The PutA flavoprotein regulates proline metabolism in Escherichia coli by performing two distinct functions. First, in the cytoplasm, PutA represses transcription of the put (proline utilization) regulon. Second, PutA associates with the membrane to oxidize proline to glutamate using discrete proline dehydrogenase and Delta(1)-pyrroline-5-carboxylate dehydrogenase domains. Here, we identify a proline analog that will be useful for testing the role substrate binding has in regulating PutA functions. L-Tetrahydro-2-furoic acid (L-THFA) was found to display simple competitive inhibition of proline dehydrogenase activity in PutA (apparent K(i)=0.2mM) and to perturb the flavin adenine dinucleotide (FAD) absorbance spectrum upon complexation to PutA. At pH 7.5, a reduction potential (E(m)) of -0.089V for the FAD/FADH(2) couple in L-THFA-complexed PutA was determined by potentiometric titrations. The E(m) value for L-THFA-complexed PutA is 12mV more negative than the E(m) for uncomplexed PutA (E(m)=-0.077V, pH 7.5) and corresponds to just a twofold increase in the dissociation constant of L-THFA with PutA upon reduction of FAD.