In Vitro Structural and Functional Studies of a Novel Cupredoxin, FtrB, from <i>Brucella abortus</i> 2308

FtrABCD is a four-component iron transporter found in several Gram-negative bacteria. Previous data confirm that FtrABCD can only utilize Fe²⁺ and the inner membrane permease, FtrC, from this system, like its eukaryotic homologue, Ftr1p, is predicted to utilize the free energy released during Fe²⁺ 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 Cu²⁺, forming an active enzyme. In this work, we report structural data for recombinant wild-type and D118A and H121A mutants from <i>Brucella abortus</i> 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 Cu²⁺ and an Fe²⁺ mimic (Mn²⁺), which facilitate the formation of the active enzyme and the enzyme-substrate complex, respectively. In contrast, the D118A mutant failed to bind Cu²⁺. Finally, the electrochemical data reported here revealed biologically accessible reduction potentials for the Cu²⁺ ion in the active enzyme which also showed a pseudozero-order rate of Fe²⁺ oxidation at pH 6.5 and could oxidize Fe²⁺ 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.