Cytochrome P450 BM3 is an important model system for mammalian P450s and has great potential for biotechnological applications owing to its high catalytic activity, facilitated by its fused diflavin reductase domain. Mutagenesis studies have shown that Ala-82 and Phe-87 are important in controlling molecular selectivity and regioselectivity in BM3 substrate oxidation, with the A82F, F87V, and A82F/F87V double mutant effectively enhancing the affinity for the human P450 drug omeprazole and related proton pump inhibitor drugs. Herein, resonance Raman spectroscopy is used to probe the heme structure and coordination environments of these BM3 mutants, which are known to afford substantial changes in omeprazole recognition. Specifically, it is demonstrated that binding of omeprazole to these BM3 mutants leads to differing degrees of low spin to high spin state conversion of the heme iron. Furthermore, the ferrous-CO adducts of these proteins are analyzed by resonance Raman to interrogate the effect of substrate binding on the distal pocket architecture, with differing values for the internal modes associated with the Fe-C-O fragment reflecting distinct interactions with distal pocket residues.
|Number of pages||11|
|Journal||Journal of Raman Spectroscopy|
|Early online date||10 Nov 2017|
|Publication status||Published - 1 Feb 2018|