Understanding efficient O-O bond cleavage chemistry from heme-copper oxidase enzymes and model complexes

Heme-copper oxidase enzymes (HCOs) catalyze the selective 4 e--/4 H+ reduction of O2 to H2O, employing a binuclear active site that consists of a heme Fe and a Cu center. Motivated by the importance of O2 reduction for renewable energy applications such as fuel cells, efforts to understand HCO chemistry span a wide range of areas including catalysis, inorganic/coordination chemistry, biochemistry, spectroscopy, and theory. This research therefore aims to elucidate how the heme-Cu active site catalyzes O2 reduction with a low kinetic barrier. The studies present in this thesis have employed a wide range of spectroscopic techniques (including magnetic, vibrational, electronic, and X-ray methods) in combination with computation (DFT), working to characterize model complexes and enzyme intermediates, understand spectral features, develop structure-function relationships, and explore possible reaction coordinates. Based on a number of synthetic and computational model studies, it is generally believed that the rate-limiting step in O2 reduction by HCOs is O-O bond cleavage, and that the reaction proceeds through a transient intermediate wherein a peroxo moiety is bridging the active site Fe(III) and Cu(II). Invoking this "bridging peroxo" species as central to the process of O2 reduction, extensive effort has aimed to understand key factors responsible for enabling a low barrier to O-O bond cleavage, particularly relying on model chemistry. As it is believed that a tyrosine residue in HCOs participates in the O-O bond breaking step by donating a H+/e-- pair, the majority of synthetic model studies have employed phenolic substrates, and investigations with separate H+ and e-- donors have been applied herein to understand the necessity and sequentiality of the net H-atom transferred. DFT evaluation of phenol-induced O-O cleavage in a heme-peroxo-Cu complex identifies two possible reaction mechanisms, which differ by whether proton transfer (PT) occurs before or after the transition state. Insights from a combination of experiment and theory determine the active mechanism and establish key factors in the chemistry involved, including the roles of the H+ and e-- in O--O bond cleavage, and the pivotal impact of H-bonding. Applying these findings to further understand their importance in O2 reduction, several subsequent studies with synthetic models have explored H-bonding effects and the PT-ET nature of this reaction. Carrying the work with synthetic models to the enzyme active site, DFT calculations were performed to consider O-O cleavage in HCOs, where only a few intermediates are observed experimentally. Compared to the phenol-reactive model complex above, it is shown how the active site structure is engineered to not only favor a different mechanism, but also perform O--O cleavage with a lower energetic barrier. While several possible mechanisms are again identified, these are found to generate different product structures. Importantly, the species following O-O cleavage is a semi-stable intermediate called "PM"), presenting an opportunity to correlate the calculations with available experimental data. To this end, a method has been developed to generate PM in a specific HCO (ubiquinol oxidase) that lacks an additional Cu redox center, making this critical intermediate accessible by a range of site-selective spectroscopic methods. These studies will contribute to a greater understanding of how HCOs accomplish efficient O2 reduction throughout aerobic biology.

Keywords: Chemistry.
Advisor: Solomon Edward I. 
Dai Hongjie
Hodgson K. O. (Keith O.)