Exploring the Copper (I) and Iron (III) Binding to Recombinant Wild-type FtrB from Brucella abortus: Role of Cu-ion in the FtrABCD Iron Transport System in Gram-Negative Pathogens

Abstract

FtrABCD is a four-component Fe²⁺ active transporter found in several Gram-negative pathogens, including Brucella and Bordetella spp. Co-expression of these proteins is essential for the survival of these organisms inside the host where these pathogens encounter iron-restricted environment due to host immune response. Based on protein homology and evolutionary data for FtrC, it is hypothesized that this permease uses the free energy (ΔG) derived from Fe²⁺ oxidation to perform the active transport of its cargo. A similar permease, Ftr1p, is found in yeast, and experiments have confirmed that Fe2+ oxidation is required for iron utilization by Ftr1p. In this yeast system, Fe2+ oxidation is done by a co-expressed multicopper oxidase (MCO), Fet3p. The proteins co-expressed with FtrC (FtrA, FtrB, and FtrD) are not MCOs and the soluble components, periplasmic FtrA and FtrB do not belong to any known families of redox proteins. Phylogenetic studies on FtrB and known single and multidomain copper redox proteins have shown that FtrB is evolutionarily related to Fet3p. Based on this, it was proposed that Brucella abortus FtrB is a novel and uncharacterized copper redox protein and can perform the following reaction—

Cu2+-FtrB + Fe2+  Cu2+-FtrB-Fe2+ (aq)  Cu+-FtrB-Fe3+ (aq)

For FtrB to be an effective redox protein and perform the above reaction, it must bind Cu2+, Cu+, Fe2+, as loss of Cu+ from FtrB will terminate its redox activity. As free Fe3+ is toxic and insoluble, it is also predicted that Fe3+ produced by Cu2+-FtrB must stay bound to reduced FtrB until it gets recognized by the permease, FtrC, and is translocated. The ability of recombinant wild-type FtrB to bind Cu2+ and a Fe2+ mimic were determined by another undergraduate researcher from my lab. This thesis describes experiments performed by me and were performed to establish if FtrB can stay bound to Cu⁺ and Fe3+. To investigate if Cu⁺ and Fe³⁺ bind to FtrB, we used Ag⁺ and Ga³⁺ as mimics for Cu⁺ and Fe³⁺, respectively. These mimics were chosen because Cu⁺ and Fe³⁺ are unstable and can oxidize quickly, making them difficult to work with directly. Ag⁺ and Ga³⁺ provide similar coordination chemistry and binding properties, allowing for accurate modeling of the interactions without these complications. Isothermal titration calorimetry (ITC) was performed by titrating Ag⁺ into apo-FtrB in water at neutral pH, showing strong binding (Kd ~ 3 µM, N ~1). Further ITC experiments with Ga³⁺ and Ag⁺-FtrB revealed two distinct binding sites for Ga³⁺. The first site showed a stoichiometry (n) of 2.844, a dissociation constant (Kd) of (1.208 ×10-5) M, a positive change in enthalpy (ΔH) of 28.94 kcal/mol, and a substantial increase in entropy (ΔS) of (1.196 × 102) cal/mol·K. The second site exhibited a stoichiometry (n) of 10, a dissociation constant (Kd) of (1 ×10-3) M, a negative change in enthalpy (ΔH) of -8.378 kcal/mol, and a decrease in entropy (ΔS) of -14.37 cal/mol·K. These findings enhance our understanding of the metal-binding properties of FtrB and its role in ion homeostasis, emphasizing the importance of these interactions for the protein's biological function. To determine if recombinant wild-type FtrB can bind Cu²⁺ specifically, I performed circular dichroism (CD) spectroscopy on as isolated wild-type FtrB as it is exposed to other divalent transition metal ions during its expression and purification. I used the signature Cu²⁺-His CD peaks to establish the presence of Cu²⁺ in as isolated wild-type FtrB.