CAREER: Experimental and Theoretical Studies of Thiolate-Heme Enzymes

Cytochrome P450, nitric oxide synthase, and chloroperoxidase are thiolate-ligated heme-enzymes that play critical roles in a number of important physiological processes (e.g. the metabolism of xenobiotics). They are unique among oxidative heme-proteins in that they catalyze the insertion of an oxygen atom, derived either from molecular oxygen or peroxide, into a variety of organic substrates, often with high degrees of regio- and stereo-selectivity. All three of these enzymes are thought to function through a highly reactive iron-oxo intermediate, called compound I. It is known that the reactivity of metal-oxos, with respect to oxygen transfer chemistry, generally scales with redox potential: The more oxidizing the metal-oxo the more reactive it is. Thus, it is somewhat surprising that Nature uses donating thiolate-ligands in its most oxidizing heme-enzymes, as thiolate-ligation can drop the reduction potential of a heme active site by several hundred millivolts. This project examines Nature's use of thiolate ligands in oxidizing heme-enzymes. Spectroscopic methods (EXAFS, EPR, Mossbauer, Raman, and UV/visible) will be coupled with theory to study reactive intermediates in naturally-occurring and isotopically-labeled chloroperoxidase. The aim is to obtain a better understanding of the electronic and geometrical structures of the high-valent intermediates found in the catalytic cycles of thiolate-ligated enzymes. The project also involves the preparation and spectroscopic characterization of Cys_Sec mutants of cytochrome P450. Understanding how changes in reactivity can be related to the different size and redox properties of selenium will give us further insight into the role of the axial-ligand in these systems. Broader Impacts: This award will support the course and curriculum development and the research training of graduate and undergraduate students at the interface of chemistry and biology. The project will broaden the participation of underrepresented groups in science and enhance the infrastructure for research at Penn State University through an increase in shared instrumentation. The societal benefits of the research include a better understanding of oxygen transfer chemistry in thiolate-ligated heme-proteins, which could be parlayed into improved catalysts for industrial applications. The enzymes to be studied use only electrons, protons, and dioxygen (or peroxide) to oxidize substrates. The only byproduct is water. Thus, these enzymes are particularly 'green' catalysts, and synthetic systems that could mimic their chemistry would be of obvious value.