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Abstract
Of the three most common Fe-S clusters found in nature, [4Fe-4S] clusters are the most abundant and account for the most diverse functions, ranging from electron transfer to regulation of gene expression and radical generation. [4Fe-4S] clusters are very sensitive to oxidative degradation resulting in loss of function upon oxidation of solvent exposed [4Fe-4S] clusters or clusters within 10 Å of the protein surface. Conversely, oxidatively labile [4Fe-4S] clusters are sometimes used as sensors of oxidative stress. The major aim of this research project is to understand the mechanism of degradation of [4Fe-4S] clusters, when exposed to oxidative stress, by air exposure or titration with H2O2, via spectroscopic characterization of stable or semi-stable breakdown intermediates. Moreover, understanding the degradation pathway has the potential for assessing how degraded [4Fe-4S] clusters are repaired in vivo. This work investigates the breakdown products of two classes of proteins containing [4Fe-4S] clusters, (de)hydratases, and radical SAM enzymes. The breakdown products of aconitase and dihydroxyacid dehydratase were characterized using resonance Raman, EPR, UV-visible absorption and circular dichroism, and Mössbauer spectroscopies. While the breakdown of aconitase proceeds through a semi-stable cubane [3Fe-4S]1+ cluster, followed by degradation to the apo product, the spectroscopic results indicate that the [4Fe-4S]2+ cluster of DHAD degrades to a semi-stable cysteine persulfide-ligated [2Fe-2S]2+ cluster intermediate. In addition, resonance Raman spectroscopy of the radical SAM enzymes MiaB and MOCS1A show that the cluster degradation products of these proteins also contain a [2Fe-2S]2+ cluster with cysteine persulfides that can be repaired on addition of Fe2+ and a reducing agent. Spectroscopic studies of the radical SAM enzyme, PFL-AE, indicate that the degradation of its [4Fe-4S]2+ cluster proceeds through first a cubane [3Fe-4S]1+ cluster, then a [2Fe-2S]2+ cluster with cysteine persulfides. All these breakdown products can be repaired to the [4Fe-4S]2+ cluster by adding Fe2+ and a reducing agent. While the breakdown intermediates are differentially stable, the breakdown of [4Fe-4S] clusters proceeds via a singular pathway and the intermediate products remain in a form that can be easily repaired. Consequently, this work affords insight into the mechanism of repair of [4Fe-4S] clusters damaged by oxidative stress.