Investigating the role of secondary coordination sphere in designed metalloenzymes and engineering protein interactions for enhanced electron transfer

Metalloproteins are essential to biological systems. They define a large class of proteins that contain a variety of metals such as copper, iron, manganese and zinc, which are commonly found in nature. The complexity of metal centers can range from single ion to extremely complex cofactors such as those found in nitrogenase. These metal sites play essential structural and/or catalytic roles that shape the protein’s natural functions. Through evolutionary processes, proteins fold in unique ways to stabilize the binding and to control the properties of their cofactors. Both primary and secondary coordination spheres control metal geometries and redox properties, thus making them powerful tools in metalloprotein engineering. With metalloproteins that are involved in catalysis, electron transfer is the key to enzyme function. Many proteins in biological systems rely on their native redox partners for electron source. Being able to control the partners’ interaction allows for regulation of electron transfer and, thus, the rate of catalysis. The first goal of this thesis is to understand the role of secondary coordination sphere in a variety of designed metalloproteins and how it regulates their function. The second goal of this work is to enhance the rate of electron transfer between the redox pair through redesign of the protein interface. This way, we explore both factors controlling catalysis in our designed metalloproteins: secondary coordination sphere at the active site and interaction between redox partners. In chapter 2, we redesign the interface between CuB Myoglobin and its redox partner, cytochrome b5, to maintain their electron transfer at various ionic strength buffers. This would then help this system overcome the ionic strength barrier it faces and broadens its potential applications. In chapter 3, we investigate the role of water network at the active site in controlling the oxidase activity of CuB Myoglobin through x-ray crystallography. In chapter 4, we apply the knowledge learned from CuB Myoglobin to designing oxidase activity in cytochrome c peroxidase. If key features identified in CuB Myoglobin are incorporated into another scaffold, we can test to see if these additions give rise to oxidase activity. The success of this work would confirm our understand of how CuB Myoglobin work. In chapters 5 and 6, we look at how secondary coordination sphere in both SiRCcP and Azurin tune the metal’s redox potentials, respectively. We will use EPR spectroscopy to help with this investigation, primarily ESEEM and HYSCORE techniques.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2022

Author dc:creator

• Lam, Quan

Contributors dc:contributor

• Lu, Yi
• Nair, Satish
• Gennis, Robert R
• Procko, Erik

• protein interaction
• redox potential
• artificial oxidase
• protein engineering
• ESEEM
• HYSCORE
• oxygen reduction
• sulfite reduction

Statement dc:rights

• Copyright 2022 Quan Lam

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/115737

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F115737