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 repressor and a membraneassociated proline dehydrogenase. PutA represses transcription 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 functions 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 flavoprotein from E. coli. At pH 7.5, an E_m(E-FAD/E-FADH₂) of -0.076 V for the twoelectron reduction of PutA-bound FAD was determined by potentiometric titrations. Stabilization of serniquinone species was not observed during potentiometric measurements. Dithionite reduction of PutA, however, caused formation of red anionic serniquinone. The E_m value for the proline/Δ₁-pyrroline-5carboxylate couple was determined to be -0.123 V; demonstrating the reduction of PutA by proline is favored by a potential difference (ΔE^°′) of more than 0.045 V. Characterization of the PutA redox properties in the presence of put intergenic DNA revealed an E_m(E_DNA-FAD/E_DNA-FADH₂) of -0.086 V. The 10 mV negative shift in Em corresponds to just a 2.3-fold increase in the dissociation constant of PutA with the DNA upon reduction of FAD. Thus, it appears the FAD redox state has little influence on the overall PutA - DNA interactions.