Substrate binding in catechol oxidase activity: biomimetic approach.
A series of dicopper(II) complexes have been investigated as model systems for the catechol oxidase active site enzyme, regarding the binding of catechol substrate in the first step of the catalytic cycle. The [Cu(2)(L(R))(mu-OH)](ClO(4))(2) and [Cu(2)(L(R))(H(2)O)(2)](ClO(4))(3) complexes are based on the L(R) ligands (2,6-bis[(bis(2-pyridylmethyl)amino)methyl]-4-R-substituted phenol) with -R = -OCH(3), -CH(3), or -F. Binding studies of diphenol substrates were investigated using UV-vis and EPR spectroscopy, electrochemistry, and (19)F NMR (fluorinated derivatives). All the complexes are able to bind two ortho-diphenol substrates (tetrachlorocatechol and 3,5-di-tert-butylcatechol). Two successive fixation steps, respectively fast and slower, were evidenced for the mu-OH complexes (the bis(aqua) complexes are inactive in catalysis) by stopped-flow measurement and (19)F NMR. From the mu-OH species, the 1:1 complex/substrate adduct is the catalytically active form. In relation with the substrate specificity observed in the enzyme, different substrate/inhibitor combinations were also examined. These studies enabled us to propose that ortho-diphenol binds monodentately one copper(II) center with the concomitant cleavage of the OH bridge. This hydroxo ligand appears to be a key factor to achieve the complete deprotonation of the catechol, leading to a bridging catecholate.
Torelli
Stéphane
S
LEDSS/Chimie Biomimétique, UMR CNRS 56160, Université J. Fourier, B.P. 53, 38041 Grenoble Cedex, France.
Belle
Catherine
C
Hamman
Sylvain
S
Pierre
Jean-Louis
JL
Saint-Aman
Eric
E
D001665:Binding Sites; D002384:Catalysis; D004156:Catechol Oxidase / Q000737:chemistry* / Q000378:metabolism; D003300:Copper / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009942:Organometallic Compounds / Q000138:chemical synthesis* / Q000737:chemistry; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship; D014867:Water / Q000737:chemistry
D008024:Ligands; D009942:Organometallic Compounds; D014867:Water; D003300:Copper; D004156:Catechol Oxidase