Proline utilization A (PutA) from Escherichia coli is a multifunctional flavoprotein that is both a transcriptional repressor of the proline utilization (put) genes and a membrane-associated enzyme which catalyzes the 4-electron oxidation of proline to glutamate. Previously, proline was shown to induce PutA-membrane binding and alter the intracellular location and function of PutA. To distinguish the roles of substrate binding and FAD reduction in the mechanism of how PutA changes from a DNA-binding protein to a membrane-bound enzyme, the kinetic parameters of PutA-membrane binding were measured under different conditions using model lipid bilayers and surface plasmon resonance (SPR). The effects of proline, FAD reduction, and proline analogues on PutA-membrane associations were determined. Oxidized PutA shows no binding to E. coli polar lipid vesicles. In contrast, proline and sodium dithionite induce tight binding of PutA to the lipid bilayer with indistinguishable kinetic parameters and an estimated dissociation constant (K(D)) of <0.01 nM (pH 7.4) for the reduced PutA-lipid complex. Proline analogues such as L-THFA and DL-P5C also stimulate PutA binding to E. coli polar lipid vesicles with K(D) values ranging from approximately 3.6 to 34 nM (pH 7.4) for the PutA-lipid complex. The greater PutA-membrane binding affinity (>300-fold) generated by FAD reduction relative to the nonreducing ligands demonstrates that FAD reduction controls PutA-membrane associations. On the basis of SPR kinetic analysis with differently charged lipid bilayers, the driving force for PutA-membrane binding is primarily hydrophobic. In the SPR experiments membrane-bound PutA did not bind put control DNA, confirming that the membrane-binding and DNA-binding activities of PutA are mutually exclusive. A model for the regulation of PutA is described in which the overall translocation of PutA from the cytoplasm to the membrane is driven by FAD reduction and the subsequent energy difference ( approximately 24 kJ/mol) between PutA-membrane and PutA-DNA binding.