Dataset Information

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

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-η² :η² -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.

SUBMITTER: Kipouros I

PROVIDER: S-EPMC9839588 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Kipouros Ioannis I Solomon Edward I EI

FEBS letters 20221006 1

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-η2 :η2 -peroxide dicopper(II) O-O bond to accept the phenolic proton, followed by monophenolate coordination to copper concomitant with ar ...[more]

PMID: 36178078

Similar Datasets

Project description:Cu(A) is a binuclear electron transfer (ET) center found in cytochrome c oxidases (CcOs), nitrous oxide reductases (N₂ORs), and nitric oxide reductase (NOR). In these proteins, the Cu(A) centers facilitate efficient ET (kET > 10⁴s⁻¹) under low thermodynamic driving forces (10-90 mV). While the structure and functional properties of Cu(A) are well understood, a detailed mechanism of the incorporation of copper into the protein and the identity of the intermediates formed during the Cu(A) maturation process are still lacking. Previous studies of the Cu(A) assembly mechanism in vitro using a biosynthetic model Cu(A) center in azurin (Cu(A)Az) identified a novel intermediate X (Ix) during reconstitution of the binuclear site. However, because of the instability of Ix and the coexistence of other Cu centers, such as Cu(A)' and type 1 copper centers, the identity of this intermediate could not be established. Here, we report the mechanism of Cu(A) assembly using variants of Glu114XCuAAz (X = Gly, Ala, Leu, or Gln), the backbone carbonyl of which acts as a ligand to the Cu(A) site, with a major focus on characterization of the novel intermediate Ix. We show that Cu(A) assembly in these variants proceeds through several types of Cu centers, such as mononuclear red type 2 Cu, the novel intermediate Ix, and blue type 1 Cu. Our results show that the backbone flexibility of the Glu114 residue is an important factor in determining the rates of T2Cu → Ix formation, suggesting that Cu(A) formation is facilitated by swinging of the ligand loop, which internalizes the T2Cu capture complex to the protein interior. The kinetic data further suggest that the nature of the Glu114 side chain influences the time scales on which these intermediates are formed, the wavelengths of the absorption peaks, and how cleanly one intermediate is converted to another. Through careful understanding of these mechanisms and optimization of the conditions, we have obtained Ix in ∼80-85% population in these variants, which allowed us to employ ultraviolet-visible, electron paramagnetic resonance, and extended X-ray absorption fine structure spectroscopic techniques to identify the Ix as a mononuclear Cu(Cys)(2)(His) complex. Because some of the intermediates have been proposed to be involved in the assembly of native Cu(A), these results shed light on the structural features of the important intermediates and mechanism of Cu(A) formation.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets