Kerkan, AlexaSantisteban Veiga, AndreaBanerjee, Sambuddha2026-05-052026-05-052025-03-22Kerkan, A., Hart, K., Martin, D. W., Pajski, J., Aidoo, B., Garcia, B. L., Roy, S., Dasgupta, S., Hematian, S., Santisteban-Veiga, A., Schaaf, N. J., & Banerjee, S. (2025). In Vitro Structural and Functional Studies of a Novel Cupredoxin, FtrB, from Brucella abortus 2308. ACS Omega, 10(12), 12653-12670. https://doi.org/10.1021/ACSOMEGA.5C00690https://hdl.handle.net/10347/47083FtrABCD 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 Brucella abortus 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.engCopyright © 2025 The Authors. Published by American Chemical SocietyAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/journal article2026-05-0410.1021/ACSOMEGA.5C006902470-1343open access2470-1343