Chemical Approaches to Understanding the Mechanisms of Iron-Sulfur Enzymes

Project Summary/Abstract Enzymes with iron-sulfur (Fe-S) clusters in their active sites catalyze myriad transformations relevant to human health and disease. Identifying disease targets and designing inhibitors requires an in-depth understanding of their reaction mechanisms, which in turn requires both molecular-level characterization of resting states and intermediates as well as comparison with structurally and functionally faithful synthetic models. This project entails both advancing the synthetic modeling chemistry of Fe-S proteins and developing new methods for improving the information content of spectroscopic data on Fe-S proteins. In the context of modeling the chemistry of Fe-S enzymes, we will synthesize clusters whose geometric structures closely resemble those of enzymatic intermediates and whose spectroscopic data can be compared with the data acquired on natural systems. In addition to using models in this manner (i.e., for structure elucidation), we will formulate hypotheses regarding the electronic structures of these intermediates and the factors that dictate their diverse reactivity patterns, and we will test these hypotheses by harnessing the exquisite tunability of synthetic chemistry to rationally altering the clusters’ properties and reactivity. Overall, this part of the project will combine synthesis, spectroscopy, and mechanistically oriented reactivity studies to shed light on the mechanisms of Fe-S enzymes. Regarding our biological work, we first note that although 57Fe Mössbauer and 57Fe ENDOR spectroscopy can be exceptionally powerful in characterizing intermediates of Fe-containing enzymes, the high nuclearity of Fe-S clusters can limit the usefulness of such techniques because the signals arising from multiple metal sites can be challenging to resolve. This is true of resting states and is especially problematic when analyzing mixtures of reaction intermediates. Moreover, it is often impossible to map spectroscopic responses to specific sites in the geometric structure, and this severely limits our understanding of the chemical bonding—and therefore the reactivity—of biological Fe-S clusters. We propose to address these challenges by developing methods for incorporating 57Fe into specific sites of biological Fe-S clusters. Doing so would make Fe-S proteins as spectroscopically tractable as mononuclear Fe-containing enzymes, and would thereby greatly enhance our mechanistic understanding of Fe-S proteins. In addition to developing the methodology, we will use the site- selectively labeled samples generated in this project to study reactive intermediates and inhibitor-bound states of Fe-S enzymes of relevance to human health and disease.