Role and spectroscopic characterization of [2Fe-2S] centers in perrochelatases
Albetel, Angela Nadia
MetadataShow full item record
Ferrochelatase (E.C. 22.214.171.124), 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.