Redox properties of the PutA protein from Escherichia coli and the influence of the flavin redox state on PutA-DNA interactions

The PutA flavoprotein from Escherichia coli is both a transcriptional repre ssor and a membrane-associated proline dehydrogenase. PutA represses transc ription of the putA and putP genes by binding to the control region DNA of the put regulon (put intergenic DNA). Previous work has shown that FAD has a role in regulating the transcriptional repressor and membrane binding fun ctions of the PutA protein. To test the influence of the FAD redox state on PutA-DNA interactions, we characterized the redox properties of the PutA f lavoprotein from E. coli. At pH 7.5, an E-m(E-FAD/E-FADH(2)) of -0.076 V fo r the two-electron reduction of PutA-bound FAD was determined by potentiome tric titrations. Stabilization of semiquinone species was not observed duri ng potentiometric measurements. Dithionite reduction of PutA, however. caus ed formation of red anionic semiquinone. The E-m value for the proline/Delt a (1)-pyrroline-5-carboxylate couple was determined to be -0.123 V, demonst rating the reduction of PutA by proline is favored by a potential differenc e (DeltaE degrees') of more than 0.045 V. Characterization of the PutA redo x properties in the presence of put intergenic DNA revealed an E-m(E-DNA-FA D/E-DNA-FADH(2)) of -0.086 V. The 10 mV negative shift in E-m corresponds t o just a 2.3-fold increase in the dissociation constant of PutA with the DN A upon reduction of FAD. Thus, it appears the FAD redox state has little in fluence on the overall PutA-DNA interactions.