) cycle is reversible, (ii) the active Fe centers are stable under the reaction conditions, and (iii) methanol can be released to gas phase without the necessity of water or water-organic medium extraction.

• Publikační typ
• časopisecké články MeSH