OXIDATIVE DEGRADATION AND REDUCTIVE REPAIR OF [4Fe-4S] CLUSTERS

Of the three most common Fe-S clusters found in nature, the [4Fe-4S] cluster is the most abundant and accounts for the most diverse functions, ranging from electron transfer to regulation of gene expression and radical generation¹. Solvent accessible [4Fe-4S] clusters are very sensitive to oxidative degradation and as such, are sometimes used as sensors of oxidative stress². 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 H₂O_2, 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 presentation will discuss 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 (DHAD) have been characterized using resonance Raman, electron paramagnetic resonance (EPR), UV-visible absorption and circular dichroism (CD) spectroscopies. While the breakdown of aconitase proceeds through a cubane [3Fe-4S]¹⁺ cluster, followed by rapid breakdown to the apo product, the spectroscopic results indicate that the [4Fe-4S]²⁺ cluster of DHAD degrades to a 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 an additional radical SAM enzyme, PFL-AE, that contains only one [4Fe-4S] cluster, 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, and these breakdown products can be repaired to the [4Fe-4S]²⁺ cluster by adding Fe²⁺ and a reducing agent. While the breakdown products are variable, they remain in a form that can easily repaired. Consequently, this work affords insight into the mechanism of repair of [4Fe-4S] clusters damaged by oxidative stress.

[1] Johnson, M. K., and Smith, A. D. (2011) Iron–Sulfur Proteins, In Encyclopedia of Inorganic and Bioinorganic Chemistry, John Wiley & Sons, Ltd.

[2] Jordan, P. A., Thomson, A. J., Ralph, E. T., Guest, J. R., and Green, J. (1997) FNR is a direct oxygen sensor having a biphasic response curve, FEBS Lett 416, 349-352.