Chemical Modification of Iron-Sulfur Cofactors

Abstract

Iron–sulfur (Fe–S) cofactors are multi-metallic clusters that are found in all living organisms. The presence of multiple metal centers enables these cofactors to perform some of the most challenging chemical transformations in biosphere, such as activation of strong C–H bonds and reduction of N2, many of which are pertinent to human health and global biogeochemical cycles. We hypothesize that these unique reactivities of FeS cofactors arise at least in part from their electronic structures, which can be described as an ensemble of magnetically coupled, locally high-spin Fe sites that can access electronic states distinct from those available to mononuclear Fe. However, there is a substantial barrier to deciphering the electronic structure of Fe–S cofactors: the complexity associated with spectroscopic analysis using ⁵⁷Fe-specific techniques (e.g., ⁵⁷Fe Mössbauer and electron-nuclear double resonance spectroscopy) due to the large number of Fe sites. This thesis reports strategies to chemically modify complex Fe–S cofactors to address this challenge and to facilitate the electronic-structure studies of FeS enzymes. In Chapter 1, selected examples of Fe–S enzymes, their chemistry, and their remaining mechanistic questions are described in addition to the discussion on advantages and limitations of ⁵⁷Fe-specific techniques. In Chapter 2, we demonstrate that Fe ions in a wide range of [Fe₄S₄] clusters exchange with exogenous Fe²⁺, and exploit this reactivity to develop a facile method for incorporating ⁵⁷Fe into the SAM-binding cluster of a radical SAM methyltransferase, RlmN. In Chapter 3, we show that two [Fe₄S₄] clusters of BtrN, a Twitch-domain-containing radical SAM enzyme, have different rates of Fe exchange, and by utilizing such difference, we demonstrate that we can selectively label either of the two clusters with ⁵⁷Fe. In Chapter 4, we apply a similar Fe exchange method to IspG, an [Fe₄S₄] enzyme involved in bacterial isoprenoid biosynthesis, to show that the Fe sites within the [Fe₄S₄] cluster can be further differentiated in the Fe exchange kinetics and consequently that site-selective ⁵⁷Fe labeling can be achieved. Using the site-selectively labeled sample, we study a unique π-complex in an inhibitor-bound state of IspG to understand its electronic structure. Lastly, in Chapter 5, extending the site-selective ⁵⁷Fe labeling strategy for the iron–molybdenum cofactor of the Mo-dependent nitrogenase, we report protocols for selectively removing an Fe from the cofactor and incorporating a closed-shell metal, Zn²⁺. We anticipate the modified clusters to serve as ‘simplified’ models for interrogating the complex electronic structure of the ironmolybdenum cofactor.