The Impact of O2 on the Fe–S Cluster Biogenesis Requirements of Escherichia coli FNR

Abstract

In this study, the functions of two established Fe–S cluster biogenesis pathways, Isc (iron–sulfur cluster) and Suf (sulfur mobilization), under aerobic and anaerobic growth conditions were compared by measuring the activity of the Escherichia coli global anaerobic regulator FNR. A [4Fe–4S] cluster is required for FNR activity under anaerobic conditions. An assay of the expression of FNR-dependent promoters in strains containing various deletions of the iscSUAhscBAfdx operon revealed that, under anaerobic conditions, FNR activity was reduced by 60% in the absence of the Isc pathway. In contrast, a mutant lacking the entire Suf pathway had normal FNR activity, although overexpression of the suf operon fully rescued the anaerobic defect in FNR activity in strains lacking the Isc pathway. Expression of the sufA promoter and levels of SufD protein were upregulated by twofold to threefold in Isc− strains under anaerobic conditions, suggesting that increased expression of the Suf pathway may be partially responsible for the FNR activity remaining in strains lacking the Isc pathway. In contrast, use of the O2-stable [4Fe–4S] cluster FNR variant FNR-L28H showed that overexpression of the suf operon did not restore FNR activity to strains lacking the Isc pathway under aerobic conditions. In addition, FNR-L28H activity was more impaired under aerobic conditions than under anaerobic conditions. The greater requirement for the Isc pathway under aerobic conditions was not due to a change in the rate of Fe–S cluster acquisition by FNR-L28H under aerobic and anaerobic conditions, as shown by 55Fe-labeling experiments. Using [35S]methionine pulse-chase assays, we observed that the Isc pathway, but not the Suf pathway, is the major pathway required for conversion of O2-inactivated apo-FNR into [4Fe–4S]FNR upon the onset of anaerobic growth conditions. Taken together, these findings indicate a major role for the Isc pathway in FNR Fe–S cluster biogenesis under both aerobic and anaerobic conditions.