Structure and Dynamics of C-H-Bond-Cleaving High-Valent Iron Intermediates
Open Access
- Author:
- Keller, Rebecca Lynn
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- November 16, 2011
- Committee Members:
- Carsten Krebs, Thesis Advisor/Co-Advisor
Joseph M Bollinger Jr., Thesis Advisor/Co-Advisor
Michael Thomas Green, Thesis Advisor/Co-Advisor - Keywords:
- C-H activation
TauD
Bleomycin
P450 - Abstract:
- C-H bond cleavage is one of the primary tenets of the chemical world. Industry has long searched for more efficient commercial processes to functionalize C-H bonds to activate such molecules as hydrocarbons. Current processes require large amounts of energy and often expensive reagents and often produce a number of increasingly less desirable pollutants such as carbon dioxide. Discovering a process that requires less energy input or is more environmentally neutral is still a very desirable research goal. The natural world already has generated a number of enzymatic processes that can activate C-H bonds at ambient temperatures and pressures. These oxygenase enzymes typically operate at temperatures less than 37 oC and atmospheric pressure. Both heme and non-heme containing oxygenases as well as certain DNA cleaving anti-cancer compounds activate C-H bonds via high valent iron-oxo compounds. A C-H bond activating organic molecule of interest to this document is bleomycin. Bleomycin is a glycopeptide macrocycle used in clinical situations as Blenoxane ® for treatment of a variety of cancers. It coordinates an iron with five nitrogen (or four nitrogen and one oxygen) ligands allowing a sixth coordination site to bind molecular oxygen in a manner similar to heme oxygenases. The clinical function of bleomycin is to bind DNA and activate the C4 hydrogen to generate single and double strand breaks in the chain and ultimately cause the death of the cancerous cell. Here, samples of the activated form of bleomycin have been cryoreduced in an attempt to observe an Fe(IV)=O containing species that has not been previously characterized. The non-heme, taurine:α-ketoglutarate (αKG) dioxygenase (TauD) has been found to activate C-H bonds by generating an Fe(IV)=O species in order to hydroxylate substrate. The ferryl species of TauD has already been well characterized, but recent observations suggested that in the ferryl state, TauD may be able to release and rebind the taurine substrate. Here, substrate exchange during the ferryl and ferrous states of TauD is one main focus of this study. The primary difference between the αKG dependent oxygenase family of enzymes and the halogenase SyrB2 is that SyrB2 contains an alanine rather than a carboxylate ligand in the iron coordinating facial triad. Recent studies have shown that under certain conditions SyrB2 is able to hydroxylate its substrate. The carboxylate residue of TauD is D101, and the D101A variant was generated for studies to determine if TauD D101A is able to bind Cl- and potentially halogenate its substrate. P450s have been studied for more than 50 years and until recently, the Fe(IV)=O intermediate in their reaction had not been characterized. The novel P450 fatty acid hydroxylating peroxygenase from Bacillus subtilis P450BSβ was believed to be a good candidate to observe P450 Compound I, the Fe(IV)=O species that is coupled to a π-cation radical. Characterization of a P450 Compound I from Sulfolobus acidocaldarius rendered the search for a P450 Compound I much less novel. Studies of P450BSβ never produced spectra of a Compound I- like species. However, other observations made through the course of study yielded further insight into the reaction and capabilities of P450BSβ that have not been previously reported.