The anaerobic global regulator FNR from Escherichia coli is a [4Fe-4S](2+) cluster containing, O-2 labile dimer that plays an important role in adapting the bacterium to its anaerobic lifestyle. Although functional significance of this global regulator has been well established, its structural and biochemical characterizations have been hindered by the intrinsic O-2 lability of FNR protein. To obtain oxygen stable FNR variants for in vitro characterizations, in this study, we utilized the approach of covalently linking two fnr alleles in tandem to promote their in situ dimerization in the absence of the [4Fe-4S](2+) cluster under aerobic conditions. By covalently linking two alleles of fnrD154A mutant, which has reduced charge repulsions between two FNR monomers, an FNR variant of FNRD154A-Linker-FNRD154A (designated as (FNRD154A)(2)) was constructed. (FNRD154A)(2) displayed significantly enhanced DNA binding affinities and transcriptional regulatory activities to various FNR dependent promoters under aerobic conditions. Further in vivo and in vitro studies demonstrated that this variant retains oxygen sensing capability and maintains a similar secondary structure as that formed by native monomers of FNRD154A. We conclude that this novel variant of FNR can be widely used in various biochemical and structural studies of FNR in the presence of O-2. (C) 2012 Elsevier Inc. All rights reserved.