Files in this item

FilesDescriptionFormat

application/pdf

application/pdfDWARAKNATH-DISSERTATION-2021.pdf (6MB)Restricted Access
(no description provided)PDF

Description

Title:Engineering an oxygen storage metalloprotein into carbon dioxide and oxygen reduction metalloenzymes
Author(s):Dwaraknath, Sudharsan
Director of Research:Lu, Yi
Doctoral Committee Chair(s):Lu, Yi
Doctoral Committee Member(s):Gennis, Robert B; Gewirth, Andrew A; Vura-Weis, Joshua
Department / Program:Chemistry
Discipline:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):artificial metalloenzymes
oxygen reduction reaction
carbon dioxide conversion
heme copper oxidase
biosynthetic modeling
protein engineering
Abstract:Metalloenzymes catalyze reactions that are among the “holy grails” of modern chemical research and do so at high rates and selectivity and low overpotential. While our understanding of the mechanistic strategies of nature’s catalysts remains an active and expanding area of study, several salient features of biocatalysis have been observed repeatedly. Natural systems climb steep energy landscapes through energy transduction, exemplified by the conversion of light energy to reducing power in photosystem I. Metalloenzymes utilize efficient proton transfer pathways to coordinate the timely movement of electrons and protons across large distances, despite the 2000-fold mass difference between the two, as in the D-channel in heme-copper oxidases (HCOs). Finally, biology’s catalysts feature elaborate active sites featuring heteronuclear metal centers and amino acid secondary coordination spheres oriented to site-specifically activate substrates and deliver protons and electrons, such as the NiFe4S4 center that interconverts CO2 and CO in carbon monoxide dehydrogenase. However, nature’s catalysts were not evolved to be exploited by human civilization, and so they fall short in several key performance areas. The large size, tedious purification, limited thermal and chemical stability, and narrow substrate scope are select examples of how native proteins are unamenable for many industrial and even research scale applications. Protein engineering seeks to reproduce and even exceed the catalytic activity of native enzymes using artificial protein scaffolds that are free of the shortcomings of their native counterparts. In Chapter 1, this thesis surveys 21st-century advances in the field of metalloprotein design and engineering. In Chapters 2-5, I report our progress in engineering the O2 storage protein, myoglobin, into artificial metalloenzymes that catalyze the reduction of O2 and CO2. Nature tasks heme-copper oxidases with the delicate job of delivering 4H+ and 4e- to O2 in order to reduce it fully to water. Failure to coordinate the timely and site-specific delivery of the two will yield reactive oxygen species that can reign havoc on the cell and bring life to an end. In almost all terminal oxidases, 3 of the 4 electrons are delivered by the copper and heme cofactors and the last electron is provided by a tyrosine (Tyr). To deconvolute the role of the heme, copper, and Tyr on oxidase chemistry, the Lu Group previously engineered myoglobin into a series of structural and functional biosynthetic models of HCO. Despite the critical role of Tyr in HCOs, there is one terminal oxidase, bd oxidase, which defies the idea that a Tyr is required for ORR, because it lacks an active site Tyr. Interestingly, instead of a Tyr, there is a highly conserved (>99%) tryptophan (Trp) residue located in the proposed O2 reduction site. This raises the possibility that biology has substituted the active site Tyr found in all other terminal oxidases for a Trp residue in bd oxidase. We reasoned that our biosynthetic model would be a facile system in which to test this possibility. To explore this possibility, and to compare and contrast the properties of Tyr and Trp residues in functional proteins, we have modified our biosynthetic models of HCOs by mutating the active site Tyr to a Trp residue. Through mutational, crystallographic, heme redox potential, EPR spectroscopic and catalytic activity analysis, we demonstrate that Trp can provide 1 of the 4 electrons required for the complete reduction of O2 to water. We discovered that a single Leu to Asp mutation transforms the O2 storage protein, myoglobin, into an oxidase with 80% selectivity for product water over-reactive oxygen species. Remarkably, the single mutation is in the proximal heme pocket, far from the O2 binding face of the heme. To explain this surprising finding, we elucidated the mechanism by which Asp89 switches on oxidase function in myoglobin. Through mutational, spectroscopic, crystallographic and activity analysis, we determined that WT Mb inhibits O2 reduction by insulating the O2 binding site from protons. In contrast, the L89D mutation facilitates proton transfer into the O2 binding site, unlocking a key proton-coupled electron transfer step (PCET) that initiates O2 reduction. Inspection of mutant myoglobin crystal structures inspired the hypothesis that the heme propionates shuttle protons from the proximal Asp89 to the O2 binding site. We rationally designed a series of mutants to test this theory and found that one variant exhibited a 4-fold increase in oxidase activity compared to the original mutant. Mutation of the Asp to an Asn, which is expected to hamper proton transfer, muffled the oxidase activity. Our results suggest that the heme propionates can mediate proton transfer for catalysis, opening up a new modality by which nature coordinates critical PCET steps. Having extended the function of myoglobin from O2 storage to O2 reduction, we sought to push its catalytic competency even further to the non-native and inert substrate, CO2. Initial investigations revealed that the low reduction potential of the native heme b makes it challenging to access low metal oxidation states that have been shown to facilitate CO2 reduction in small molecule catalysts. We replaced the native Fe porphyrin with its Co analogue and demonstrated that Co myoglobin performs homogenous CO2 reduction to CO. The enzyme exhibits both high substrate selectivity for CO2 over protons and product selectivity for CO over hydrocarbons. By borrowing active site mutations shown to organize a second metal ion above the heme cofactor, we explored whether a heterobimetallic center could outperform the catalytic turnover frequency of the mononuclear cobalt enzyme. In addition, we studied whether secondary sphere residues designed to organize a hydrogen-bonding network in the active site could enhance catalysis. Our first-generation biocatalysts exceed the turnover frequency of all other reported artificial CO2-to-CO conversion metalloenzymes.
Issue Date:2021-02-24
Type:Thesis
URI:http://hdl.handle.net/2142/110770
Rights Information:Copyright 2021 Sudharsan Dwaraknath
Date Available in IDEALS:2021-09-17
Date Deposited:2021-05


This item appears in the following Collection(s)

Item Statistics