The cytochromes P450 (CYPs) are a superfamily of biological catalysts that are ubiquitous throughout the biological domain. CYPs are heme-b containing monooxygenases which oxidize substrates with the help of accessory redox partners. CYP substrates include endogenous compounds required for many biological functions and homeostasis, such as steroids, as well as the majority of clinically used drugs and environmental xenobiotics. The majority of studies that have been performed to date are on P450cam (CYP101) from Pseudomonas putida. Of the numerous reactions catalyzed by CYPs, unactivated carbon-carbon bond cleavage, is one of particular versatility. Being unique in their catalytic mechanisms, the C-C bond cleaving enzymes and in particular CYP51 from Mycobacterium tuberculosis are though to be capable of utilizing multiple reactive oxygen intermediates. During the process of C-C bond cleavage, CYP51 catalyzes two classical hydroxylation reactions. The final reaction culminates in an enigmatic third step which cleaves a C-C bond, liberates formate, and installs a 14,15 double bond within its steroid substrate. The mechanism of CYP51s final step is still unclear and the exact activated oxygen species has yet to be observed. CYP51 is also distinct from most CYPs owing to the fact that the acid functionality of the conserved active site “acid-alcohol pair” found in most CYPs, is replaced by a histidine. This study aimed to trap and characterize dioxygen reactive intermediates, and elucidate the role of the unique acid-alcohol pair in the formation and stabilization of these intermediates. This study demonstrates our success in generating, stabilizing, and spectroscopically characterizing reactive dioxygen intermediates in Mtb CYP51. As the life-time of the oxyferrous intermediate in Mtb CYP51 is extremely short at ambient temperatures, this work has shown the laboratory’s expertise in being able to generate reduced oxyferrous intermediates at cryogenic temperatures. These intermediates have only been generated in a handful of cytochromes P450 and as such this work adds critical information to the small body of work currently reported.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-1440 |
| Date | 23 October 2012 |
| Creators | Jennings, Gareth Kent |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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