Biogenesis of iron-sulfur clusters by the ISC machinery in Azotobacter vinelandii.
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Iron-sulfur (Fe-S) clusters represent one of the most ancient, ubiquitous and versatile type of cofactors found in nature. The biogenesis of Fe-S clusters is a tightly regulated process owing to the cellular toxicity of free iron and sulfide, and specialized Fe-S cluster assembly systems have been identified for fabrication of the cofactors in vivo. The objective of the research presented here was to understand the assembly of Fe-S clusters by one of the assembly machineries, the ISC system, in the aerobic nitrogen-fixing bacterium Azotobacter vinelandii. The approach involved in vitro biochemical and biosphysical spectroscopic studies (UV-visible absorption/CD/MCD, EPR, resonance Raman, and Mossbauer) of purified recombinant ISC 2+proteins. The results reveal rapid and quantitative formation of [4Fe-4S] clusters on IscU by 2+reductive coupling of two [2Fe-2S] centers using dithionite as the electron donor. Moreover, reduced IscFdx was shown to be competent and approximately isopotential electron donor for reductive coupling and therefore is a candidate for the physiological reductant. The importance 2+2+of [4Fe-4S] cluster assembly on IscU was demonstrated by the observation that the [4Fe-4S] 2+cluster-loaded form of IscU, but not the [2Fe-2S] cluster-loaded form, is competent for 2+ activation of apo-aconitase via intact cluster transfer. Intact [2Fe-2S]cluster transfer from IscU to apo-IscFdx was found to be greatly enhanced by the HscA and HscB co-chaperones in an ATP-dependent reaction. These results provide the first in vitro evidence that the role of HscA/HscB is to enhance the rate of cluster transfer from IscU to acceptor proteins and that cluster transfer from IscU can be an ATP-driven process. Finally, the regulatory protein IscR, 2+,+2+,+has been shown to accommodate both [4Fe-4S] and [2Fe-2S] clusters. Both IscS-mediated cluster assembly and cluster transfer from [2Fe-2S]-loaded IscU were found to entail sequential 2+2+assembly of [2Fe-2S] and [4Fe-4S] cluster on IscR, with the final product containing 0.5 2+[4Fe-4S] clusters per IscR monomer. The results support a feedback mechanism for IscR 2+repression of the isc operon involving [2Fe-2S] cluster transfer from IscU to apo-IscR and raise 2+the possibility that the [4Fe-4S] cluster on IscR is used in sensing oxidative stress.