Sutton Victoria R, Mettert Erin L, Beinert Helmut, Kiley Patricia J
Program in Cellular & Molecular Biology, University of Wisconsin, Madison, WI 53706, USA.
J Bacteriol. 2004 Dec;186(23):8018-25. doi: 10.1128/JB.186.23.8018-8025.2004.
The ability of FNR to sense and respond to cellular O(2) levels depends on its 4Fe-4S cluster. In the presence of O(2), the 4Fe-4S cluster is converted to a 2Fe-2S cluster, which inactivates FNR as a transcriptional regulator. In this study, we demonstrate that approximately 2 Fe(2+) ions are released from the reaction of O(2) with the 4Fe-4S cluster. Fe(2+) release was then used as an assay of reaction progress to investigate the rate of 4Fe-4S to 2Fe-2S cluster conversion in vitro. We also found that there was no detectable difference in the rate of O(2)-induced cluster conversion for FNR free in solution compared to its DNA-bound form. In addition, the rate of FNR inactivation was monitored in vivo by measuring the rate at which transcriptional regulation by FNR is lost upon the exposure of cells to O(2); a comparison of the in vitro and in vivo rates of conversion suggests that O(2)-induced cluster conversion is sufficient to explain FNR inactivation in cells. FNR protein levels were also compared for cells grown under aerobic and anaerobic conditions.
FNR感知并响应细胞内氧气水平的能力取决于其4Fe-4S簇。在有氧存在的情况下，4Fe-4S簇会转变为2Fe-2S簇，这会使作为转录调节因子的FNR失活。在本研究中，我们证明约2个Fe(2+)离子会从氧气与4Fe-4S簇的反应中释放出来。然后将Fe(2+)的释放用作反应进程的测定方法，以研究体外4Fe-4S向2Fe-2S簇转化的速率。我们还发现，与结合DNA的形式相比，溶液中游离的FNR在氧气诱导的簇转化速率上没有可检测到的差异。此外，通过测量细胞暴露于氧气后FNR转录调节丧失的速率，在体内监测FNR的失活速率；体外和体内转化速率的比较表明，氧气诱导的簇转化足以解释细胞中FNR的失活。还比较了在有氧和无氧条件下生长的细胞的FNR蛋白水平。
