The catalytic cycle of tyrosinase: peroxide attack on the phenolate ring followed by O-O bond cleavage

Siegbahn, Per E. M.

doi:10.1007/s00775-003-0449-4

Cited by 85 publications

(89 citation statements)

References 45 publications

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“…The pK a s of the BNC aquo-cofactors have been calculated by Siegbahn and colleagues using the hybrid density functional method (44). Since no other residues are allowed to change ionization, their pK a s are equivalent to pK′ 7 here.…”

Section: Resultsmentioning

confidence: 99%

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

2006

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Cytochrome c oxidase is a transmembrane proton pump that builds an electrochemical gradient using chemical energy from the reduction of O 2 . Ionization states of all residues were calculated with Multi-Conformation Continuum Electrostatics (MCCE) in seven anaerobic oxidase redox states ranging from fully oxidized to fully reduced. One long-standing problem is how proton uptake is coupled to the reduction of the active site binuclear center (BNC). The BNC has two cofactors: heme a 3 and Cu B . If the protein needs to maintain electroneutrality, then 2 protons will be bound when the BNC is reduced by 2 electrons in the reductive half of the reaction cycle. The effective pK a s of ionizable residues around the BNC are evaluated in Rhodobacter sphaeroides cytochrome c oxidase. At pH 7, only a hydroxide coordinated to Cu B shifts its pK a from below 7 to above 7 and so picks up a proton when heme a 3 and Cu B are reduced. Glu I-286, Tyr I-288, His I-334, and a second hydroxide on heme a 3 all have pK a s above 7 in all redox states, although they have only 1.6-3.5 ∆pK units energy cost for deprotonation. Thus, at equilibrium, they are protonated and cannot serve as proton acceptors. The propionic acids near the BNC are deprotonated with pK a s well below 7. They are well stabilized in their anionic state and do not bind a proton upon BNC reduction. This suggests that electroneutrality in the BNC is not maintained during the anaerobic reduction. Proton uptake on reduction of Cu A , heme a, heme a 3 , and Cu B shows ≈2.5 protons bound per 4 electrons, in agreement with prior experiments. One proton is bound by a hydroxyl group in the BNC and the rest to groups far from the BNC. The electrochemical midpoint potential (E m ) of heme a is calculated in the fully oxidized protein and with 1 or 2 electrons in the BNC. The E m of heme a shifts down when the BNC is reduced, which agrees with prior experiments. If the BNC reduction is electroneutral, then the heme a E m is independent of the BNC redox state.Heme-copper oxidases are the terminal electron acceptors in anaerobic organisms. These transmembrane proteins reduce dioxygen and convert the released chemical energy into an electrochemical gradient, across the eukaryotic mitochondrial membrane or the bacterial cell membrane (1-4). Cytochrome c oxidases are the most prevalent hemecopper oxidases. In this protein 4 electrons, provided by 4 cytochromes c, are used to reduce dioxygen to water. The 4 protons needed to make water are taken from the negative cytoplasmic side of the membrane, adding to the electrochemical gradient. Four additional protons are pumped across the membrane (5). Thus, each dioxygen molecule reduced by cytochrome c oxidase is coupled to the transfer of 8 charges across the membrane.The first step of electron transfer is from cytochrome c to Cu A , a dicopper center in subunit II which extends beyond the membrane on the proton release side of the protein (Figure 1). After receiving the electron, Cu A reduces the 6-coordinate, low-spin, bis-Hi...

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Section: Resultsmentioning

confidence: 99%

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

2006

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“…Therefore, we cannot propose the reliable binding mode of the substrate. As proposed previously using molecular simulation (43,44), the binding of phenolic oxygen to Cu B may be necessary as the first process for the tyrosinase reaction.…”

Section: Resultsmentioning

confidence: 99%

Crystallographic Evidence That the Dinuclear Copper Center of Tyrosinase Is Flexible during Catalysis

Matoba

Kumagai

Yamamoto

et al. 2006

Journal of Biological Chemistry

727

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At high resolution, we determined the crystal structures of copper-bound and metal-free tyrosinase in a complex with ORF378 designated as a "caddie" protein because it assists with transportation of two Cu(II) ions into the tyrosinase catalytic center. These structures suggest that the caddie protein covers the hydrophobic molecular surface of tyrosinase and interferes with the binding of a substrate tyrosine to the catalytic site of tyrosinase. The caddie protein, which consists of one six-stranded ␤-sheet and one ␣-helix, has no similarity with all proteins deposited into the Protein Data Bank. Although tyrosinase and catechol oxidase are classified into the type 3 copper protein family, the latter enzyme lacks monooxygenase activity. The difference in catalytic activity is based on the structural observations that a large vacant space is present just above the active center of tyrosinase and that one of the six His ligands for the two copper ions is highly flexible. These structural characteristics of tyrosinase suggest that, in the reaction that catalyzes the ortho-hydroxylation of monophenol, one of the two Cu(II) ions is coordinated by the peroxide-originated oxygen bound to the substrate. Our crystallographic study shows evidence that the tyrosinase active center formed by dinuclear coppers is flexible during catalysis.

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“…It should be noticed that the structure of the transition state is very similar to that previously described by Siegbahn in a DFT-computed mechanism of tyrosinase. [53] In addition, intramolecular arene hydroxylation by a m-1,1-hydroperoxide also finds precedent in the literature. [54] Subsequent O À O bond cleavage and rearomatization should follow to form the catechol product, but computational details for these steps are still under study.…”

Section: Mechanistic Scenariomentioning

confidence: 96%

Electrophilic Arene Hydroxylation and Phenol OH Oxidations Performed by an Unsymmetric μ‐η¹:η¹‐O₂‐Peroxo Dicopper(II) Complex

Garcia‐Bosch

Ribas

Costas

2012

Chemistry A European J

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Reactions of the unsymmetric dicopper(II) peroxide complex [Cu(II)(2)(μ-η(1):η(1)-O(2))(m-XYL(N3N4))](2+) (1 O(2), where m-XYL is a heptadentate N-based ligand), with phenolates and phenols are described. Complex 1 O(2) reacts with p-X-PhONa (X = MeO, Cl, H, or Me) at -90 °C performing tyrosinase-like ortho-hydroxylation of the aromatic ring to afford the corresponding catechol products. Mechanistic studies demonstrate that reactions occur through initial reversible formation of metastable association complexes [Cu(II)(2)(μ-η(1):η(1)-O(2))(p-X-PhO)(m-XYL(N3N4))](+) (1 O(2)⋅X-PhO) that then undergo ortho-hydroxylation of the aromatic ring by the peroxide moiety. Complex 1 O(2) also reacts with 4-X-substituted phenols p-X-PhOH (X = MeO, Me, F, H, or Cl) and with 2,4-di-tert-butylphenol at -90 °C causing rapid decay of 1 O(2) and affording biphenol coupling products, which is indicative that reactions occur through formation of phenoxyl radicals that then undergo radical C-C coupling. Spectroscopic UV/Vis monitoring and kinetic analysis show that reactions take place through reversible formation of ground-state association complexes [Cu(II)(2)(μ-η(1):η(1)-O(2))(X-PhOH)(m-XYL(N3N4))](2+) (1 O(2)⋅X-PhOH) that then evolve through an irreversible rate-determining step. Mechanistic studies indicate that 1 O(2) reacts with phenols through initial phenol binding to the Cu(2)O(2) core, followed by a proton-coupled electron transfer (PCET) at the rate-determining step. Results disclosed in this work provide experimental evidence that the unsymmetric 1 O(2) complex can mediate electrophilic arene hydroxylation and PCET reactions commonly associated with electrophilic Cu(2)O(2) cores, and strongly suggest that the ability to form substrate⋅Cu(2)O(2) association complexes may provide paths to overcome the inherent reactivity of the O(2)-binding mode. This work provides experimental evidence that the presence of a H(+) completely determines the fate of the association complex [Cu(II)(2)(μ-η(1):η(1)-O(2))(X-PhO(H))(m-XYL(N3N4))](n+) between a PCET and an arene hydroxylation reaction, and may provide clues to help understand enzymatic reactions at dicopper sites.

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The catalytic cycle of tyrosinase: peroxide attack on the phenolate ring followed by O-O bond cleavage

Cited by 85 publications

References 45 publications

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

Crystallographic Evidence That the Dinuclear Copper Center of Tyrosinase Is Flexible during Catalysis

Electrophilic Arene Hydroxylation and Phenol OH Oxidations Performed by an Unsymmetric μ‐η¹:η¹‐O₂‐Peroxo Dicopper(II) Complex

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The catalytic cycle of tyrosinase: peroxide attack on the phenolate ring followed by O-O bond cleavage

Cited by 85 publications

References 45 publications

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

Crystallographic Evidence That the Dinuclear Copper Center of Tyrosinase Is Flexible during Catalysis

Electrophilic Arene Hydroxylation and Phenol OH Oxidations Performed by an Unsymmetric μ‐η1:η1‐O2‐Peroxo Dicopper(II) Complex

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Electrophilic Arene Hydroxylation and Phenol OH Oxidations Performed by an Unsymmetric μ‐η¹:η¹‐O₂‐Peroxo Dicopper(II) Complex