Burkholderia pseudomallei rubrerythrin promiscuously binds metals in a structurally pre-formed bimetallic binding site

Rubrerythrins are a group of proteins within the Ferritin-like superfamily that display a defining four-helix bundle domain. They also show multiple structural features that are crucial to their functionality as iron storage proteins and in detoxification and oxidative stress response. Here we investigate rubrerythrin (Rbr) in multiple metalated states, from the pathogenBurkholderia pseudomallei(Bp). We use X-ray crystallography for structure determination of Rbr to probe the capacity and specificity of metal binding.BpRbr lacks the rubredoxin moiety found in canonical Rbrs from anaerobic lineages, and we demonstrate thatBpRbr also possesses a domain-swapped dimer, which has functional implications for its putative role in oxidative stress response. We also carry outin crystallospectroscopic assessment ofBpRbr with various metals, using energy dispersive X-ray (EDX) spectroscopy. We observe that samples can contain metals other than those supplied in crystallization conditions, and developed a strategy of utilizing EDX spectroscopy to select those samples with single metal incorporation for downstream diffraction data collection. Our work underscores the importance of spectroscopic probing for definitive metal identification and characterization.