Currently, mammalian plasma is the main source of pure HRG protein. This has allowed only limited systematic investigation of metal ion binding. Furthermore, since we presently cannot produce natively glycosylated HRG recombinantly to sufficient yield, routine modifications performed with in vitro protein production such as site-directed mutagenesis and selective or uniform labelling with e.g., isotopes or spin labels (see below) are out of reach for the time being. 10 EPR is an important tool for structural analysis and characterisation of biomacromolecules. In contrast to other structural biology methods EPR is not limited by the size, shape, or complexity of the system and does not require protein crystallization. Compared to techniques such as ITC, less material is required, and samples can be stored frozen and re-used. EPR measurements require the presence of paramagnetic species, which can be either endogenous, such as paramagnetic metal centres or cofactors, or deliberately introduced to the site(s) of interest. The latter is commonly achieved by incorporating stable nitroxide radicals via site-specific mutagenesis and site-directed spin labelling (SDSL), or by site-specifically engineering artificial metal ion binding sites. Another option is substituting endogenous diamagnetic metal ions (e.g., Zn II ) with paramagnetic ones (e.g., Cu II ). Different EPR techniques provide access to local structural information around paramagnetic centres, nanometre distances between those centres employing pulse dipolar EPR spectroscopy (PDS), and binding constants. Continuous wave (CW) EPR can be used to characterise the binding geometry around the paramagnetic metal centre and to resolve interactions with nearby nuclear spins, including superhyperfine (SHF) interactions with directly coordinating nitrogen atoms. Hyperfine spectroscopy can identify the orbitals harbouring the unpaired electron and their distance to close-by magnetic nuclei; this is useful to investigate whether different binding sites are sequentially populated (in contrast to all sites having similar binding constants). One hyperfine method is EDNMR (ELDOR-detected NMR), which provides information on magnetic nuclei directly coordinated to (and thus strongly coupled to) the paramagnetic centre. Complementarily, ESEEM (electron spin echo envelope modulation) is sensitive to weakly coupled nuclei, and can therefore help elucidate interactions between paramagnetic centres and more distant nuclear spins. These experiments allow for example, assessment of the number of remote amino nitrogen atoms attributed to histidine residues present at metal ion binding sites. The corresponding HYSCORE (hyperfine sublevel correlation) experiment essentially adds a second dimension to the ESEEM experiment, and resulting cross-peaks can attribute electron-nuclear couplings to electron spin centres and ease data interpretation. To complement these hyperfine techniques, PDS experiments, such as the PELDOR method (pulsed electron-electron double resonance, or DEER), are commonly used to determine distances between paramagnetic centres on the nanometre scale. Furthermore, the number of spins or paramagnetic centres present per complex is encoded in the PELDOR modulation depth (), which is also the basis for determination of binding constants. In the present work, we employed EPR toward the characterisation of metal ion binding sites and their local environment in HRG purified from rabbit plasma, to enhance our understanding of the role of metal ions in HRG-regulated biological processes. We first established that Cu II can be used as a proxy for Zn II to enable CW and pulse EPR studies employing ITC and binding assays. Importantly, a recent report found that HRR-derived peptides not only bind Cu II but may influence copper homeostasis, indicating the potential physiological relevance of Cu II binding to HRG. Using a suite of EPR techniques including CW and hyperfine spectroscopy, we then characterised the local environment and topology of Cu II ions bound to native HRG. PDS measurements were performed to investigate the global arrangement and potential preferential order of the Cu II binding sites within the HRG protein. Here, a speciation model for the occupation of high-and low-affinity metal ion binding sites was developed based on PELDOR modulation depths. Together with structure predictions a holistic picture of HRG binding to divalent metal ions emerged with potential regulatory role on HRG function.