Characterization and catalytic application of an engineered cupredoxin possessing a potential in excess of 1 volt

Kearl, Bryant

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https://hdl.handle.net/2142/32028 Copy

Description

Title

Characterization and catalytic application of an engineered cupredoxin possessing a potential in excess of 1 volt

Author(s)

Kearl, Bryant

Issue Date

2012-06-27T21:28:57Z

Director of Research (if dissertation) or Advisor (if thesis)

Lu, Yi

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

M.S.

Degree Level

Thesis

Keyword(s)

• Azurin
• Laccase
• Cupredoxin
• High Potential
• 1 Volt
• Catalyst
• Polymerization

Abstract

An engineered variant of the electron transfer catalyst Azurin - a cupredoxin cloned from Pseudomonas aeruginosa - has recently been created, in an attempt to demonstrate the power of the secondary coordination sphere constituents in determining the redox potential of a metal center. This new variant, M44F/N47S/F114N/G116F/M121L (henceforth referred to as 5mAz, for brevity’s sake), is an amalgamation of previously characterized secondary coordination sphere mutations known to increase the reduction potential of the active site copper by (primarily) modifying the surrounding hydrogen bonding network. This ensemble of mutations alters bonding interactions and strengths between the C112 and H117 ligands and the copper atom, and in doing so alters both the electron density contributed by the ligand in such a way as to stabilize the reduced, Cu(I) state of the active site. What follows are the results of extensive characterization of this new mutant. While certain aspects of the wild-type protein have not survived such extensive modification (EPR and visual spectroscopy suggests that the active site geometry has been thoroughly altered), the original goal of the construct has been achieved. Years of characterization have yielded data suggesting that this cupredoxin’s heavily engineered active site has a reduction potential now well in excess of 1V, opening up a wide range of new possible roles for this protein, including roles not typically ascribed to a mononuclear, type I cupredoxin. This same cupredoxin, whose reduction potential has been characterized as in excess of 1 volt vs. NHE, appears to promote radical polymerization (in olefins) and oxidative polymerization (in monomers used for the production of conductive polymers). Generation of polymers based on styrene and pyrrole are discussed herein, as well as characterization of those polymers that have proved promising thus far. The protein used appears to have several advantages over a conventional inorganic or organometallic polymerization catalyst, including: activity in the presence of oxygen, activity in water, and activity at room temperature, as well as potentially tactic polymerization capability. These advantages may make a protein-based catalyst of interest when aqueous functionality is necessitated. Moreover, other possible catalytic chemistries of interest are described that have only been discovered in recent weeks, and while not thoroughly characterized, do suggest possibly fascinating new functionalities and redox states imparted by the effects of the highly altered reduction potential and geometry of the active site of this protein. A separate project involving the production of a laccase protein (Streptomyces coelicolor) was undertaken, beginning from a synthetic gene. A protocol for the growth, harvest, and purification of this protein was developed, though optimization of the process is still needed. As laccases are potent oxidizers of organic substrates, show the potential to be used as fuel cell anodes, and contain all 3 common types of cupredoxin active site (type I, II, and III), the effective large-scale production of a modifiable laccase would be useful. This particular laccase is both small (allowing it to have high electrode densities), and multimeric (forming a ring of 3 monomers), with 3 active sites per trimer generated (active sites present at each interface of the individual monomers). Modifications to the interfacial residues and their hydrogen bonding network, hydrophobic interface, and electron transfer network, may prove useful in the design of both binding sites for specific substrates and in improving electron transfer rate for use in fuel cells, both by optimization of the electron transfer route and the reduction potential of the gateway type I copper site.

Graduation Semester

2012-05

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http://hdl.handle.net/2142/32028

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Copyright Bryant Kearl 2012

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