In earlier work, de novo designed peptides with a helix-loop-helix motif and 63 residues have been synthesized as potential scaffolds for stabilization of the [Ni-II-X-Fe4S4] bridged assembly that is the spectroscopically deduced structure of the A-Cluster in clostridial carbon monoxide dehydrogenase. The 63mers contain a consensus tricysteinyl ferredoxin domain in the loop for binding an Fe4S4 cluster and Cys and His residues proximate to the loop for binding Ni(II), with one Cys residue designed as the bridge X. The metallopeptides HC4H2-[Fe4S4]-Ni and HC5H-[Fe4S4]-M, containing three His and one Cys residue for Ni(II) coordination and two His and two Cys residues for binding M = Ni(II) and Co(II), have been examined by Fe-, Ni-, and Co-K edge spectroscopy and EXAFS. All peptides bind an [Fe4S4](2+) cubane-type cluster. Interpretation of the Ni and Co data is complicated by the presence of a minority population of six-coordinate species with low Z ligands, designated for simplicity as [M(OH2)6]2+. Best fits of the data were obtained with ca. 20% [M(OH2)(6)](2+) and ca. 80% M(II) with mixed N/S coordination. The collective XAS results for HC4H2-[Fe4S4]-Ni and HC5H-[Fe4S4]-M demonstrate the presence of an Fe4S4 cluster and support the existence of the distorted square-planar coordination units [Ni-II(S.Cys)(N.HiS)(3)] and [Ni-II(S.Cys)(2).(N.His)(2)] in the HC4H2 and HC5H metallopeptides, respectively. In the HC5H metallopeptide, tetrahedral [Co-II(S-Cys)(2)(N.HiS)(2)] is present. We conclude that the designed scaffolded binding sites, including Ni-(mu(2)-Cys)-Fe bridges, have been achieved. This is the first XAS study of a de novo designed metallopeptide intended to stabilize a bridged biological assembly, and one of a few XAS analyses of metal derivatives of designed peptides. The scaffolding concept should be extendable to other bridged metal assemblies.