Diagnostic radical clock probes for heme and non-heme monooxygenases

Heme monooxygenases (cytochrome P450s) are ubiquitous enzymes found in all forms of life, with a variety of roles. The goal of this thesis was the exploration of the reactions of cytochrome P450s, along with models for their reaction---metalloporphyrins---and non-heme monooxygenases, using radical clock probes to study the mechanism. In the oxidation of the radical clock tetramethylcyclopropane (TMCP) with various metalloporphyrins, an oxygen-trapped product and desaturation products were identified. Although desaturation by cytochrome P450s and non-heme iron monooxygenases is common, it is a rarely reported reactivity for metalloporphyrins. The structural and steric constraints of desaturation by metalloporphyrins were explored by looking at a variety of branched alkanes and unbranched alkanes. The branched alkanes were desaturated and unbranched ones were not. The steric component thus seems important, and the iso-propyl motif seems especially vulnerable to desaturation.;The new fluorine-containing probe 1,1-diethyl-2,2-difluorocyclopropane (F2-DECP) and expected products were synthesized. The radical ring-opening of the difluorinated cyclopropyl ring occurs 100 times faster than that of unfluorinated analogues, while fluorine is the same size as hydrogen and therefore does not change the sterics. In the reaction of F2-DECP with the non-heme iron enzyme toluene-4-monooxygenase, the major product was the unrearranged alcohol, along with 1.7% radical rearranged alcohol. The extent of rearrangement was less than expected compared to the unfluorinated analogue, which argues for an effect of the fluorines beyond the acceleration of ring-opening.;TMCP was reacted with three drug-metabolizing cytochrome P450 enzymes (human CYP3A4 and CYP2E1 and rat CYP2B1). The results show that there are definite differences between P450s.;The non-heme enzyme AlkB was investigated (for the first time in a purified form) using the diagnostic radical clock probe norcarane and the deuterated analog 3,3,4,4-norcarane-d4. The deuteration revealed a kinetic isotope effect of 20, making significant contribution from hydrogen tunneling likely for the mechanism of C-H abstraction. The product distribution included desaturation products and showed that desaturation occurred only from the C3 radical. The results were consistent with whole cell studies from the literature.