Work

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

Year: 2025

Type: article

Abstract: High Resolution Image Download MS PowerPoint Slide FtrABCD is a four-component iron transporter found in several Gram-negative bacteria. Previous data confirm that FtrABCD can only utilize Fe 2+ and the inner membrane permease, FtrC, from this system, like its eukaryotic homologue, Ftr1p, is predicted to utilize the free energy released during Fe 2+ 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 2+, 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 Cu 2+ and an Fe 2+ mimic (Mn 2+ ), which facilitate the formation of the active enzyme and the enzyme–substrate complex, respectively. In contrast, the D118A mutant failed to bind Cu 2+ . Finally, the electrochemical data reported here revealed biologically accessible reduction potentials for the Cu 2+ ion in the active enzyme which also showed a pseudozero-order rate of Fe 2+ oxidation at pH 6.5 and could oxidize Fe 2+ 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.

Source: ACS Omega

DOI: 10.1021/acsomega.5c00690