The electronic structures of Copper (II)-substrate activating sites in biology

Copper active sites catalyze a wide range of oxygen reactions in biology. While the majority of enzymes utilize Cu(I) to directly reduce O2, two families of enzymes instead use mononuclear Cu(II) sites to enhance the reactivity of organic molecules toward O2, which is a process known as Cu(II)-substrate activation. These are quercetin 2,4-dioxygenase (QD), which catalyzes the dioxygenation of flavones, and amine oxidase (AO), which utilizes Cu(II) to activate an active site tyrosine residue for a 6e- oxidation reaction to form its redox active cofactor, TPQ. Given the negligible reactivities of the free substrates and of Cu(II) toward O2, mechanistic proposals for these reactions invoke Cu(I)-substrate radical character as the basis for these sites reactivities. In order to evaluate these as well as alternate mechanisms, the electronic structures of these enzymes were first determined along with a number of closely related model complexes with combination of spectroscopic, computational, and kinetic techniques. These experimentally determined electronic structures were then used to calibrate DFT calculations to generate accurate electronic structure descriptions of the QD and AO enzyme-substrate complexes. Finally, these calculated active site models were used to evaluate possible mechanisms for their reactions with O2. Chapter 2 details the electronic structures of the QD enzyme-substrate complexes and a number of related model complexes. Unlike QD, these model complexes decrease the reactivity of bound flavones, and differ from QD primarily by their flavone binding mode. In the model complexes, the flavones bind to the metal in a bidentate fashion with the metal coordinated within the flavone molecular plane, while in QD the flavones bind monodentate with the metal out of the flavone molecular plane. This leads to considerably different electronic structures between the QD enzyme-substrate complexes and the model complexes. In the model complexes, flavone binding to Cu(II) primarily stabilizing the σ-bonding flavone orbitals, while in QD, flavone binding to Cu(II) stabilizes the flavone π-bonding molecular orbitals. Both types of binding geometries lead to moderate flavone-Cu(II) covalencies, but minimal ground state Cu(I)-flavone radical character and no thermally accessible charge transfer states. Calculations of the free flavone, model complex, and enzyme reaction mechanisms show that the models and enzyme increase the barrier for the reaction of O2 directly on the flavone by increasing the flavone ionization potentials. Given the low energy barrier for the O2 reaction performed by the enzyme, the active site Cu(II) must play a direct role in activating O2. In chapter 3, geometric and electronic structures are provided for the Cu(II)-loaded, pre-biogenesis AO active site (preAO). It was first shown that Cu(II) loading into the active site is slow, and that a second, non-catalytic, kinetically favored Cu(II) binding site affects the rate of Cu(II)-active site binding. Cu(II) stably binds to the active site, and prior to the biogenesis reaction is in a thermal equilibrium between two forms. The major form is entropically favored, and is a 4-coordinate site with 3-histidines and a hydroxide as the Cu(II) ligands. The minor form (which is present at 7% at room temperature) is a 5-coordinate site with three histidines, water, and the tyrosinate which processes to TPQ as its ligands. We showed that the minor form is active in biogenesis, and furthermore that this form has a low covalency interaction between the tyrosinate and Cu(II), no discernible Cu(I)-tyrosyl radical character, and no thermally accessible Cu(I)-tyrosyl radical states. Thus, the O2 activation step in biogenesis is performed by a fairly ionic Cu(II)-tyrosinate site. Chapter 4 provides spectroscopic and kinetic analyses of the AO biogenesis reaction. Four intermediates are observed during the biogenesis reaction. First, an intermediate with a 350nm absorbance band grows at the same rate that the minor, preprocessed form disappears. This band has an ε of 2800M-1cm-1, O2-dependent growth, and an O2-independent decay that allow its possible assignment as a dopaquinone intermediate. The 350nm species decays at a rate that is commensurate with the growth of a second intermediate, which has a set of 390nm/410nm bands in absorbance and an organic radical signal in EPR. The small amount of this intermediate that builds up during the biogenesis reaction places it as the minor species of an equilibrium with a Cu(II) form of the active site, and allows its likely assignment as the Cu(I)-organic radical form of trihydroxyphenylalanine, a species which undergoes a 2e- oxidation by reducing a molecule of O2 to H2O2 to become TPQ. Finally, a 330nm band forms at a rate that is commensurate with TPQ formation and then decays at a similar rate. Possible assignments for this species are discussed. A comprehensive kinetic model for the biogenesis reaction provides a lower limit of 1.5s-1 for the initial O2 activating step, giving its energy barrier an upper limit of ∆G^‡=11kcal/mol. This provides a key bench mark for evaluating possible mechanisms of tyrosinate activation for O2 reaction by a Cu(II) center.

Keywords: Chemistry.
Advisor: Solomon Edward I. 
Hodgson K. O. (Keith O.)
Karunadasa Hemamala