De novo design and characterization of copper centers in synthetic four-helix-bundle proteins.

The design and chemical synthesis of de novo metalloproteins on cellulose membranes with the structure of an antiparallel four-helix bundle is described. All possible combinations of three different sets of amphiphilic helices were assembled on cyclic peptide templates which were bound by a cleavable linker to the cellulose. In the hydrophobic interior, the four-helix bundle proteins carry a cysteine and several histidines at various positions for copper ligation. This approach was used successfully to synthesize, for the first time, copper proteins based on a four-helix bundle. UV-vis spectra monitored on the solid support showed ligation of copper(II) by about one-third out of the 96 synthesized proteins and tetrahedral complexes of cobalt(II) by most of these proteins. Three of the most stable copper-binding proteins were synthesized in solution and their structural properties analyzed by spectroscopic methods. Circular dichroism, one-dimensional NMR, and size-exclusion chromatography indicate a folding into a compact state containing a high degree of secondary structure with a reasonably ordered hydrophobic core. They displayed UV-vis absorption, resonance Raman, and EPR spectra intermediate between those of type 1 and type 2 copper centers. The present approach provides a sound basis for further optimizing the copper binding and its functional properties by using combinatorial protein chemistry guided by rational principles.     

Spectroscopic identification of different types of copper centers generated in synthetic four-helix bundle proteins

Using a combined rational-combinatorial approach, stable copper binding sites were implemented in template-assembled synthetic four-helix bundle proteins constructed by three different helices with only 16 amino acid residues. These peptides include two histidines and one cysteine at positions appropriate for coordinating a copper ion. Sequence variations of the helices were made in the second coordination shell or even more remote from the copper binding site (i) to increase the overall stability of the metalloproteins and (ii) to fine-tune the structure and properties of the copper center. As a result, ca. 90% of the 180 proteins that were synthesized were capable to bind copper with a substantially higher specificity than those obtained in the first design cycle (Schnepf, R.; Horth, P.; Bill, E.; Wieghardt, K.; Hildebrandt, P.; Haehnel, W. J. Am. Chem. Soc. 2001, 123, 2186-2195). Furthermore, the stabilities of the copper protein complexes were increased by up to 2 orders of magnitude and thus allowed a UV-vis absorption, resonance Raman, electron paramagnetic resonance, and (magnetic) circular dichroism spectroscopic identification and characterization of three different types of copper binding sites. It could be shown that particularly steric perturbations in the vicinity of the His(2)Cys ligand set control the formation of either a tetragonal (type II) or a tetrahedral (type I) copper binding site. With the introduction of two methionine residues above the histidine ligands, a mixed-valent dinuclear copper binding site was generated with spectroscopic properties that are very similar to those of Cu(A) sites in natural proteins. The results of the present study demonstrate for the first time that structurally different metal binding sites can be formed and stabilized in four-helix bundle proteins.     

Response of a designed metalloprotein to changes in metal ion coordination, exogenous ligands, and active site volume determined by X-ray crystallography

The de novo protein DF1 is a minimal model for diiron and dimanganese metalloproteins, such as soluble methane monooxygenase. DF1 is a homodimeric four-helix bundle whose dinuclear center is formed by two bridging Glu side chains, two chelating Glu side chains, and two monodentate His ligands. Here, we report the di-Mn(II) and di-Co(II) derivatives of variants of this protein. Together with previously solved structures, 23 crystallographically independent four-helix bundle structures of DF1 variants have been determined, which differ in the bound metal ions and size of the active site cavity. For the di-Mn(II) derivatives, as the size of the cavity increases, the number and polarity of exogenous ligands increases. This collection of structures was analyzed to determine the relationship between protein conformation and the geometry of the active site. The primary mode of backbone movement involves a coordinated tilting and sliding of the first helix in the helix-loop-helix motif. Sliding depends on crystal-packing forces, the steric bulk of a critical residue that determines the dimensions of the active site access cavity, and the intermetal distance. Additionally, a torsional motion of the bridging carboxylates modulates the intermetal distance. This analysis provides a critical evaluation of how conformation, flexibility, and active site accessibility affect the geometry and ligand-binding properties of a metal center. The geometric parameters defining the DF structures were compared to natural diiron proteins; DF proteins have a restricted active site cavity, which may have implications for substrate recognition and chemical stability.     

De novo design of a single-chain diphenylporphyrin metalloprotein

We describe the computational design of a single-chain four-helix bundle that noncovalently self-assembles with fully synthetic non-natural porphyrin cofactors. With this strategy, both the electronic structure of the cofactor as well as its protein environment may be varied to explore and modulate the functional and photophysical properties of the assembly. Solution characterization (NMR, UV-vis) of the protein showed that it bound with high specificity to the desired cofactors, suggesting that a uniquely structured protein and well-defined site had indeed been created. This provides a genetically expressed single-chain protein scaffold that will allow highly facile, flexible, and asymmetric variations to enable selective incorporation of different cofactors, surface-immobilization, and introduction of spectroscopic probes.     

Three-dimensional structure and dynamics of a de novo designed, amphiphilic, metallo-porphyrin-binding protein maquette at soft interfaces by molecular dynamics simulations

The three-dimensional structure and dynamics of de novo designed, amphiphilic four-helix bundle peptides (or "maquettes"), capable of binding metallo-porphyrin cofactors at selected locations along the length of the core of the bundle, are investigated via molecular dynamics simulations. The rapid evolution of the initial design to stable three-dimensional structures in the absence (apo-form) and presence (holo-form) of bound cofactors is described for the maquettes at two different soft interfaces between polar and nonpolar media. This comparison of the apo- versus holo-forms allows the investigation of the effects of cofactor incorporation on the structure of the four-helix bundle. The simulation results are in qualitative agreement with available experimental data describing the structures at lower resolution and limited dimension.     

Computational de novo design and characterization of a four-helix bundle protein that selectively binds a nonbiological cofactor

We report the complete de novo design of a four-helix bundle protein that selectively binds the nonbiological DPP-Fe(III) metalloporphyrin cofactor (DPP-Fe(III) = 5, 15-Di[(4-carboxymethyleneoxy)phenyl]porphinato iron(III)). A tetrameric, D2-symmetric backbone scaffold was constructed to encapsulate two DPP-Fe(III) units through bis(His) coordination. The complete sequence was determined with the aid of the statistical computational design algorithm SCADS. The 34-residue peptide was chemically synthesized. UV-vis and CD spectroscopy, size-exclusion chromatography, and analytical ultracentrifugation indicated the peptide undergoes a transition from a predominantly random coil monomer to an alpha-helical tetramer upon binding DPP-Fe(III). EPR spectroscopy studies indicated the axial imidazole ligands were oriented in a perpendicular fashion, as defined by second-shell interactions that were included in the design. The 1-D 1H NMR spectrum of the assembled protein displayed features of a well-packed interior. The assembled protein possessed functional redox properties different from those of structurally similar systems containing the heme cofactor. The designed peptide demonstrated remarkable cofactor selectivity with a significantly weaker binding affinity for the natural heme cofactor. These findings open a path for the selective incorporation of more elaborate cofactors into designed scaffolds for constructing molecularly well-defined nanoscale materials.     

A De Novo Heterodimeric Due Ferri Protein Minimizes the Release of Reactive Intermediates in Dioxygen‐Dependent Oxidation

Metalloproteins utilize O₂ as an oxidant, and they often achieve a 4‐electron reduction without H₂O₂ or oxygen radical release. Several proteins have been designed to catalyze one or two‐electron oxidative chemistry, but the de novo design of a protein that catalyzes the net 4‐electron reduction of O₂ has not been reported yet. We report the construction of a diiron‐binding four‐helix bundle, made up of two different covalently linked α₂ monomers, through click chemistry. Surprisingly, the prototype protein, DF‐C1, showed a large divergence in its reactivity from earlier DFs (DF: due ferri, two iron). DFs release the quinone imine and free H₂O₂ in the oxidation of 4‐aminophenol in the presence of O₂, whereas Fe^III‐DF‐C1 sequesters the quinone imine into the active site, and catalyzes inside the scaffold an oxidative coupling between oxidized and reduced 4‐aminophenol. The asymmetry of the scaffold allowed a fine‐engineering of the substrate binding pocket, that ensures selectivity.     

Rational De Novo Design of a Cu Metalloenzyme for Superoxide Dismutation

Superoxide dismutases (SODs) are highly efficient enzymes for superoxide dismutation and the first line of defense against oxidative stress. These metalloproteins contain a redox-active metal ion in their active site (Mn, Cu, Fe, Ni) with a tightly controlled reduction potential found in a close range around the optimal value of 0.36 V versus the normal hydrogen electrode (NHE). Rationally designed proteins with well-defined three-dimensional structures offer new opportunities for obtaining functional SOD mimics. Here, we explore four different copper-binding scaffolds: H₃ (His₃), H₄ (His₄), H₂DH (His₃Asp with two His and one Asp in the same plane) and H₃D (His₃Asp with three His in the same plane) by using the scaffold of the de novo protein GRα₃D. EPR and XAS analysis of the resulting copper complexes demonstrates that they are good Cu^II-bound structural mimics of Cu-only SODs. Furthermore, all the complexes exhibit SOD activity, though three orders of magnitude slower than the native enzyme, making them the first de novo copper SOD mimics.     

De novo and inverse folding predictions of protein structure and dynamics

Summary In the last two years, the use of simplified models has facilitated major progress in the globular protein folding problem, viz., the prediction of the three-dimensional (3D) structure of a globular protein from its amino acid sequence. A number of groups have addressed the inverse folding problem where one examines the compatibility of a given sequence with a given (and already determined) structure. A comparison of extant inverse protein-folding algorithms is presented, and methodologies for identifying sequences likely to adopt identical folding topologies, even when they lack sequence homology, are described. Extension to produce structural templates or fingerprints from idealized structures is discussed, and for eight-membered β-barrel proteins, it is shown that idealized fingerprints constructed from simple topology diagrams can correctly identify sequences having the appropriate topology. Furthermore, this inverse folding algorithm is generalized to predict elements of supersecondary structure including β-hairpins, helical hairpins and α/β/α fragments. Then, we describe a very high coordination number lattice model that can predict the 3D structure of a number of globular proteins de novo; i.e. using just the amino acid sequence. Applications to sequences designed by DeGrado and co-workers [Biophys. J., 61 (1992) A265] predict folding intermediates, native states and relative stabilities in accord with experiment. The methodology has also been applied to the four-helix bundle designed by Richardson and co-workers [Science, 249 (1990) 884] and a redesigned monomeric version of a naturally occurring four-helix dimer, rop. Based on comparison to the rop dimer, the simulations predict conformations with rms values of 3–4 ? from native. Furthermore, the de novo algorithms can asses the stability of the folds predicted from the inverse algorithm, while the inverse folding algorithms can assess the quality of the de novo models. Thus, the synergism of the de novo and inverse folding algorthhm approaches provides a set of complementary tools that will facilitate further progress on the protein-folding problem.     

Product bound structures of the soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): protein motion in the alpha-subunit

The soluble methane monooxygenase hydroxylase (MMOH) alpha-subunit contains a series of cavities that delineate the route of substrate entrance to and product egress from the buried carboxylate-bridged diiron center. The presence of discrete cavities is a major structural difference between MMOH, which can hydroxylate methane, and toluene/o-xylene monooxygenase hydroxylase (ToMOH), which cannot. To understand better the functions of the cavities and to investigate how an enzyme designed for methane hydroxylation can also accommodate larger substrates such as octane, methylcubane, and trans-1-methyl-2-phenylcyclopropane, MMOH crystals were soaked with an assortment of different alcohols and their X-ray structures were solved to 1.8-2.4 A resolution. The product analogues localize to cavities 1-3 and delineate a path of product exit and/or substrate entrance from the active site to the surface of the protein. The binding of the alcohols to a position bridging the two iron atoms in cavity 1 extends and validates previous crystallographic, spectroscopic, and computational work indicating this site to be where substrates are hydroxylated and products form. The presence of these alcohols induces perturbations in the amino acid side-chain gates linking pairs of cavities, allowing for the formation of a channel similar to one observed in ToMOH. Upon binding of 6-bromohexan-1-ol, the pi helix formed by residues 202-211 in helix E of the alpha-subunit is extended through residue 216, changing the orientations of several amino acid residues in the active site cavity. This remarkable secondary structure rearrangement in the four-helix bundle has several mechanistic implications for substrate accommodation and the function of the effector protein, MMOB.     

Nonnatural amino acid ligands in heme protein design

The de novo design, synthesis, and characterization of a four-alpha-helix bundle scaffold containing heme ligated by 4-beta-(pyridyl)-l-alanine (Pal) is presented. The protein scaffold is highly helical, stable, and conformationally specific in the apo-state. Incorporation of heme using the designed bis-Pal axial ligation is shown using UV-visible and EPR spectroscopies. The observed heme midpoint reduction potential, +58 mV versus SHE, is 287 mV (6.8 kcal/mol) higher than the analogous bis-histidine-ligated heme protein.     

De Novo Design,Synthesis and Characterisation of MP3, A New Catalytic Four‐Helix Bundle Hemeprotein

A new artificial metalloenzyme, MP3 (MiniPeroxidase 3), designed by combining the excellent structural properties of four‐helix bundle protein scaffolds with the activity of natural peroxidases, was synthesised and characterised. This new hemeprotein model was developed by covalently linking the deuteroporphyrin to two peptide chains of different compositions to obtain an asymmetric helix–loop–helix/heme/helix–loop–helix sandwich arrangement, characterised by 1) a His residue on one chain that acts as an axial ligand to the iron ion; 2) a vacant distal site that is able to accommodate exogenous ligands or substrates; and 3) an Arg residue in the distal site that should assist in hydrogen peroxide activation to give an HRP‐like catalytic process. MP3 was synthesised and characterised as its iron complex. CD measurements revealed the high helix‐forming propensity of the peptide, confirming the appropriateness of the model procedure; UV/Vis, MCD and EPR experiments gave insights into the coordination geometry and the spin state of the metal. Kinetic experiments showed that Fe^III–MP3 possesses peroxidase‐like activity comparable to R38A–hHRP, highlighting the possibility of mimicking the functional features of natural enzymes. The synergistic application of de novo design methods, synthetic procedures, and spectroscopic characterisation, described herein, demonstrates a method by which to implement and optimise catalytic activity for an enzyme mimetic.     

Zinc-bacteriochlorophyllide dimers in de novo designed four-helix bundle proteins. A model system for natural light energy harvesting and dissipation

Photosynthetic organisms utilize interacting pairs of chlorophylls and bacteriochlorophylls as excitation energy donors and acceptors in light harvesting complexes, as photosensitizers of charge separation in reaction centers, and maybe as photoprotective quenching centers that dissipate excess excitation energy under high light intensities. To better understand how the pigment's local environment and spatial organization within the protein tune its ground- and excited-state properties to perform different functions, we prepared and characterized the simplest possible system of interacting bacteriochlorophylls within a protein scaffold. Using HP7, a high-affinity heme-binding protein of the HP class of de novo designed four-helix bundles, we incorporated 13(2)-OH-zinc-bacteriochlorophyllide-a (ZnBChlide), a water-soluble bacteriochlorophyll derivative, into specific binding sites within the four-helix bundle protein core. We capitalized on the rich and informative optical spectrum of ZnBChlide to rigorously characterize its complexes with HP7 and two variants, in which a single heme-binding site is eliminated by replacing histidine residues at positions 7 or 42 by phenylalanine. Surprisingly, we found the ZnBChlide binding capacity of HP7 and its variants to be higher than for heme: up to three ZnBChlide pigments bind per HP7, or two per each single histidine variant. The formation of dimers within HP7 results in dramatic quenching of ZnBChlide fluorescence, reducing its quantum yield by about 80%, and the singlet excited-state lifetime by 2 orders of magnitudes compared to the monomer. Thus, HP7 and its variants are the first examples of a simple protein environment that can isolate a self-quenching pair of photosynthetic pigments in pure form. Unlike its complicated natural analogues, this system can be constructed from the ground up, starting with the simplest functional element, increasing the complexity as needed.     

A Histidine Residue and a Tetranuclear Cuprous-thiolate Cluster Dominate the Copper Loading Landscape of a Copper Storage Protein from Streptomyces lividans

The chemical basis for protecting organisms against the toxic effect imposed by excess cuprous ions is to constrain this through high-affinity binding sites that use cuprous-thiolate coordination chemistry. In bacteria, a family of cysteine rich four-helix bundle proteins utilise thiolate chemistry to bind up to 80 cuprous ions. These proteins have been termed copper storage proteins (Csp). The present study investigates cuprous ion loading to the Csp from Streptomyces lividans (SlCsp) using a combination of X-ray crystallography, site-directed mutagenesis and stopped-flow reaction kinetics with either aquatic cuprous ions or a chelating donor. We illustrate that at low cuprous ion concentrations, copper is loaded exclusively into an outer core region of SlCsp via one end of the four-helix bundle, facilitated by a set of three histidine residues. X-ray crystallography reveals the existence of polynuclear cuprous-thiolate clusters culminating in the assembly of a tetranuclear [Cu₄(μ₂-S-Cys)₄(Ν^δ1-His)] cluster in the outer core. As more cuprous ions are loaded, the cysteine lined inner core of SlCsp fills with cuprous ions but in a fluxional and dynamic manner with no evidence for the assembly of further intermediate polynuclear cuprous-thiolate clusters as observed in the outer core. Using site-directed mutagenesis a key role for His107 in the efficient loading of cuprous ions from a donor is established. A model of copper loading to SlCsp is proposed and discussed.     

Electrochemically driven reversible solid state metal exchange processes in polynuclear copper complexes

The electrochemical characteristics of polynuclear di-copper and tetra-copper complexes of an expanded "Robson-type" macrocyclic ligand are explored by solid state voltammetry in aqueous media. When adhered to a graphite electrode surface in the form of microcrystalline powders and immersed in aqueous buffer solution, these water-insoluble polynuclear copper complexes show well-defined voltammetric reduction and re-oxidation responses. The di-copper metal complexes [Cu₂(H₃L)(OH)][BF₄]₂ and the tetra-copper complexes [Cu₄(L)(OH)][NO₃]₃ with an O₄N₄ octadentate macrocyclic ligand L are shown to exhibit inter-related and proton concentration sensitive solid state voltammetric characteristics. At sufficiently negative potential, copper is extracted from the complexes to form a solid copper deposit and the neutral form of the insoluble free ligand. Upon re-oxidation of the copper deposit, Cu²⁺ undergoes facile re-insertion into the ligand sphere to re-form solid di- and tetra-copper complexes at the electrode surface. The reduction process occurs in two stages, with two Cu²⁺ cations being extracted in each step. The ability of the macrocyclic ligand to efficiently release and accumulate copper is demonstrated. Electronic Publication     

Conformationally constrained sequence designs to bias monomer-dimer equilibriums in TASP systems

We have designed template-assembled synthetic proteins (TASPs) with the intent of controlling their oligomeric state by stabilizing specific helical tertiary structures via histidine metal ion chelation or disulfide incorporation. In solution, cavitein Q4 was previously determined to interconvert between a four-helix bundle monomer and an eight-helix bundle dimer. In this paper, we show that judicious mutation of cavitein Q4 can stabilize either the monomeric parallel four-helix bundle or the dimeric antiparallel eight-helix bundle structure. Cavitein Q4-E3H, designed to be dimeric, is indeed biased toward dimerization as a result of incorporation of histidines. Moreover, the addition of nickel was found to further increase the association constant of dimerization. Similarly, a cavitein designed to stabilize the monomeric structure via histidine metal ion chelation (Q4-H) was found to favor a monomer in solution upon addition of nickel. Lastly, a cavitein intended to stabilize a monomeric structure via disulfide incorporation (Q4-C2) is reported. Surprisingly, this disulfide cavitein yielded two products upon oxidation suggesting disulfide formation both above the cavitand template and below may be possible. Nevertheless, the two disulfide caviteins were shown to exist as monomers as per their design.     

A designed well-folded monomeric four-helix bundle protein prepared by Fmoc solid-phase peptide synthesis and native chemical ligation

The design and total chemical synthesis of a monomeric native-like four-helix bundle protein is presented. The designed protein, GTD-Lig, consists of 90 amino acids and is based on the dimeric structure of the de novo designed helix-loop-helix GTD-43. GTD-Lig was prepared by the native chemical ligation strategy and the fragments (45 residues long) were synthesized by applying standard fluorenylmethoxycarbonyl (Fmoc) chemistry. The required peptide-thioester fragment was prepared by anchoring the free gamma-carboxy group of Fmoc-Glu-allyl to the solid phase. After chain elongation the allyl moiety was orthogonally removed and the resulting carboxy group was functionalized with a glycine-thioester followed by standard trifluoroacetic acid (TFA) cleavage to produce the unprotected peptide-thioester. The structure of the synthetic protein was examined by far- and near-UV circular dichroism (CD), sedimentation equilibrium ultracentrifugation, and NMR and fluorescence spectroscopy. The spectroscopic methods show a highly helical and native-like monomeric protein consistent with the design. Heat-induced unfolding was studied by tryptophan absorbance and far-UV CD. The thermal unfolding of GTD-Lig occurs in two steps; a cooperative transition from the native state to an intermediate state and thereafter by noncooperative melting to the unfolded state. The intermediate exhibits the properties of a molten globule such as a retained native secondary structure and a compact hydrophobic core. The thermodynamics of GuHCl-induced unfolding were evaluated by far-UV CD monitoring and the unfolding exhibited a cooperative transition that is well-fitted by a two-state mechanism from the native to the unfolded state. GTD-Lig clearly shows the characteristics of a native protein with a well-defined structure and typical unfolding transitions. The design and synthesis presented herein is of general applicability for the construction of large monomeric proteins.     

Design of a calcium-binding protein with desired structure in a cell adhesion molecule

Ca2+, "a signal of life and death", controls numerous cellular processes through interactions with proteins. An effective approach to understanding the role of Ca2+ is the design of a Ca2+-binding protein with predicted structural and functional properties. To design de novo Ca2+-binding sites in proteins is challenging due to the high coordination numbers and the incorporation of charged ligand residues, in addition to Ca2+-induced conformational change. Here, we demonstrate the successful design of a Ca2+-binding site in the non-Ca2+-binding cell adhesion protein CD2. This designed protein, Ca.CD2, exhibits selectivity for Ca2+ versus other di- and monovalent cations. In addition, La3+ (Kd 5.0 microM) and Tb3+ (Kd 6.6 microM) bind to the designed protein somewhat more tightly than does Ca2+ (Kd 1.4 mM). More interestingly, Ca.CD2 retains the native ability to associate with the natural target molecule. The solution structure reveals that Ca.CD2 binds Ca2+ at the intended site with the designed arrangement, which validates our general strategy for designing de novo Ca2+-binding proteins. The structural information also provides a close view of structural determinants that are necessary for a functional protein to accommodate the metal-binding site. This first success in designing Ca2+-binding proteins with desired structural and functional properties opens a new avenue in unveiling key determinants to Ca2+ binding, the mechanism of Ca2+ signaling, and Ca2+-dependent cell adhesion, while avoiding the complexities of the global conformational changes and cooperativity in natural Ca2+-binding proteins. It also represents a major achievement toward designing functional proteins controlled by Ca2+ binding.     

An engineered azurin variant containing a selenocysteine copper ligand

Modulating the properties of proteins through de novo design or redesign of existing proteins has been a longstanding goal in protein chemistry. Over the past two decades, site-directed mutagenesis has been a powerful tool to probe the role of certain residues and to fine-tune the activity of proteins. A limitation of this approach has been the accessibility of only a restricted number of functional groups through the 20 amino acids in the genetic code. The more recent technique of expressed protein ligation (EPL) provides an alternative route that allows efficient incorporation of nonnatural residues into proteins. We report here the preparation and spectroscopic characterization of an azurin variant in which a cysteine ligand to the blue copper center has been replaced by EPL with selenocysteine (Sec). This reports marks the first time that selenocysteine is artificially incorporated into the active site of a metalloprotein. The variant displays a significantly increased A(parallel) (from 56 to 104 G) and red-shifted CT band (from 625 to 677 nm), while maintaining the general type 1 copper characteristics, including similarity in reduction potentials. This study illustrates that iso-structural substitution using EPL can fine-tune the structural and functional properties of a metal-binding site without loss of most of its characteristics. Further spectroscopic and X-ray crystallographic studies of this and other EPL variants will provide new insights into the fine-control of the structure and function of metalloproteins.     

Copper–Oxygen Dynamics in the Tyrosinase Mechanism

The dinuclear copper enzyme, tyrosinase, activates O₂ to form a (μ‐η²:η²‐peroxido)dicopper(II) species, which hydroxylates phenols to catechols. However, the exact mechanism of phenolase reaction in the catalytic site of tyrosinase is still under debate. We herein report the near atomic resolution X‐ray crystal structures of the active tyrosinases with substrate l ‐tyrosine. At their catalytic sites, CuA moved toward l ‐tyrosine (CuA1 → CuA2), whose phenol oxygen directly coordinates to CuA2, involving the movement of CuB (CuB1 → CuB2). The crystal structures and spectroscopic analyses of the dioxygen‐bound tyrosinases demonstrated that the peroxide ligand rotated, spontaneously weakening its O?O bond. Thus, the copper migration induced by the substrate‐binding is accompanied by rearrangement of the bound peroxide species so as to provide one of the peroxide oxygen atoms with access to the phenol substrate's ? carbon atom.