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Redox Properties of the PutA Protein from Escherichia coli and the Influence of the Flavin Redox State on PutA−DNA Interactions

2001; American Chemical Society; Volume: 40; Issue: 15 Linguagem: Inglês

10.1021/bi0019491

1943-295X

Donald Becker, Elizabeth A. Thomas,

Enzyme Structure and Function

The PutA flavoprotein from Escherichia coli is both a transcriptional repressor and a membrane-associated 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 Em(E−FAD/E−FADH2) of −0.076 V for the two-electron reduction of PutA-bound FAD was determined by potentiometric titrations. Stabilization of semiquinone species was not observed during potentiometric measurements. Dithionite reduction of PutA, however, caused formation of red anionic semiquinone. The Em value for the proline/Δ1-pyrroline-5-carboxylate 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 Em(EDNA−FAD/EDNA−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.