Metallomechanisms: Investigation of organometallic catalysts and metalloprotein mechanisms through the use of electron paramagnetic resonance

The E2 domain of amyloid precursor protein (APP) is a copper binding domain of unknown function. We perform spectroscopic analyses, including extended X-ray absorption fine structure (EXAFS), UV-Vis, and electron paramagnetic resonance (EPR) to investigate the E2 domain. The combination of these spectroscopies revealed a labile water ligand in the primary coordination sphere of the Cu(II) cofactor, suggesting E2 has the ability to interact with small molecules in solution. Previous studies have debated the ability of E2 to behave as a ferroxidase, oxidizing ferrous iron into ferric iron, in physiologically relevant conditions. Recent data has suggested that E2 does not bind to Fe(II) nor does it catalyze its oxidation under multiple turnover conditions. We reexamine these results under single turnover conditions using both stopped-flow absorption and EPR to determine that Cu(I)-E2 can catalyze the oxidation of Fe(II) to Fe(III), likely through an outer sphere electron transfer mechanism. This may have been obscured in studies that utilize multiple turnover conditions as Cu(I)-E2 has reacts with oxygen significantly slower than the single turnover rate for ferroxidase activity. In search of oxidants that could more quickly perform the oxidation of Cu(I)-E2 into Cu(II)-E2, we found that Cu(I)-E2 can react with oxidants linked to Alzheimer’s disease, including superoxide and peroxynitrite, with a second order rate constant in the range of 10⁵ M^-1s^-1 while maintaining the integrity of its active site over multiple turnovers. This suggests that E2 plays a crucial role in mitigating oxidative stress through its solvent exposed copper-binding site.

The reduction of peroxynitrite by Cu(I)-enzymes is poorly understood. We utilize the colorometric Griess assay to determine reduction of peroxynitrite forms an equal amount of both nitrite and nitrate in solution indicating the reduction of peroxynitrite occurs via a single electron transfer mechanism. Further, we had interest in understanding the effect that secondary sphere residues have on the ability of E2 to remove peroxynitrite. Mutagenesis at Lys435 impacted the rate of peroxynitrite reduction from solution. Results obtained highlight the impact of secondary sphere interactions, with electrostatic and steric factors modulating substrate recruitment.

While we extensively studied the E2 domain of APP, we have begun to examine the role of other rigidly folded domains in APP, as well as full-length APP. While the D1 and D2 subdomains of APP are reported to bind Cu(II) with similar affinities, we show that the Cu(II)-D2 survives a PD-10 desalting column but Cu(II) is separated from D1 under the same conditions. We have spectroscopically characterized the D2 domain of APP and examined its potential reactivity. In addition to work on the subdomains, we report that APP is capable of binding to three equivalents of copper. Cellular and organismal studies demonstrate that APP potentially acts as a ferroxidase, aiding iron efflux. In Vitro studies on the protein system do not have this activity. The lack of activity could be due to APP being purified in its apo-form. We report that holo-APP has ferroxidase activity with a steady state turnover rate that is similar to the known serum ferroxidase, ceruloplasmin.

In addition to our protein work, we utilized EPR as a tool to study organometallic systems. We utilize EPR to determine the nature of the electronic structure of a series of organometallic complexes including organic radicals, d-block metals, and f-block metals. Additionally, we utilize EPR to study transient intermediates in organocopper systems. We highlight the mechanism of an organocopper catalyst where EPR reveals the presence of a transient paramagnetic Cu(II) species formed after treatment of the starting Cu(I) catalyst with a diazonium salt. This signal instantly disappeared after addition of an alkyl iodide to solution, forming a catalytically relevant organo-Cu(III) species that our collaborators characterize by crystallography and computational work. This study demonstrated the critical pathway for stepwise oxidative addition of substrate to an organocopper catalyst and provided insight into the electronic and structural properties that influence reactivity and stability.