Copper(I)-phenolate complexes as models of the reduced active site of galactose oxidase: Synthesis, characterization, and O2 reactivity

Brian A. Jazdzewski, Anne M. Reynolds, Patrick L. Holland, Victor G. Young, Susan Kaderli, Andreas D. Zuberbühler, William B. Tolman

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Abstract

The Cu(I)-phenolate complexes 1LCu and 2LCu and the Cu(I)-phenol complex [H2LCu(CNC6H3Me2)] BArF4 were prepared and structurally characterized by X-ray crystallography, where 1L- and 2L- are ligands comprised of a 2,4-di-tert-butylphenolate linked to 1-isopropyl-1,5-diazacyclooctane or 1,4-diisopropyl-1,4,7-triazacyclononane, respectively. The reduced galactose oxidase (GAO) structural models 1LCu and 2LCu were found to be highly reactive with O2, and through combined stopped-flow kinetic and EPR, UV-vis, and resonance Raman spectroscopic studies of the oxygenation of 2LCu at low temperature, new intermediates relevant to those postulated for the active site oxidation step of the GAO catalytic cycle were identified. The oxygenation was shown by kinetics experiments to proceed via initial binding of O2 to yield a green, unusually thermodynamically stable 1:1 adduct, 2LCu(O2). Symmetric (η2) binding of a superoxo ligand was indicated by oxygen-isotope-sensitive features in resonance Raman spectra obtained in batch experiments; peaks at v(16O2) = 1120 cm-1, v(18O16O) = 1093 cm-1, and v(18O2) = 1058 cm-1 were assigned as O-O stretching vibrations. These data represent the first experimental evidence for such superoxide coordination in complexes of tetradentate tripodal ligands and provide new precedent for how O2 may bind at the reduced GAO active site. The 1: 1 Cu/O2 adduct subsequently evolves into a metastable purple species that is only observable under conditions of substoichiometric O2. The kinetics of formation of this transient species are second order overall (rate = k2[2LCu(O2)] [2L1Cu]). It exhibits an absorption band with λmax = 565 nm (ε = 17,900 M-1 cm-1) and multiple oxygen-isotope-sensitive v(Cu-O) and v(O-O) features in the respective regions 500-550 cm-1 and 700-850 cm-1 in Raman spectra, with excitation-wavelength-dependent intensities that correlate with the 565 nm absorption feature. On the basis of the combined data available, the presence of multiple isomeric peroxodicopper species in the transient purple solution is postulated.

Original languageEnglish (US)
Pages (from-to)381-393
Number of pages13
JournalJournal of Biological Inorganic Chemistry
Volume8
Issue number4
DOIs
StatePublished - Apr 2003

Bibliographical note

Funding Information:
Acknowledgements We thank the NIH (GM47365 to W.B.T.), the NSF (predoctoral fellowship to A.M.R.), and the Swiss National Science Foundation (20-58958.99 to A.D.Z.) for funding this research. Also, we thank Mark Mehn for assistance with obtaining Raman spectra, Prof. Doyle Britton and Dr. Neil Brooks for helping to interpret X-ray crystallographic data, and Profs. Lawrence Que, Jr. and John Lipscomb for providing access to the Raman and EPR instrumentation, respectively.

Keywords

  • Copper
  • Cryogenic stopped flow
  • Dioxygen
  • Galactose oxidase
  • Superoxo complex

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