Flavin-containing siderophore-interacting protein of Shewanella putrefaciens DSM 9451 reveals substrate specificity in ferric-siderophore reduction

Shewanella are bacteria widespread in marine and brackish water environments and emergent opportunistic pathogens. Their environmental versatility is highly dependent on the ability to produce an abundance of iron-rich proteins, mainly multiheme c -type cytochromes. Although iron plays a vital role in the ability of Shewanella species to survive in various environments, very few studies exist regarding the strategies by which these bacteria scavenge iron from the environment. Small molecule siderophore-mediated iron transport is a strategy commonly employed for iron acquisition, and it was identified amongst Shewanella spp. over two decades ago. Shewanella species produce hydroxamate-type siderophores and iron removal from these compounds can occur in the cytoplasm via Fe(III)-siderophore reduction mediated by siderophore-interacting proteins (SIPs). The genome of Shewanella putrefaciens DSM 9451 isolated from an infected child contains representatives of the two different cytosolic families of SIPs: the flavin-containing siderophore interacting protein family (SIP) and the iron−sulfur cluster-containing ferric siderophore reductase family (FSR). Here, we report the expression and purification of the flavin-containing ( Sb SIP) and iron-sulfur cluster-containing ( Sb FSR) Fe(III)-siderophore reductases of Shewanella putrefaciens DSM 9451. The structural and functional characterization of Sb SIP shows distinct features from the highly homologous SIP from Shewanella frigidimarina ( Sf SIP). These include significant structural differences, different binding affinities for NADH and NADPH, and lower rates of Fe(III)-siderophore reduction, results which consolidate in the putative identification of the binding pocket for these proteins. Overall our work highlights NADH and NADPH specificity and the different Fe(III)- siderophore reduction abilities of the SIP family suggesting a tailoring of these enzymes towards meeting different microbial iron requirements. ### Competing Interest Statement The authors have declared no competing interest.