Date of Award
1-1-2024
Document Type
Dissertation
Degree Name
Ph.D. in Chemistry
First Advisor
Saumen Chakraborty
Second Advisor
Susan Pedigo
Third Advisor
JONNAH JURSS
Relational Format
dissertation/thesis
Abstract
Metalloenzymes catalyze various redox reactions in biogeochemical cycles involved in energy conversion. Our research focuses on Cu-based metalloenzymes, which perform diverse functions in nature and play a crucial role in substrate activation, energy conversion such as oxidizing methane to methanol by particulate methane monooxygenases (pMMOs), and biomass degradation by lytic polysaccharide monooxygenases (LPMOs), useful in biofuel production. However, much is unknown about how the active site and secondary/tertiary-sphere interactions in these enzymes determine their function. De novo protein design enables the creation of simpler model systems that closely resemble the structure and function of native enzymes. Using de novo protein design, we have created trimeric (3SCC) and tetrameric (4SCC) artificial Cu proteins (ArCuPs), determined their x-ray structures, and investigated the reactivity toward O2 and H2O2. The kinetics of O2/H2O2 activation along with resonance Raman, and EPR suggest that 3SCC produces the putative Cu-OOH species, while the reactivity with 4SCC is much slower. The reaction is accelerated under reducing conditions, and the reactivity is faster with H2O2 than with O2. The 3SCC catalyzes peroxygenation and oxidation of C-H bonds by electrochemically generated Cu-oxygen species. The catalytic proficiency is significantly improved by modulating the outer-sphere steric that impacted the catalytic outcome by tuning the kinetics, the nature of the intermediate produced, redox potentials, and the stability of the constructs. QM/MM studies support homolytic O-O bond cleavage and helped us elucidate the overall mechanism for CuII-O• formation and its subsequent H-atom abstraction capability from C-H substrates. Electrochemistry was used to determine the reorganization energies of these constructs, which shows that 4SCC has a higher reorganization energy that is primarily governed by water-mediated H-bonding. Importantly, this outer-sphere solvent contribution can be tuned by the deletion of H-bonding interactions. The chemistry of ArCuPs draws close relevance to LPMO and pMMO CuB sites. Single-chain helical bundles were developed using deep-learning tools such as Alphafold2, ProteinMPNN, and Rosetta, followed by high-throughput methods to evaluate their folding and catalytic properties. This work leverages the capabilities of de novo metalloprotein design toward developing an artificial class of enzymes that advances bioinorganic chemistry and protein design research.
Recommended Citation
Prakash, Divyansh, "De Novo Design Approaches Toward the Development of Artificial Cu Proteins (ArCuPs) for Activation of Small Molecules with Functional Relevance to Cu Monooxygenases" (2024). Electronic Theses and Dissertations. 2861.
https://egrove.olemiss.edu/etd/2861