The high-affinity nickel-binding site of the Escherichia coli [NiFe]-hydrogenase accessory protein HypB was localized to residues at the immediate N-terminus of the protein. Modification of a metal-binding fusion protein, site-directed mutagenesis experiments, and DFT calculations were used to identify the N-terminal amine as a ligand as well as the three cysteine residues in the CXXCGCXXX motif. This sequence can be removed from the protein and both a synthesized peptide and a protein fusion bind nickel with a similar affinity and the same structure as the parent metalloprotein, indicating the self-sufficiency of this high-affinity nickel-binding sequence.