L-Penicillamine (Pen) has been investigated as a ligand for metalloprotein design by examining the binding of Co(II) to the sequence NH2-KL(Pen)EGG.(Pen)IG(Pen)GA(Pen).GGW-CONH2. For comparison, we have studied Co(II) binding to the analogous sequence with Cys ligands, the ferredoxin maquette ligand IGA that was originally designed to bind a [4Fe-4S] cluster. The Co(II) affinity and UV-vis spectroscopic properties of IGA indicate formation of a pseudotetrahedral tetrathiolate ligated Co(II). In contrast, IGA-Pen showed formation of a pseudotetrahedral complex with Co(II) bound by three Pen ligands and an exogenous H2O. EXAFS data on both Co(II) complexes confirms not only the proposed primary coordination spheres but also shows six Co(II)-C-beta methyl group distances in Co(II)-IGA-Pen. These results demonstrate that ligand sterics in simple peptides can be designed to provide asymmetric coordination spheres such as those commonly observed in natural metalloproteins.