SlyD is a Ni(II)-binding protein that contributes to nickel homeostasis in Escherichia coli. The Cterminal domain of SlyD contains a rich variety of metal-binding amino acids, suggesting broader metal-binding capabilities, and previous work demonstrated that the protein can coordinate several types of first row transition metals. However, the binding of SlyD to metals other than Ni(II) has not been previously characterized. To further our understanding of the in vitro metal-binding activity of SlyD and how it correlates with the in vivo function of this protein, the interactions between SlyD and the series of biologically relevant transition metals Mn(II), Fe(II), Co(II), Cu(I) and Zn(II) were examined by using a combination of optical spectroscopy and mass spectrometry. Tables S1-S3 containing a summary of metal concentrations used for metal toxicity studies, a list of primers used for qRT-PCR analysis and summary of Mn(II), Co(II) and Ni(II) stoichiometries of SlyD; Figure S2 shows representative spectra of a Ni(II) titrations analyzed via ESI-MS; Figures S3-S4 shows a graphical representation of the PAR competition assay used for determining the average K D (Zn(II)-SlyD) and a comparison of CD spectra for Ni(II) and Zn(II) bound SlyD. Figure S5 is a representative data set for a Cu(I) titration of SlyD analyzed via electronic absorption spectroscopy. Figure S6-S7 are competition titrations with Bca and Bcs used for determining copper stoichiometry and affinity, respectively. Figure S8-S10 are a graphical representation of Co(II) titrations analyzed via ESI-MS, competition titrations with Fura2 for determining average K D (Co(II)-SlyD) and titration of a cobalt saturated SlyD sample with Zn(II). Figure S11 is spectroscopy data obtained for titration of Ni(II) compared with a similar titration carried out in the presence of excess Fe(II). Figure S12 depicts representative growth curves of WT and ΔslyD strains conducted in minimal media supplemented with various amounts of metal. Figure S13 is relative mRNA levels measured for various metal transporters under aerobic conditions. Figure S14-S15 are relative mRNA levels measured for various metal transporters under anaerobic conditions. This material is available free of charge via the Internet at http://pubs.acs.org.