Publication Date


Document Type


First Advisor

Erman, James E.

Degree Name

M.S. (Master of Science)

Legacy Department

Department of Chemistry and Biochemistry


Peroxidase; Enzymes--Oxidation; Hemoproteins; Hydrogen peroxide


Heme enzymes catalyze the oxidation of a wide variety of substrates utilizing either molecular oxygen or hydrogen peroxide as oxidants. There is some interest in utilizing heme enzymes in the synthetic laboratory to introduce oxygen functionalities into organic structures due to their specificity and low levels of side reactions. The most versatile oxygenation catalysts are a group of monooxygenases called the cytochrome P450s. However, the cytochrome P450s are not ideal synthetic catalysts since the monooxygenase reaction is quite complex, requiring a co-factor, either NADH or NADPH, and an additional enzyme called P450 reductase. In addition, the cytochrome P450 reactions are typically slow and the enzymes prone to oxidative degradation. Attention has been given to the peroxygenase activity of heme proteins, the incorporation of an oxygen atom from hydrogen peroxide into an organic substrate. The peroxygenase reaction eliminates the need for NADH or NADPH and the P450 reductase. Although peroxygenation is not a typical reaction catalyzed by heme enzymes, many heme proteins have low levels of peroxygenase activity. This thesis explores the peroxygenase activity of the well-studied heme protein, cytochrome c peroxidase (CcP). CcP is an ideal candidate for exploring the peroxygenase activity of heme proteins and for the development of robust peroxygenation catalysts since it reacts rapidly with hydrogen peroxide and is very stable toward oxidative degradation. Protein engineering studies allow us to enhance the peroxygenase activity of CcP by creating a more apolar environment to facilitate the binding of organic substrates near the heme. Three CcP mutants with apolar heme pockets were constructed by converting Arg48, Trp51, and His52 simultaneously to all alanines (CcP(triAla)), all valines (CcP(triVal)), or all leucines (CcP(triLeu)). Styrene and acrylonitrile epoxidation reactions and 1-methoxynaphthalene hydroxylation reactions were studied using wild-type CcP and CcP mutants to investigate the peroxygenase activity of these enzymes. CcP Compound I oxidizes styrene and acrylonitrile with observed bimolecular rate constants of (4.6 ± 4.1) x 10 ⁻⁴ M⁻¹ s⁻¹ and (4.3 ± 0.3) x 10⁻³ M⁻¹ s⁻¹, respectively. The rate of the peroxygenation of acrylonitrile catalyzed by CcP is similar to that of the monooxygenase oxidation reaction of acrylonitrile catalyzed by rat liver microsomal P450. The CcP triple mutants hydroxylate 1-methoxynapthalene at rates that are about 30 times faster than wild-type CcP. However, the turnover rates of 0.13 to 0.15 min⁻¹ are small when compared with the 10³ min⁻¹ rates of the monoxygenase activity of some bacterial P450s. A fourth CcP mutant, meant to mimic the heme coordination of cytochrome P450, CcP(H175C/D235L), had a smaller rate of naphthalene hydroxylation than the CcP triple mutants. Unfortunately, all of the CcP mutants had increased rates of heme degradation during the hydroxylation reaction compared to wild-type CcP. Further development of CcP as a peroxygenation catalyst will need to focus on the stability of the enzyme as well as increasing the rate of substrate turnover.


Advisors: James E. Erman.||Committee members: Gary M. Baker; James E. Erman; James R. Horn.||Includes bibliographical references.||Includes illustrations.


ix, 94 pages




Northern Illinois University

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