Mettert Erin L, Outten F Wayne, Wanta Brendan, Kiley Patricia J
Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, USA.
J Mol Biol. 2008 Dec 26;384(4):798-811. doi: 10.1016/j.jmb.2008.09.080. Epub 2008 Oct 10.
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 O(2)-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 (55)Fe-labeling experiments. Using [(35)S]methionine pulse-chase assays, we observed that the Isc pathway, but not the Suf pathway, is the major pathway required for conversion of O(2)-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.
在本研究中，通过测定大肠杆菌全局厌氧调节因子FNR的活性，比较了两种已确立的铁硫簇生物合成途径，即Isc（铁硫簇）和Suf（硫动员）在有氧和厌氧生长条件下的功能。厌氧条件下FNR活性需要一个[4Fe-4S]簇。对含有iscSUAhscBAfdx操纵子各种缺失的菌株中FNR依赖性启动子的表达进行检测发现，在厌氧条件下，缺失Isc途径时FNR活性降低60%。相比之下，一个缺失整个Suf途径的突变体具有正常的FNR活性，尽管suf操纵子的过表达完全挽救了缺失Isc途径的菌株中FNR活性的厌氧缺陷。在厌氧条件下，Isc(-)菌株中sufA启动子的表达和SufD蛋白水平上调了两倍至三倍，这表明Suf途径表达的增加可能部分解释了缺失Isc途径的菌株中仍保留的FNR活性。相比之下，使用对O₂稳定的[4Fe-4S]簇FNR变体FNR-L28H表明，在有氧条件下，suf操纵子的过表达并不能恢复缺失Isc途径的菌株的FNR活性。此外，FNR-L28H在有氧条件下的活性比在厌氧条件下受到的损害更大。如⁵⁵Fe标记实验所示，有氧条件下对Isc途径的更大需求并非由于FNR-L28H在有氧和厌氧条件下获取铁硫簇的速率发生变化。使用[³⁵S]甲硫氨酸脉冲追踪分析，我们观察到，在厌氧生长条件开始时，将O₂失活的脱辅基FNR转化为[4Fe-4S]FNR所需的主要途径是Isc途径，而非Suf途径。综上所述，这些发现表明Isc途径在有氧和厌氧条件下FNR铁硫簇生物合成中起主要作用。
