Role and spectroscopic characterization of [2Fe-2S] centers in perrochelatases
Albetel, Angela Nadia
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Ferrochelatase (E.C. 188.8.131.52), the terminal enzyme of the heme biosynthetic pathway, catalyzes the insertion of ferrous iron into protoporphyrin IX. All metazoan, Actinobacterial, and a few Gram negative bacterial ferrochelatases contain a labile [2Fe-2S]2+ center that is crucial for activity, but its specific role has yet to be determined. In this work the role and spectroscopic properties of [2Fe-2S]2+ centers in ferrochelatase have been investigated by using a combination of analytical and spectroscopic studies. Detailed spectroscopic characterization of various [2Fe-2S]2+,+ cluster-containing ferrochelatases reveals major differences in the coordination environment and electronic properties of the [2Fe-2S]2+,+ clusters that are ligated by distinct primary sequence arrangements of conserved cysteine residues. Parallel EPR and UV-visible studies indicate that anaerobic incubation of different recombinant ferrochelatases with the NO-donor, diethylamine NONOate, results in rapid loss of the [2Fe-2S]2+ cluster concomitant with the formation of a dinitrosyl-iron-cysteine complex (DNIC). The rates for the NO-induced [2Fe-2S]2+ cluster degradation/DNIC formation are comparable among all ferrochelatases studied, suggesting that the cluster is unlikely to play a primary role as a NO-sensor in eukaryotes, but rather serves as part of a general oxidative stress response to degrade the cluster, thereby decreasing heme biosynthesis, in response to cellular conditions in both eukaryotes and prokaryotes. Furthermore, investigations on the role of [2Fe-2S]2+ clusters from Arabidopsis thaliana sirohydrochlorin ferrochelatase (AtSirB Fc), the terminal enzyme of siroheme biosynthesis in chloroplast, demonstrate that monothiol glutaredoxins, which play a crucial role in both Fe-S cluster and heme biosynthesis in eukaryotes, play a vital role in regulating siroheme/heme biosynthesis by functioning as the [2Fe-2S] cluster donor for the maturation of AtSirB Fc. Detailed analytical and spectroscopic studies reveal that the [2Fe-2S]2+ center in AtSirB Fc undergoes a conformational change as a function of pH that is associated with a dimer/tetramer interconversion and that the cluster can only be inserted in the tetrameric form. Thus, this work provides direct evidence for an intimate connection between Fe-S clusters biogenesis and heme biosynthesis, by indicating that the [2Fe-2S] cluster-binding site in eukaryotic ferrochelatases functions in sensing the cellular Fe-S cluster status via the extent of cluster loading of the monothiol glutaredoxin [2Fe-2S] cluster donor.