Date of Award

Spring 2009

Document Type

Dissertation - Restricted

Degree Name

Doctor of Philosophy (PhD)


Biomedical Engineering

First Advisor

Kincaid, James R.

Second Advisor

Sem, Daniel S.

Third Advisor

Ryan, Michael D.


Substrate binding in Cytochrome P450 results in the activation of a new mode near 367 cm-1 in the low frequency resonance Raman spectrum. This substrate-activated mode had been previously assigned as a second "propionate bending" mode, arising in addition to the single propionate bending mode observed for the substrate-free form at 380 cm-1. In the present work, results of resonance Raman studies of the substrate-free and camphor-bound cytochrome P450cam and its isotopically labeled analogues that have been reconstituted with protoheme derivatives that bear -CD3 groups at the 1,3 ,5 and 8-positions (dl2-protoheme) or deuterated methine carbons (d4-protoheme) are discussed. This newly activated mode is observed to shift by 8 cm-1 to lower frequency in the d12-protoheme reconstituted enzyme (i.e., the same shift as that observed for the higher frequency "propionate bending" mode) and is therefore consistent with the suggested assignment. However, the newly acquired data for the d4-protoheme substituted analogue also support an earlier alternate suggestion that substrate binding activates several heme out-of-plane modes, one of which ( Y6) is accidentally degenerate with the 367 cm-1 propionate bending mode. In its catalytic cycle, Cytochrome P450 binds and cleaves molecular oxygen to generate a yet to be characterized potent intermediate capable of hydroxylating even relatively inert hydrocarbon substrates. Characterization of this fleeting intermediate by resonance Raman spectroscopy has proven elusive. Utilization of a chemically inert substrate for Cytochrome P450cam (P450cam) might perhaps increase the lifetime of certain fleeting intermediates in the P450cam enzymatic catalytic cycle, possibly facilitating their subsequent characterization by resonance Raman spectroscopy. In this work the binding ofF-adamantanol as an inert substrate for Cytochrome P450cam are summarized.



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