Sutton Victoria R, Stubna Audria, Patschkowski Thomas, Münck Eckard, Beinert Helmut, Kiley Patricia J
Program in Cellular & Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Biochemistry. 2004 Jan 27;43(3):791-8. doi: 10.1021/bi0357053.
The oxygen sensing ability of the transcription factor FNR depends on the presence of a [4Fe-4S]2+ cluster. In the presence of O2, conversion of the [4Fe-4S]2+ cluster to a [2Fe-2S]2+ cluster inactivates FNR, but the fate of the [2Fe-2S]2+ cluster in cells grown under aerobic conditions is unknown. The present study shows that the predominant form of FNR in aerobic cells is apo-FNR (cluster-less FNR) indicating that the [2Fe-2S]2+ cluster, like the [4Fe-4S]2+ cluster, is not stable under these conditions. By quantifying the amount of [2Fe-2S]2+ cluster in 2Fe-FNR in vitro in the presence of various reductants and oxidants (GSH, DTT, cysteine, O2, hydrogen peroxide, and superoxide), we found that superoxide, a byproduct of aerobic metabolism, significantly destabilized the [2Fe-2S]2+ cluster. Mössbauer spectroscopy was used to monitor the effects of superoxide on 2Fe-FNR in vivo; under cellular conditions that favored superoxide production, we observed the disappearance of the signal representative of the [2Fe-2S]2+ cluster. We conclude that the [2Fe-2S]2+ cluster of FNR is labile to superoxide both in vitro and in vivo. This lability may explain the absence of the [2Fe-2S]2+ cluster form of FNR under aerobic growth conditions.
转录因子FNR的氧感应能力取决于[4Fe-4S]2+簇的存在。在有氧条件下，[4Fe-4S]2+簇转化为[2Fe-2S]2+簇会使FNR失活，但在有氧条件下生长的细胞中[2Fe-2S]2+簇的命运尚不清楚。本研究表明，有氧细胞中FNR的主要形式是脱辅基FNR（无簇FNR），这表明[2Fe-2S]2+簇与[4Fe-4S]2+簇一样，在这些条件下不稳定。通过在各种还原剂和氧化剂（谷胱甘肽、二硫苏糖醇、半胱氨酸、氧气、过氧化氢和超氧化物）存在的情况下，对体外2Fe-FNR中[2Fe-2S]2+簇的量进行定量，我们发现超氧化物（有氧代谢的副产物）会显著破坏[2Fe-2S]2+簇的稳定性。穆斯堡尔光谱用于监测超氧化物对体内2Fe-FNR的影响；在有利于超氧化物产生的细胞条件下，我们观察到代表[2Fe-2S]2+簇的信号消失。我们得出结论，FNR的[2Fe-2S]2+簇在体外和体内对超氧化物都不稳定。这种不稳定性可能解释了在有氧生长条件下FNR不存在[2Fe-2S]2+簇形式的原因。
