Expanding the chemistry of a cupredoxin by designing nonnative active sites and using abiological metals

Metalloproteins are an important class of proteins, comprising nearly half of all native proteins. Due to their wide variety of chemical properties, metals are utilized as cofactors for electron transfer, oxidative or reductive transformations, acid-base reactions, and maintaining protein structural stability. Copper is found in both electron transfer proteins, most notably cupredoxins, and enzymes that catalyze oxidative reactions, such as copper-dependent monooxygenases. Cupredoxins are single-domain β-barrel proteins that contain a mononuclear type 1 copper binding site and typically have a strong blue color in their oxidized form due to the LMCT between a cysteine sulfur and copper(II). Cupredoxins make efficient electron transfer partners because of their low reorganization energy imparted by rigged tertiary structure of the protein. Copper-dependent monooxygenases, such as lytic polysaccharide monooxygenase (LPMO) and particulate methane monooxygenase (pMMO), catalyze the hydroxylation of organic substrate using a copper cofactor and a small molecule oxidant, such as dioxygen or hydrogen peroxide. In contrast with iron-dependent monooxygenases, copper monooxygenases such as LPMO and pMMO are not as well characterized and there is still debate over reactive intermediate responsible for the oxidative transformation. As a supplementary method to studying these native proteins, designing artificial metalloproteins can serve two important functions: 1) allowing characterization and study of recreated metalloenzyme active sites in ways not possible their native protein and 2) using nonnative and abiological amino acids, metals, or cofactors to expand the possible chemistry beyond what can be done in nature. In an effort to explore new electron transfer properties of artificial cupredoxin-like metalloproteins, a mutant of the blue copper protein azurin (Az) was metalated with chromium and its novel properties were studied. Kinetic studies were performed on electron transfer reactions between Cr-Az, Cu-Az, and metal complexes such as hexaaquachromium(II) chloride and potassium ferricyanide. Cr-Az was found to retain the efficient electron transfer properties of native azurin, with faster electron transfer rates compared to the aqueous chromium(II) ion and activation parameters similar to Cu-Az. Evidence suggests that the reaction between Cr-Az and ferricyanide occurs through an inner-sphere electron transfer mechanism in contrast to native azurin which is thought to only react through outer-sphere mechanisms. Cr-Az was also found to react with small molecules such as sulfite, nitrite, and azide and with organic halides, showing that Cr-Az retains the reactivity of aqueous chromium(II) despite having a higher reduction potential. Copper coordination by an N-terminal histidine through the side chain and amine along with another histidine side chain was coined a “histidine-brace” and is found in the active site of LPMO and a metal binding site of pMMO. The His-brace site in pMMO was once thought to be the active site, but new evidence shows it is not. The function of the His-brace site in pMMO is still remains unknown, however. In order to study a unique His-brace copper binding site found in both LPMO and pMMO, azurin was engineered to mimic the copper sites of these proteins and its structure and functions were characterized. His-brace azurin with three histidines (HB3Az) was found to bind two equivalents of copper, with one in the engineered site and one in the native type 1 site, and have an EPR spectrum consistent with a type 2 copper center as seen in pMMO and LMPO. Cu-HB3Az is not reactive towards C-H activation but rather has peroxidase-like activity. Treating Cu(II)-HB3Az with hydrogen peroxide and organic substrate, such as the dyes and thioethers, results in oxidation of the substrate. Short chains of polyaniline were produced when aniline was used as substrate. Another mutant that mimics the site in LPMO with two histidines (HB2Az) was found to not react with peroxidase-like activity but did form a stable peroxo-adduct when treated with hydrogen peroxide. Metals are typically coordinated by proteins through amino acid side chains of histidine, aspartic acid, glutamic acid, cysteine, and methionine. Coordination by tyrosine, arginine, lysine, or amide backbone has also been observed but is not as common. The diversity of biological ligands pales in comparison to the numerous ligands used in synthetic inorganic chemistry. One type of ligand that is notably absent from the biological repertoire is carbon-coordinating ligands found in organometallic chemistry. It has been theorized that histidine can coordinate to a metal through carbon to form a metal-N-heterocyclic carbene (NHC) complex, based on experimental results with imidazole analogs and DFT studies. Efforts were made towards the goal of characterizing a metal-NHC complex in a protein using UV-Vis spectroscopy, X-ray crystallography, EXAFS, mass spectrometry, vibrational spectroscopy, and NMR.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2020

Author dc:creator

• Harnden, Kevin Allen

Contributors dc:contributor

• Lu, Yi
• Fout, Alison R
• Vura-Weis, Joshua
• Olshansky, Lisa

• Artificial metalloprotein
• Bioinorganic chemistry
• Protein design
• Cupredoxin
• Azurin

Statement dc:rights

• Copyright 2020 Kevin Harnden

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/108266

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/108266