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Volume 597, Issue 1 p. 65-78
Perspective
Free Access

New mechanistic insights into coupled binuclear copper monooxygenases from the recent elucidation of the ternary intermediate of tyrosinase

Ioannis Kipouros

Ioannis Kipouros

Department of Chemistry, Stanford University, CA, USA

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Edward I. Solomon

Corresponding Author

Edward I. Solomon

Department of Chemistry, Stanford University, CA, USA

Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, CA, USA

Correspondence

Edward I. Solomon, Department of Chemistry, Stanford University, Stanford, CA 94305, USA

Tel: (650) 723-9104

E-mail: [email protected]

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First published: 30 September 2022
Citations: 5
Edited by Martin Högbom

Abstract

Tyrosinase is the most predominant member of the coupled binuclear copper (CBC) protein family. The recent trapping and spectroscopic definition of the elusive catalytic ternary intermediate (enzyme/O2/monophenol) of tyrosinase dictates a monooxygenation mechanism that revises previous proposals and involves cleavage of the μ-η22-peroxide dicopper(II) O–O bond to accept the phenolic proton, followed by monophenolate coordination to copper concomitant with aromatic hydroxylation by the non-protonated μ-oxo. Here, we compare and contrast previously proposed and current mechanistic models for monophenol monooxygenation of tyrosinase. Next, we discuss how these recent insights provide new opportunities towards uncovering structure–function relationships in CBC enzymes, as well as understanding fundamental principles for O2 activation and reactivity by bioinorganic active sites.

Abbreviations

3,4-COOCH3, methyl 3,4-dihydroxybenzoate

4-COOCH3, methyl 4-hydroxybenzoate

AOx, o-aminophenol oxidase

CaOx, catechol oxidase

CBC, coupled binuclear copper

CT, charge transfer

DBED, N,N'-ditert-butyl-ethylenediamine

DFT, density functional theory

EAS, electrophilic aromatic substitution

EPR, electron paramagnetic resonance

Hc, hemocyanin

HOMO, highest occupied molecular orbital

LUMO, lowest unoccupied molecular orbital

MO, molecular orbital

PCET, proton-coupled electron transfer

QM, quantum mechanics

QM/MM, quantum-mechanics/molecular-mechanics

RFQ, Rapid freeze quench

rR, resonance Raman

SF-Abs, stopped-flow absorption

TD-DFT, time-dependent density functional theory

TS, transition state

Ty, tyrosinase

Due to its physiologically accessible I/II redox couple and its bioavailability, copper is an essential active-site cofactor in several major classes of metalloenzymes [[1]]. These biological copper sites exhibit different redox states (I, II, and possibly III), nuclearities (Cun, n = 1–4), ligations, and coordination modes, that prime the copper site for performing their corresponding catalytic functions, which include the binding, activation, and/or reduction of small molecules (O2, NO2, N2O, etc) or larger organic substrates in anabolic (e.g., C–H functionalization) and catabolic (e.g., proton pumping in respiratory chains) processes [[1, 2]]. One of the major Cu protein families is comprised of the coupled binuclear copper (CBC) proteins, which include hemocyanin (Hc), catechol oxidase (CaOx), tyrosinase (Ty), and o-aminophenol oxidase (AOx) [[1, 3]].

All CBC proteins contain a characteristic four-alpha helix motif that binds two copper sites via histidine Nε-ligation, and brings them in close proximity to each other (< 5.0 Å), enabling magnetic coupling in the dicopper(II) states (Fig. 1A). The fully reduced state (deoxy-form; Fig. 1B) contains a dicopper(I) active site, where each Cu(I) is trigonally ligated and separated from each other by ~ 4.5 Å [[4, 5]]. This deoxy-CBC active site binds O2 reversibly [[6]], exergonically (Kd = 16–40 μm) [[7]], and reductively to form the oxy-intermediate (Fig. 1B), where the peroxide ligand is bound in a μ-η22 (side-on bridged) configuration by the two distorted square-pyramidal Cu(II) sites, which are now separated by only ~ 3.6 Å from each other (Fig. 1B) [[4, 8]]. In Ty, the oxy-intermediate can perform the monooxygenation of monophenols to catechols and generate the resting enzyme (met-form) that contains a μ-hydroxo dicopper(II) site [[4, 9, 10]], which can further oxidize catechols to quinones, and return to the deoxy form (Fig. 1B) [[11]]. Extensive studies on the oxy-intermediate have revealed the origin of its unique spectroscopic features and correlated them to its distinct electronic structure (Fig. 2A, left) [[2, 12]]. In particular, the oxy-intermediate exhibits: (a) a characteristic UV–Vis absorption spectrum (Fig. 3C, blue spectrum) with an intense high-energy (ε~350nm = 14 000–20 000 m−1·cm−1) and a weak low-energy band (ε~650nm = 800–1000 m−1·cm−1), each corresponding to a peroxide(π*σ,v)→Cu(II) charge-transfer (CT) transition (Fig. 2A, blue arrows) [[13]], the high intensity of the high-energy CT band reflecting the strong σ donor interactions of the μ-η22-peroxide with both Cu(II) centers, (b) a set of characteristic resonance Raman (rR) features (Fig. 3D, blue spectral regions) that correspond to its Cu–Cu stretch (A1g; νCu–Cu ~ 280 cm−1) [[14]], the first overtone of its Cu–O stretch (A1g; νCu–O(overtone) ~ 1180 cm−1), which is allowed and present at double the frequency of its formally rR forbidden, and thus weak or absent fundamental B3u mode (i.e., the 0→1 vibrational level transition), and its O–O stretch (A1g; νO–O ~ 740 cm−1) [[15]], the latter indicating the exceptionally weak peroxide O–O bond due to the back-bonding interaction of the Cu into the peroxide σ* orbital (Fig. 2A, left), (c) lack of any electron paramagnetic resonance (EPR) spectroscopic features [[16, 17]], due to the strong antiferromagnetic coupling (2 J > 600 cm−1) between the two Cu(II) sites associated with the large splitting between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) due to their bridging peroxide ligand, as shown in Fig. 2A (left) [[2]].

Details are in the caption following the image
Structure and catalytic cycle of tyrosinase. (A) Overall structure of tyrosinase (PDB: 2AHL) coordinating two copper ions (brown spheres) within the four-alpha helix bundle shown in color (CuA coordinated by helices shown in blue and CuB via helices in red). (B) The catalytic cycle of Ty showing the active site structures for deoxy-Ty (2AHL), oxy-Ty (1WX2), and met-Ty (2AHK) from reference [[4]], with only key residues/ligands shown for clarity (first-sphere coordination histidines as sticks, the μ-η22-peroxide in oxy-Ty as a red stick, and the O-atom of the μ-hydroxide in met-Ty as a red sphere). (C) The reactivity of different members of the CBC protein family.
Details are in the caption following the image
Summary of key insights from Cu2O2 model complex studies. (A) Molecular orbital diagrams for the μ-η22-peroxide dicopper(II) (left) and bis-μ-oxo dicopper(III) (right) isomers, including schematic representations of the lowest unoccupied frontier molecular orbitals, and with the CT transitions for their characteristic absorption features indicated as blue and pink arrows, respectively. (B) The μ-η22-peroxide dicopper(II) to bis-μ-oxo dicopper(III) equilibrium (top), and their general reactivity with monophenol (red box) and monophenolate (blue box) substrates. (C) The competitive inhibition binding of mimosine to met-Ty. (D) The previous generally proposed monophenol monooxygenation mechanism of Ty, with ‘B’ indicating the putative active site base that serves as the phenolic proton acceptor site.
Details are in the caption following the image
Spectroscopic elucidation of the ternary intermediate structure of tyrosinase in complex with monophenol and O2. (A) Kinetic scheme for the catalytic cycle of Ty in the presence of borate, with the ternary intermediate including previous proposals for either a μ-η22-peroxide dicopper(II) or a bis-μ-oxo dicopper(III) active site via reductive cleavage of the O–O bond (indicated with a dashed line). (B) Summary of the previously proposed structures for the ternary intermediate, with different proton acceptor sites, substrate coordination, and Cu2O2 isomerization. (C, D) Spectroscopic definition of the (Ty/4-COOCH3/O2) ternary intermediate (red spectra) by (C) UV–Vis absorption and (D) resonance Raman spectroscopies, including oxy-Ty (blue spectra), and the absorption spectrum of 4-COOCH3 (black spectrum in C). (E, F) Experimentally validated QM/MM structures for oxy-Ty (E) and the ternary intermediate (F), with key residues and the substrate shown as sticks, the copper sites and the μ-η22-peroxide as brown and red spheres respectively, and H-bonds as yellow dashes. (G) Structural alignment of the ternary intermediate (from F) with the caddie-bound oxy-Ty (PDB:1WX4), with the caddie protein shown in red and oxy-Ty in cyan color. Figure reproduced from Ref.[[33]].

Even though all CBC enzymes bind O2 in an equivalent fashion and form a similar oxy-intermediate, they exhibit different reactivities that facilitate their respective biological functions (Fig. 1C) [[1]]. Ty, the most ubiquitous CBC enzyme representing the phylogenetic ancestral member of this protein family [[18]], catalyzes the regioselective monooxygenation of the amino acid l-tyrosine to 3,4-dihydroxyphenylalanine (l-DOPA), as well as the subsequent two-electron oxidation of l-DOPA to l-DOPAquinone [[11]], the initial and rate-limiting steps in melanin biosynthesis found across a wide range of diverse organisms [[19, 20]]. Hc lacks any enzymatic activity and serves exclusively as a multimeric O2-carrier protein in arthropods and mollusks. The complete loss of any enzymatic reactivity in Hc is a consequence of the restricted access of aromatic substrates to its [Cu(II)2O2]2+ active site, which is covered by the extended N-terminus in arthropods and C-terminus in mollusks [[1, 5, 21]], reflecting their independent evolution pathways [[18]]. Consistent with this explanation for the structure–function-evolution relations in Hc, denaturation or cleavage of its extended terminal recovers its Ty-like activity towards monophenols and catechols [[22-24]]. The CaOx enzymes are predominately found in natural product biosynthetic pathways and immune defense mechanisms in plants, where they perform the oxidation of various catechol substrates to quinones, but lack any monooxygenation reactivity. The structural and functional origins of the loss of monooxygenation reactivity in CaOx remain unknown and an area of active investigation [[25-29]]. Finally, the newest members of the CBC family, the AOx, evolved to further catalyze the four-electron monooxygenation of o-aminophenols to their o-nitrosophenol products, utilizing the same μ-η22-peroxide dicopper(II) oxy-intermediate [[29]], with these structural and functional origins also remains unknown. Notably, the AOx members were initially annotated as Ty, due to their close sequence similarity, but were subsequently reclassified as a novel CBC member following the discovery and characterization of the NspF gene in a siderophore biosynthetic pathway which established their unique reactivity [[29, 30]].

Uncovering the sequence-structure–function relationships in the CBC protein family, as well as elucidating the mechanisms of their enzymatic reactions remain a long-standing challenge with critical implications in biocatalysis [[26, 28, 29, 31, 32]]. A major limitation in these efforts is the lack of direct experimental evidence on elusive key reaction intermediates. In this Perspective, we summarize important insights into [Cu2O2]2+ chemistry from previous model complex and enzyme studies. We then discuss the recent trapping and spectroscopic definition of the elusive ternary (tyrosinase/monophenol/O2) intermediate under single-turnover conditions, which resulted in important mechanistic implications for the subsequent monooxygenation reaction [[33]]. In our concluding remarks, we present current challenges and future directions in the study of CBC enzymes that involve elucidating their reaction mechanisms and uncovering structure–function relationships within this protein family.

Insights from model complexes, enzymology, and computational studies

Although CBC proteins were known to bind O2 since the late 19th century [[34, 35]], the geometric structure description of their Cu2O2 active site became known through the first reported μ-η22-peroxide dicopper(II) model complex in a tris(pyrazolyl)borate ligand scaffold [[36]]. This structurally-defined μ-η22-peroxide dicopper(II) complex exhibits spectroscopic features that are equivalent to those of the oxy-intermediate in CBC proteins (vide supra) [[37]], providing strong evidence for their shared μ-η22-peroxide dicopper(II) structure, later confirmed by the crystallographic resolution of oxy-Hc [[8]], and oxy-Ty [[4]]. Several μ-η22-peroxide dicopper(II) complexes with different ligand scaffolds and physical properties have been reported since then, which have been the focus of previous comprehensive reviews [[38-41]]. An important insight obtained from the study of μ-η22-peroxide dicopper(II) model complexes is that these species exist in equilibrium with their bis-μ-oxo dicopper(III) isomers (Fig. 2B, equilibrium) [[42]], and this equilibrium depends on several factors, including the ligand scaffold, solvent, counter anions, and temperature [[38]]. The bis-μ-oxo dicopper(III) isomers exhibit spectroscopic features (Abs: ε~300nm = 20 000 m−1·cm−1, ε~400nm = 25 000 m−1·cm−1; rR: ν ~ 600 cm−1) that are distinct from their μ-η22-peroxide dicopper(II) counterparts, and reflect the electronic structure changes upon the reductive cleavage of the peroxide O–O bond, (red dashed line in Fig. 2A, showing that elongation of the O–O bond brings the energy of the peroxide σ* LUMO below the Cu-centric HOMO to cleave the O–O bond and oxidize the Cu(II) centers). Although Cu(III) has not been observed in biology, these model complex insights have opened the possibility that the μ-η22-peroxide dicopper(II) active site of oxy-CBC enzymes could convert to its bis-μ-oxo dicopper(III) isomer [[43]].

Investigations into the reactivity of these model complexes have established a general mechanistic framework for the diverse [Cu2O2]2+ chemistry. The reaction of the μ-η22-peroxide dicopper(II) complex in N,N′-ditert-butyl-ethylenediamine (DBED) ligation with monophenolate substrates results in the equatorial monophenolate coordination to Cu and O–O cleavage via bis-μ-oxo dicopper(III) isomerization, which is followed by aromatic hydroxylation of the bound monophenolate substrate to form the catechol product (Fig. 2B, blue box) [[43]]. Based on its Hammett plot parameters (ρ = −2.2) and the observation of a small inverse Ar-CH/D KIE, this monooxygenation reaction was proposed to proceed via an electrophilic aromatic substitution (EAS) mechanism, with DFT calculations suggesting a σ-attack pathway involving the LUMO Cu2O2 σ* (Fig. 2A, schematic representation of LUMO in top right) and the HOMO phenolate π orbital [[43]]. In another μ-η22-peroxide dicopper(II) complex within an aromatic xylyl ligand scaffold (NO2-XYL), intramolecular arene monooxygenation has been reported to proceed by the μ-η22-peroxide, without bis-μ-oxo isomerization [[44]], via direct electron donation from the aromatic ring into the σ* orbital of the μ-η22-peroxide [[45]]. Interestingly, the reaction of monophenols with either μ-η22-peroxide dicopper(II) or bis-μ-oxo dicopper(III) complexes proceeds without the formation of a substrate-bound intermediate, via abstraction of the phenolic H-atom by the Cu2O2 core and generation of free phenoxyl radicals and their coupling products (Fig. 2B, red box) [[46]]. Based on the dependence of its reaction rate on the monophenol one-electron reduction potentials and the observed small normal Ar-OH/D KIE, this monophenol oxidation reaction was proposed to proceed via a proton-coupled electron-transfer (PCET) mechanism [[46]].

In parallel to model complex studies and in the absence of related crystallographic evidence for Ty, the use of substrate analogue inhibitors provided insights into the binding of monophenol(ate) substrates to the enzyme pocket. In particular, the competitive inhibitor mimosine was found to bind in met-Ty via direct coordination to a Cu(II) site (Fig. 2C), based on the appearance of a 425 nm absorption feature assigned as a mimosine→Cu(II) CT transition [[47]]. Probing the binding of mimosine and benzoic acid inhibitors to a mixed valent Cu(I)Cu(II) half-met-Ty (S = 1/2 ground state) by EPR indicated significant dz2 mixing to the dx2-y2 ground state, suggesting a trigonal bipyramidal distortion of the Cu due to its interaction with or coordination of the bound inhibitor [[48]]. An increase of the forbidden 1 s→3d transition in the Cu K-edge X-ray absorption spectrum for the mimosine-bound Ty further supports this copper site distortion upon inhibitor coordination [[49]]. These studies on the binding of aromatic inhibitors to Ty suggested that the enzyme pocket could similarly facilitate monophenolate coordination to a Cu(II) of oxy-Ty, although direct experimental evidence on this ternary intermediate (Ty/monophenol(ate)/O2) has only recently become available (vide infra) [[33]].

Experimental insights into the enzymatic monooxygenation mechanism of Ty have been mostly limited to steady-state kinetics. Although these studies have been complicated by the presence of an enzyme lag phase [[50, 51]], inactivation pathways [[52-54]], and inhibition reactions [[48]], the steady-state rate constants (kcat) for the reaction of Ty with monophenols exhibit Hammett plot parameters (ρ = −1.8 to −2.4) [[55, 56]] that are similar to those for the EAS monooxygenation reaction between Cu2O2 model complexes and monophenolate substrates. Based on this Hammett plot correlation and the results of inhibitor binding to Ty (vide supra), a generally accepted monophenol monooxygenation mechanism of Ty was developed (Fig. 2D), which invokes the formation of a ternary intermediate with the bound monophenolate substrate coordinated to the μ-η22-peroxide dicopper(II) active site, that proceeds to aromatic hydroxylation via EAS by the peroxide [[1, 57]]. Since Ty functions under physiological conditions (pH = 7), this monooxygenation mechanism (Fig. 2D) requires an initial deprotonation step of the monophenol substrate (pKa = 10). However, the identity of the putative proton acceptor site remained unknown and was a subject of debate, with proposed candidates including (a) a conserved Glu/Asn/water site [[27, 58]], (b) one of the first-coordination sphere histidine residues [[59, 60]], and (c) the μ-η22-peroxide ligand to form a hydroperoxide dicopper(II) active site [[61, 62]].

Parallel computational studies on Ty evaluated different mechanistic possibilities but did not converge on an unanimously accepted proposal. Initial computational studies found that the bis-μ-oxo dicopper(III) isomer of oxy-Ty is thermodynamically inaccessible relevant to μ-η22-peroxide dicopper(II) [[63, 64]], although this equilibrium is dependent on the functional employed [[65]]. A quantum-mechanics (QM) density functional theory (DFT) evaluation starting from the μ-η22-peroxide dicopper(II) monophenolate-bound ternary intermediate invoked in the generally accepted mechanism (Fig. 2D) predicted that the subsequent monooxygenation involves peroxide attack on the aromatic ring, without O–O cleavage, via a thermodynamically accessible barrier (12.3 kcal·mol−1) [[66]]. However, this computational mechanism assumed that the substrate binds to oxy-Ty as a monophenolate and did not consider the initial deprotonation step. In fact, this QM-only DFT study proposed that the mechanism starting from a ternary intermediate with a docked monophenol in the vicinity of the μ-η22-peroxide dicopper(II) active site proceeds via proton transfer to the peroxide and monophenolate coordination to CuA to form a weakly endergonic (5 kcal·mol−1) μ-η22-hydroperoxide dicopper(II) monophenolate-bound intermediate via a low barrier (8 kcal·mol−1), but with a high subsequent barrier for aromatic monooxygenation (> 20 kcal·mol−1), and was thus excluded for further consideration [[66]]. However, a quantum-mechanics molecular-mechanics (QM/MM) evaluation including the entire Ty enzyme and starting from the same monophenol bound μ-η22-peroxide dicopper(II) ternary intermediate proposed initial Cu–O dissociation to form a μ-η12-peroxide dicopper(II) active site that abstracts the phenolic H-atom from the docked monophenol substrate via a rate-limiting barrier (+16.3 kcal·mol−1) that proceeds to subsequent exergonic monooxygenation via radical coupling and with low barriers (< 5 kcal·mol−1) [[62]]. Although the discrepancies between the QM-only and QM/MM studies were investigated [[67]], the lack of strong experimental evidence on both the structure of the catalytically relevant ternary intermediate and the nature of the TS of monooxygenation, precluded the development of a robust computational coordinate, that is experimentally validated, and can then provide reliable mechanistic insights.

Elucidating the ternary intermediate of tyrosinase

Extending the insights from model complex and early enzyme studies towards the elucidation of the monophenol monooxygenation mechanism of Ty has been limited by the lack of direct experimental evidence on the ternary intermediate (Ty/monophenol(ate)/O2) formed via the monophenol substrate binding to the oxy-Ty. Several crystallographic studies have focused on providing insights into the structure of this elusive intermediate. The first crystal structure of oxy-Ty (PDB: 1WX2/4) was complex with its caddie protein, where a tyrosine caddie residue (Y98) occupied the protein pocket in the vicinity of the μ-η22-peroxide dicopper(II) active site [[4]]. Importantly, although the protonation state of Y98 was unclear (i.e., monophenol vs. monophenolate) [[4, 31, 59]], it is clearly not coordinated with CuA (CuA–OY98 = 2.7–3.0 Å; CuA is coordinated by His residues closer to the N-terminus, while CuB coordinated by His residues closer to the C-terminus). However, since Y98 is tethered to the caddie protein, the relevance of the caddie-bound oxy-Ty structure to the catalytic ternary intermediate with a free monophenol substrate was unclear. In fact, the caddie-bound oxy-Ty was proposed to reflect the structure of a possible pre-ternary intermediate that under catalytically relevant conditions would convert to the ternary intermediate via coordination of the monophenolate substrate to CuA [[59, 68]]. Although extended kinetic investigations on the caddie monooxygenation by oxy-Ty have been limited due to the protein–protein interactions in the complex formation, time-resolved crystallographic investigations reported the CuA movement towards the bound caddie along with the formation of the hydroxylated product, but without accumulating or resolving a distinct ternary intermediate [[68]]. Equivalent crystallographic studies on the pro-form of Ty mutants (i.e., an extended Ty terminus occupying the protein pocket similarly to the bound caddie protein) also proposed movement of CuA towards the residue occupying the substrate binding pocket, with similar limitations to catalytic relevance [[59]].

Crystallographic attempts to resolve the structure of Ty with a free monophenol bound to its active site pocket resulted in the structure of Ty bound to the hydroxylated catechol product [[58]]. To overcome this reactivity issue, the crystal structure of the Zn-substituted Ty with the l-tyrosine (PDB: 4P6R) was resolved instead [[58]], which indicated that the native monophenol substrate docks to the protein pocket via π-π interaction with a first coordination histidine residue, similar to Y98 in the caddie-bound oxy-Ty structure [[4]]. However, unlike the Y98 residue, free l-tyrosine appears to coordinate with ZnA as a monophenolate (ZnA–Otyrosine = 1.9–2.0 Å), with its phenolic proton proposed to be accepted by a crystallographically observed nearby water molecule (Owater–Otyrosinate = 5.9 Å) that is H-bonded by the side chains of the conserved Glu and Asn residues [[58]]. It should be noted that the use of Zn in place of Cu, as well as the absence of the μ-η22-peroxide ligand significantly limits the interpretation of these results in terms of their relevance to the catalytic ternary intermediate. Finally, previous efforts to trap and spectroscopically define the ternary intermediate of Ty with the free monophenol substrate 3,5-difluorophenol under cryogenic steady-state conditions did not produce definitive results [[69]].

On the basis of the above model complex and enzyme studies, a number of possible structures for the ternary structures have been proposed, which are shown in Fig. 3B. These proposed structures exhibit: (a) different sites for the phenolic proton, which include the conserved Glu/Asn/water cluster (structure 3 in Fig. 3B), the axial histidine ligand of CuA (structure 4 in Fig. 3B), the μ-η22-peroxide (structure 2 in Fig. 3B) or remain to the bound monophenol substrate (structure 1 in Fig. 3B), (b) monophenolate substrate coordination to CuA, or lack thereof (Fig. 3B), and for each of these possibilities (c) preservation or cleavage of the peroxide O–O bond via μ-η22-peroxide dicopper(II) to bis-μ-oxo dicopper(III) isomerization (Fig. 2B, equilibrium). In the absence of direct experimental evidence, it is not feasible to conclusively assign any of these proposed structures as the catalytically relevant ternary intermediate, which further precludes the elucidation of the aromatic monooxygenation mechanism of CBC enzymes.

To resolve this critical issue, we recently reported the trapping and spectroscopic definition of the elusive ternary (tyrosinase/monophenol/O2) intermediate under catalytically relevant conditions by employing the slow-reacting monophenol substrate, methyl 4-hydroxybenzoate (4-COOCH3) [[33]]. The monooxygenation of 4-COOCH3 by oxy-Ty in borate buffer (0.5 m, pH 9.0) and in the absence of any external reductants, proceeds as a single-turnover reaction due to the fast dissociation (Kd3) of the catechol product (methyl 3,4-dihydroxybenzoate; 3,4-COOCH3) from the resultant met-Ty active site and its condensation-driven trapping by borate [[55]], relative to its slower oxidation to quinone by met-Ty (k3), which would close the catalytic cycle and result in multiple turnovers (Fig. 3A). Under these conditions, stopped-flow absorption (SF-Abs) monitoring of the reaction between deoxy-Ty and O2-saturated solutions of 4-COOCH3 showed the immediate formation (< 2 ms) of the two characteristic absorption features of oxy-Ty, consistent with the fast (k1 = 19–23 μm·s−1) and exergonic (Kd1 = 40 μm) binding of O2 to deoxy-Ty [[7]], followed by their first-order exponential decay to met-Ty. The observed rate constant for this decay increases linearly and eventually saturates with increasing 4-COOCH3 concentration, indicating the presence of the ternary intermediate via the fast and reversible binding (Kd2 = 13 mm) of 4-COOCH3 to oxy-Ty followed by its slow and irreversible monooxygenation reaction (k2 = 0.56 s−1).

Therefore, at early times (100 ms) and high 4-COOCH3 concentration (10 mm) the reaction of oxy-Ty with 4-COOCH3 contains 57% oxy-Ty, 43% ternary intermediate, and < 1% products. Using this early-time speciation, the UV–Vis absorption spectrum of the ternary intermediate was obtained from the SF-Abs data (Fig. 3C). The presence of the two characteristic μ-η22-peroxide→Cu(II) CT bands in the ternary intermediate absorption spectrum clearly demonstrated that binding of the monophenol substrate to oxy-Ty does not result in cleavage of the peroxide O–O bond, consistent with the absence of the characteristic intense 400 nm for the bis-μ-oxo dicopper(III) [[42]]. In fact, the absence of any additional bands in the 350–550 nm region that would correspond to a phenolate→Cu CT [[70]], is consistent with non-coordinative binding of the monophenol(ate) substrate.

To define the ternary intermediate at a higher resolution we proceeded to characterize it by rR spectroscopy [[33]]. First, we obtained the reference rR spectrum of oxy-Ty under our reaction conditions. Since previous studies were mostly focused on oxy-Ty either in its pro-form (i.e., the latent enzyme form that is activated by post-translational proteolytic cleavage of its extended terminal) or its complex with the caddie protein, in all of which the surface-exposed active site pocket is fully occupied by terminal or caddie residues, we proceeded to obtain the rR spectrum of the catalytic and monomeric oxy-Ty. Interestingly, upon solvent isotopic perturbations (H2O→D2O), the rR of oxy-Ty exhibited frequency upshifts in its fundamental (ΔνCu–O = +4 cm−1), and first-overtone Cu–O modes (ΔνCu–O(overtone) = +7 cm−1), suggesting solvent interactions with the μ-η22-peroxide dicopper(II) active site [[71]]. These H2O→D2O frequency upshifts were not observed in rR studies for oxy-Hc [[72]], consistent with the fact that its μ-η22-peroxide dicopper(II) active site is buried within the protein matrix, precluding solvent access. Molecular dynamics (MD) simulations predicted the presence of active site solvent water molecules in close proximity to the μ-η22-peroxide of oxy-Ty, with subsequent QM frequency calculations indicating that H-bonding interaction between active site water molecules (W1, W2) and the μ-η22-peroxide (Fig. 3E) is required to reproduce the spectroscopically observed frequency upshift of the Cu–O mode [[71]]. This frequency upshift of the Cu–O mode upon solvent deuteration is due to its mode coupling with close-in-energy W1 and W2 modes since in H2O the water modes are higher in energy than the Cu–O mode, and thus mixing decreases the Cu–O frequency, while in D2O the energy of the water modes are below the energy of the Cu–O mode and mixing increases the Cu–O frequency [[71]]. Having defined the reference rR spectra and active site structure for the monomeric oxy-Ty, we proceed to trap the ternary intermediate via 100-ms rapid freeze quench (RFQ) for the reaction of deoxy-Ty with an O2-saturated solution of 4-COOCH3 (10 mm). The rR spectrum of the trapped ternary intermediate (43% accumulation) was obtained by 351 nm laser excitation and corrected for the spectral contributions of oxy-Ty (57%) and the borate buffer, and intensity renormalization (Fig. 3D). The rR spectrum of the ternary intermediate exhibits the characteristic vibrational features of the μ-η22-peroxide dicopper(II) core, consistent with the UV–Vis absorption results (Fig. 3C,D). Interestingly, upon binding of the monophenol substrate to oxy-Ty the μ-η22-peroxide dicopper(II) site appears to be only moderately perturbed based on the frequency changes for the O–O (ΔνO–O = +3 cm−1) and first overtone of the Cu–O (Δν(Cu–O)overtone = +24 cm−1) modes (Fig. 3D).

These spectroscopic (UV–Vis abs, rR) perturbations upon binding of the monophenol substrate to oxy-Ty (i.e., oxy-Ty vs. ternary intermediate; Fig. 3C,D) were correlated to time-dependent DFT (TD-DFT) and QM-frequency calculations on QM/MM optimized structures of oxy-Ty and the previous proposals for the ternary intermediate (Fig. 3B). These results showed that only one of the ternary intermediate structures (Fig. 3F), and not the other candidates (Fig. 3B), closely reproduces our spectroscopic results. This ternary intermediate (Fig. 3F) shows that the substrate binds to the oxy-Ty protein pocket fully protonated (as a monophenol) by displacing the active site water molecules in oxy-Ty (W1, W2) and replacing their H-bonding to the μ-η22-peroxide (Fig. 3E) via a single H-bond with its hydroxyl group, without coordination to CuA. The protonation state of the monophenol substrate in the ternary intermediate is further supported by (a) the pH dependence of Kd2, indicating that the substrate binding affinity to oxy-Ty decreases with increasing pH above the monophenol pKa, and (b) the small normal solvent KIE of 1.6 on the monooxygenation rate constant (k2) indicating that the phenolic proton is involved in the TS of the rate-limiting step, and thus has not been transferred prior to the formation of the ternary intermediate.

Comparison of the catalytic ternary intermediate (Fig. 3F) with the caddie-bound oxy-Ty structure reveals a similar substrate binding mode (Fig. 3G). The Operoxide–Omonophenol distance in the ternary intermediate (2.7 Å) is similar to those observed in the caddie-bound oxy-Ty structures (2.7–3.0 Å), suggesting that the caddie Y98 residue is also likely protonated and H-bonded to the μ-η22-peroxide. Interestingly, the rR spectra changes from the WT to Y98F caddie-bound to oxy-Ty (ΔνO–O ~ +3 cm−1, ΔνCu–O(overone) ~ +17 cm−1), [[73]] are also similar to those observed between oxy-Ty and the ternary intermediate (Fig. 3D). Thus, a retrospective interpretation of the rR of caddie-bound oxy-Ty suggests that the removal of the H-bond between caddie Y98 and the μ-η22-peroxide of oxy-Ty via its site-selective mutagenesis (Y98F) appears to increase the frequency of the O–O and Cu–O modes similarly to the removal of the H-bond between W2 and the μ-η22-peroxide of oxy-Ty upon binding of the monophenol substrate to form the ternary intermediate (Fig. 3E,F). Overall, the recent report of the experimentally-defined structure of the long elusive catalytic ternary intermediate of Ty (Fig. 3F) [[33]], resolves longstanding debates in the field and indicates that the monophenol substrate binds to the protein pocket without coordinating to CuA but rather H-bonding to the μ-η22-peroxide. This structure of the ternary intermediate in Ty has unique mechanistic implications for the subsequent monooxygenation reaction that are discussed below.

New mechanistic insights into the enzymatic monooxygenation of monophenols

The elucidation of the geometric and electronic structure of the elusive ternary intermediate (Fig. 3F) along with the direct probing of its subsequent monooxygenation TS via single-turnover kinetics, allowed the generation of an experimentally validated computational mechanism for the key monooxygenation steps in the catalytic cycle of Ty [[33]]. A systematic evaluation of the monooxygenation reaction coordinates from the ternary intermediate to the hydroxylated product by QM/MM calculations indicated that the previously assumed mechanism based on early enzyme experiments and model complex studies (Fig. 2D) is not thermodynamically favorable. Instead, the most thermodynamically accessible pathway reflects a fundamentally different monooxygenation mechanism that involves the initial cleavage of the μ-η22-peroxide O–O bond to accept the phenolic proton (along with 0.6 electron spin density) via a low barrier (+7 kcal·mol−1) to form a weakly endergonic μ-oxo-μ-hydroxo dicopper intermediate (2O in Fig. 4A), followed by substrate coordination to CuA concomitant with C–O bond formation via the rate-limiting barrier (+14.1 kcal·mol−1). This rate-limiting barrier is in agreement with the observed single-turnover monooxygenation rate, and its corresponding TS structure (TS2 in Fig. 4A) reproduces the experimental Ar-OH/D and Ar-C-H/D KIE values of 1.6 and 1.0, respectively [[33]].

Details are in the caption following the image
The monooxygenation mechanism of tyrosinase. (A) The key steps in the monooxygenation mechanism of Ty defined by experimentally-validated QM/MM calculations [[33]]. The copper atoms are shown as pink spheres (CuA on the left, CuB on the right), the imidazole rings of the first-sphere coordination histidines as thin lines (with the N atom of the axial histidine of CuA shown as a blue sphere in the TS2 and 3O structures), the atoms of the 4-COOCH3 monophenol substrate as spheres (C = gray, O = red, H = white) connected with thick lines, H-bonds as dashed lines, and the remaining atoms of the QM and MM parts are omitted for clarity. Energies were calculated from single-point higher-level optimizations (TPSS/def2-TZVPD) upon the QM/MM-optimized geometries (QM/MM/TPSS/def2-SVP) in a homogeneous dielectric continuum with εr = 8. (B) The revised catalytic cycle of Ty, with the new monophenol monooxygenation mechanism shown in the black box, is fundamentally different from the previously proposed mechanism based on model complex studies (Fig. 2D). Figure reproduced from Ref.[[33]].

An interesting consideration in this new mechanism relates to the extent and concertedness of electron transfer along with the initial proton transfer, associated with an EAS vs. radical coupling description of the subsequent monooxygenation step. Additional experimental and computational investigations are necessary to fully resolve this mechanistic consideration. It should also be noted that although these calculations evaluating 2D scans of reaction coordinates significantly improve upon the previous 1D computational efforts (vide supra), a more concerted mechanism along all four reaction coordinates (4D scan) is possible, but remains challenging to evaluate due to its high computational requirements. Irrespective of these mechanistic considerations, this QM/MM reaction coordinate (Fig. 4A) constitutes a new experimentally validated monooxygenation mechanism of Ty that revises all previous proposals and provides key mechanistic insights. In fact, this new mechanism identifies the μ-η22-peroxide of the Cu2O2 active site as the proton acceptor site, thus resolving a longstanding debate in the field. This proton transfer step is coupled to the cleavage of the μ-η22-peroxide O–O bond, which extends the bis-μ-oxo equilibrium beyond model complexes to the CBC enzyme active site. This mechanism also highlights the critical importance of second-sphere interactions in the Ty protein pocket to control the reactivity of its Cu2O2 active site and facilitate a monooxygenation mechanism that is different from their model complex counterparts. Indeed, although model complexes also accept the monophenolic H-atom via PCET, the absence of significant stabilizing interactions between the ligand scaffold and the monophenol substrate results in phenoxyl radical escape and subsequent radical coupling. Alternatively for Ty, its protein pocket allows both formation of a ternary intermediate and prevents premature substrate escape during the monooxygenation reaction.

Future perspectives and concluding remarks

The recent elucidation of the ternary intermediate in Ty has advanced the longstanding goal of defining the monooxygenation mechanism of CBC enzymes. Further studies are required to explore the considerations regarding the timing of ET and the concertedness of the reaction coordinates (Fig. 4B) in monophenol monooxygenation, which were discussed above. Given the critical significance of trapping and spectroscopically defining reaction intermediates in mechanistic enzymology, recently demonstrated for Ty monooxygenation [[33]], equivalent investigations need to extend to CaOx and AOx with the goal of defining their respective ternary intermediates with para-substituted monophenols, catechols, and/or o-aminophenols.

Overall, the novel mechanistic insights obtained for the monooxygenation reaction of Ty open new opportunities for a critical challenge in CBC enzymology. This relates to the fact that the diverse reactivities in different members of the CBC family (Fig. 1C) cannot be predicted or rationalized by simple correlations to their gene sequence or protein structures [[28]]. Instead, this would require a causal framework derived from direct mechanistic insights. Towards this goal, the recent identification of the key second-sphere residues involved in enabling substrate binding and/or lowering the TS monooxygenation barrier in Ty [[33]] serves as the main reference for uncovering the elusive structure–function relationships within the CBC protein family. Future efforts to obtain a complete understanding of the active site factors that control the reactivity of [Cu2O2]2+ in different CBC enzymes would enable both their accurate classification based on gene sequence, as well as the discovery of putative new CBC enzymes capable of performing novel chemical transformations.

Acknowledgements

We would like to thank all previous members of the Solomon group who contributed to the studies on coupled binuclear copper systems. We are also thankful to all our previous and current collaborators and their research groups. In particular, we want to acknowledge the collaborative research groups of Prof Kenneth Karlin (Johns Hopkins University) for model complex studies, Prof Lubomir Rulisek (IOCB) for QM/MM calculations, and Prof Timothy Machonkin (Whitman College) for enzyme expression and purification protocols. We are grateful for the funding of our work by the U.S. National Institutes of Health (DK31450).

    Data accessibility

    All data related to this Perspective are included in the main text.