Iron is essential for various pivotal biological processes, such as oxygen transport, DNA synthesis, and ATP production. In humans, dysregulation in iron metabolism leads to severe disorders due to its intrinsic toxicity. Thus, iron homeostasis has to be tightly regulated to ensure its safe handling. A crucial role in recycling and systemic iron homeostasis is played by a system formed by the unique iron transporter ferroportin and a ferroxidase, that mediate safe export of iron across cellular membranes. Despite its importance, several aspects of this system still have to be unveiled. This work focuses on addressing different unanswered questions of the iron export mechanism. The precise modality in which the transporter ferroportin operates is still under debate. To enable its systematic characterization, site-specific fluorescent labeled versions of FPN were generated. Employing the genetic incorporation of unnatural amino acid technique we successfully incorporated the fluorescent probe dansylalanine in four different positions within the structure of the transporter. Fluorescence spectroscopy confirmed the ability of the fluorophore to sense conformational changes upon metal binding by ferroportin. Thus, a reliable experimental system to systematically probe the translocation mechanism of the transporter has been established. Moreover, the peculiar two metal-binding site feature of ferroportin has been widely investigated through site-directed mutagenesis and fluorescence spectroscopy. The different affinity of the two metal binding sites has been assessed allowing for the reconciliation of numerous conflicting data reported in literature. Based on these novel findings, an innovative metal translocation model for ferroportin has been proposed. Also, the two ferroxidases ceruloplasmin and hephaestin were investigated. Specifically, different mutants of ceruloplasmin were produced and purified from a heterologous expression system for the first time. The potential of obtaining a high-quality recombinant form of this protein for unlocking in-depth analysis has been demonstrated. Similarly, a protocol has been established for the production and purification of full-length hephaestin, enabling future structural and functional characterization. The potential of this protocol has been tested through functional investigation of two cancer-related variants of this protein. Finally, the interaction between ferroportin and ceruloplasmin has been demonstrated using the Proximity Ligation Assay, resolving a long-standing question about this system. In vitro trials have been conducted to handle and stabilize this protein complex, enabling its future structural characterization.