Date of Award
8-1-2022
Document Type
Dissertation
Degree Name
Ph.D. in Chemistry
First Advisor
Saumen Chakraborty
Second Advisor
Charles L. Hussey
Third Advisor
Susan Pedigo
School
University of Mississippi
Relational Format
dissertation/thesis
Abstract
Energy crisis, energy demand and climate change are the major challenges of this century. As we face these challenges, it’s important that we depend on alternate energy forms like renewable energy sources and at the same devise ways to tackle the greenhouse effect. Therefore, it is important to move towards a carbon neutral energy economy where we can convert atmospheric carbon dioxide back into organic carbon and depend on clean energy sources like Hydrogen. These energy concerns can be tackled well if we understand the role of enzymes responsible for various biological reactions.Metals play a variety of unexpected roles in biological systems and are essential to the existence of life. Enzymes with metal bound active site that catalyzes reactions are called as metalloenzymes. We are interested in learning about two such complex metalloenzymes, Hydrogenases and Acetyl Coenzyme A Synthases (ACS) which is part of an enzyme complex CODH/ACS. Hydrogenases can catalyze the interconversion of protons into H2 and vice versa at a very low overpotential (<100 >mV). The CODH/ACS enzymes together can reduce CO2 into CO and couple two carbon compounds along with Coenzyme A (CoA) to make an important metabolite Acetyl-CoA. However, the complex nature of these enzymes along with poor yield and oxygen sensitivity restrict their study under normal laboratory conditions. To understand the structural and functional details of these enzymes and to use them for environmental purposes, we use protein engineering and de novo protein design approaches to make small biological model systems called Artificial Metalloenzymes (ArMs). The ArMs are often designed based on the metal active center of the metalloenzymes. We use various spectroscopic techniques to understand metal binding and the hidden mechanism of the catalytic center and electrochemical and photochemical techniques to evaluate the catalytic efficiencies of these systems. This dissertation focuses on the protein reengineering and de novo design approaches to build Artificial Hydrogenases (ArHs) and Artificial Acetyl Coenzyme A Synthases (Ar-ACSs). In, chapter 3, we have redesigned a copper storage protein into a nickel binding protein (NBP) and proved that Ni-NBP is an ArH through electrochemical and photochemical techniques. In chapter 4, we studied the oxygen sensitivity of the NBP through electrochemistry and found that it catalyzes the complete 4e- oxygen reduction reaction at low pH. In chapter 5, we designed a de novo peptide with NiS4 at the active site and studied the incorporation of a synthetic Fe complex to build a bimetallic active site similar to the [NiFe] hydrogenase. In chapter 6, we designed trimeric peptide assembly based on de novo design that makes a NiS3 active site, which binds and couples two carbon compounds (CO and -CH3) making it an Ar ACS enzyme. In chapter 7, we pursued the original research proposal on redesigning a ferredoxin scaffold to build the active center of ACS enzyme and studied the metal and ligand binding through various techniques.
Recommended Citation
Selvan, Dhanashree, "Protein Redesign and De Novo Design Approaches to Create Artificial Nickel-Dependent Metalloenzymes" (2022). Electronic Theses and Dissertations. 2401.
https://egrove.olemiss.edu/etd/2401