Flavin Redox State Triggers Conformational Changes in the PutA Protein from Escherichia coli

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Abstract
The regulation of proline utilization in Escherichia coli involves the proline-dependent translocation of the PutA flavoprotein from the cytoplasm to a peripheral position on the membrane. In the cytoplasm, PutA represses transcription of the proline utilization (put) genes while membrane-bound PutA catalyzes the oxidation of l-proline to glutamate. The mechanism by which PutA switches from a DNA-binding protein to a membrane-bound enzyme involves a proline-induced conformational change that is characterized by the appearance of a 119-kDa fragment during limited proteolysis of proline-reduced PutA. To establish whether the FAD redox state is responsible for the proline-induced conformational change in PutA, we distinguished the effects that FAD reduction and proline analogue binding have on PutA conformation by limited chymotrypsin proteolysis. Controlled potentiometric proteolysis of PutA demonstrated that the formation of the 119-kDa band occurs at an Em(conf) value of −0.058 V (pH 7.5), which is within 20 mV of the Em value for FAD bound to PutA. The manipulation of the Em(conf) value by reconstitution of PutA with the FAD analogue, 5-deazaFAD, confirmed that the conformational change observed in the presence of proline is solely dependent on the FAD redox state. The proline analogue, l-tetrahydro-2-furoic acid (l-THFA), failed to elicit the formation of the 119-kDa fragment during chymotrypsin cleavage of PutA. Instead, a unique fragment of about 93-kDa was observed, indicating that a distinct PutA conformer is stabilized by l-THFA. Reduction of l-THFA-complexed PutA, however, regenerated the 119-kDa fragment showing that reduction of the FAD cofactor overrides conformational changes induced by l-THFA. Mapping of the protease susceptibility sites in PutA revealed that the conformational changes caused by FAD reduction and l-THFA binding are transmitted to domains outside the proline dehydrogenase active site.

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