NifL of Klebsiella pneumoniae: redox characterization in relation to the nitrogen source

In Klebsiella pneumoniae, NifL modulates the activity of the transcriptiona l activator NifA in response to combined nitrogen or external molecular oxy gen. We recently showed that K. pneumoniae NifL is a flavoprotein which app arently senses oxygen through a redox-sensitive, conformational change. In order to study whether the nitrogen signal might be transmitted to NifA thr ough a stable modification of NifL we characterized the redox properties of NifL synthesized in Escherichia coli in the presence of different nitrogen sources. FAD analyses showed that purified NifL, carried FAD as cofactor i ndependent of nitrogen and oxygen availability. The redox potential of NifL synthesized in the presence of ammonium was -277 +/- 5 mV at pH 8.0 and 25 degrees C, as determined by reduction with dithionite or with enzymatic re duction by xanthine oxidase in the presence of methyl viologen as redox med iator. When synthesized under nitrogen-limiting conditions, NifL showed a r edox potential of -274 +/- 6 mV at pH 8.0 and 25 degrees C. Fully reduced N ifL fractions, synthesized under either condition listed above, reoxidized rapidly in the presence of molecular oxygen. These results indicate that fo r NifL synthesized in E. coli, the redox potential of the NifL-bound FAD is not influenced by the nitrogen source. The two NifL fractions differed, ho wever, in that a non-flavin specific absorbance at 420 nm was found only in NifL synthesized in the presence of ammonium. (C) 1999 Published by Elsevi er Science B.V. All rights reserved.