Brucella spp. FtrB copper binding thermodynamics and iron oxidase activity

Abstract

FtrABCD is a four-component iron uptake system found in several Gram-negative bacteria, including Brucella spp. This four-component transporter consists of two soluble periplasmic subunits (FtrA and FtrB), a putative membrane embedded terminal electron acceptor (FtrD), and a membrane spanning iron permease (FtrC). Based on the evolutionary relationship and sequence homology between FtrC and eukaryotic Ftr1p, this bacterial permease is predicted to be an oxidase dependent Fe2+ transporter. Unlike the eukaryotic Ftr1p permease, which can only function when co-expressed with the multicopper oxidase Fet3p, FtrC does not co-express homologs of any known ferrous oxidase. However, based on its evolutionary relationship with cupredoxins and ferroxidases, periplasmic FtrB is proposed to bind and oxidize Fe2+ during its transport through FtrC. This oxidase property of FtrB can be achieved if it binds a single copper ion using conserved D118 and H121 residues. This non-classical copper ion binding site in FtrB and its ability to form the active enzyme substrate complex (Cu2+ -FtrB-Fe2+) producing the ferrous oxidase function has not been verified experimentally, creating a knowledge gap in the bacterial iron uptake and redox enzyme literature. Data presented in this thesis demonstrates that a) recombinant wild-type Brucella FtrB can form a predominantly β-sheet containing structure; b) the ability of this protein to form this expected native secondary structure and coordinate Cu2+ (Kd = 3.0 ± 1.0 μM in 50 mM bis-tris, 100 mM NaCl, pH 7.3) is dependent on the presence of the conserved residue D118; and c) the recombinant wild-type FtrB can effectively oxidize Fe2+ at pH 7.3 under in vitro conditions. The results described in this thesis are significant as they demonstrate FtrB as a novel ferrous oxidase and open future research opportunities to investigate the importance of this Fe2+ oxidation during iron transport through FtrABCD under in vivo conditions.