In Vitro Structural and Functional Studies of a Novel Cupredoxin, FtrB, from 2308.

FtrABCD is a four-component iron transporter found in several Gram-negative bacteria. Previous data confirm that FtrABCD can only utilize Fe and the inner membrane permease, FtrC, from this system, like its eukaryotic homologue, Ftr1p, is predicted to utilize the free energy released during Fe oxidation for the transport. Periplasmic FtrB from this system is coancestral with known copper oxidases, and the conserved D118 and H121 are predicted to bind to Cu, forming an active enzyme. In this work, we report structural data for recombinant wild-type and D118A and H121A mutants from 2308 which confirm a β-sheet-rich structure which is distinct from known cupredoxins. Calorimetric studies on the wild-type protein show μM affinities for Cu and an Fe mimic (Mn), which facilitate the formation of the active enzyme and the enzyme-substrate complex, respectively. In contrast, the D118A mutant failed to bind Cu. Finally, the electrochemical data reported here revealed biologically accessible reduction potentials for the Cu ion in the active enzyme which also showed a pseudozero-order rate of Fe oxidation at pH 6.5 and could oxidize Fe 3.5-times faster than its rate of autoxidation. Taken together, this report provides experimental data that support structural and functional predictions of FtrB under in vitro conditions.