Electronic Theses and Dissertations

Date of Award

1-1-2022

Document Type

Dissertation

Degree Name

Ph.D. in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Dr. Saumen Chakraborty

Second Advisor

Dr. Susan Pedigo

Third Advisor

Dr. Walter Cleland

Relational Format

dissertation/thesis

Abstract

Metals play a crucial role in biology. Metalloenzymes perform various functions in various life sustaining processes. Global energy crisis is a major challenge that the world is facing. The depleting fossil fuels and increasing carbon footprint calls for a need of alternate clean energy sources. Researchers have been working to help alleviate this challenge for decades. Hydrogen gas comes up as a potential candidate which can be used as a biofuel in a hydrogen fuel cell. Hydrogenase is a metalloenzyme that does a two-electron reversible conversion of protons to hydrogen, making it an excellent hydrogen source. However, this native enzyme on its own suffers from poor yield, oxygen sensitivity and present complex experimental challenges. Protein re-engineering and de novo protein design are powerful tools which can be employed to construct Artificial Metalloenzymes (ArM’s) which can mimic the active site and the functionality of these native enzymes. These ArM’s offer an excellent advantage over the native enzymes since they present simpler functional models of the native enzymes which mainly focuses on the active site, are environmentally friendly, comparatively air stable and easier to handle. These ArM’s also help gain deeper understanding of the native enzyme and help elucidate mechanistic details which are sometime hard to understand in the native enzyme.

This dissertation focuses on the construction of functional Artificial Hydrogenases (ArH) using rational protein engineering and protein design approach. Using these techniques, we have constructed a re-engineered protein, a tetrameric coiled coil assembly and a trimeric coiled coil assembly which provide a proper metal binding local environment and act as functional artificial hydrogenases. Mimicking the reductive half of photosynthesis, these ArH’s are studied for photocatalytic hydrogen evolution.

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