Heme proteins incorporate iron to carry out complex chemistries, such as transfer of electrons, carrying or storage of oxygen, and synthetic modifications of hydrocarbons. Essential to these processes is iron’s ability to transition between various oxidation states. As iron cycles through these states, the heme and protein active site geometry and chemistry is changed, which has large implications for enzyme stability and lifetime. Little is known about the impact of oxidation state in Cytochrome P450s (CYPs), which play a role in many important chemical transformations, including steroid synthesis and xenobiotic metabolism in mammals. The stability of CYPs is commonly assessed in the Fe(III) resting state, but the enzyme must be reduced to the Fe(II) state to initiate catalysis. This study focused on determining if iron oxidation state plays a role in the differences in stability for selective vs. promiscuous P450s. Cytochrome P450BM3 (CYP102A) was used as a model system as it is a selective enzyme, but can become highly promiscuous with only a few mutations. A range of biophysical techniques, including UV/Vis, circular dichroism, and pulse proteolysis were used to investigate active site, secondary, and global protein stability. It was found that the stability of the Fe(III) and Fe(II) states converged as the variant became more promiscuous. In comparison, the more selective variants exhibited significant changes in stability based on oxidation state. This finding has implications for understanding why CYPS with similar global structures are able to perform such different types of chemistry, and suggests that the stability of the Fe(II) state should be evaluated in order to develop better enzymes for biotechnical purposes.

Impact of Fe oxidation state on the stability of Cytochrome P450BM3

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.