Metallopeptides designed as scaffolds of the spectroscopic A-Cluster in carbon monoxide dehydrogenase

Four helix-loop-helix 63mer peptides were designed and synthesized in order to assess the utility of peptides as scaffolds for the stabilization of biologically relevant bridged assemblies. The target bridged assembly was {lcub}NiII (μ₂-S·Cys)-[Fe₄S ₄]2+{rcub}, consistent with spectroscopic information on the A-Cluster of carbon monoxide dehydrogenase. The peptides consist of two helices with ca. 20 residues connected by a flexible loop containing the ferredoxin consensus sequence Cys-Ile-Ala-Cys-Gly-Ala-Cys to bind the Fe₄S ₄ cluster. For the binding of Ni(II), one of the peptides was designed with a N₃S (His₃Cys) site and each of the other three with N₂S₂ (His₂Cys₂) sites. The synthesis and characterization of the apopeptides and metallopeptides are described. All the peptides are highly stable, as assessed by guanidine hydrochloride-induced denaturation studies.; As the [Fe₄S₄]1+ cluster could not be stabilized by the 63mer peptides, it was hypothesized that the two helix-loop helix bundles of each 63mer dimeric unit are oriented in a parallel fashion and that upon reduction the two [Fe₄S₄]2+ clusters within each four-helix-bundle rearrange into one cluster of higher nuclearity. The effect of loop flexibility in the stabilization of [Fe ₄S₄]1+ was investigated with a newly designed 67mer helix-loop-helix peptide. As the [Fe₄S₄] 1+ cluster could not be obtained within the 67mer either, the findings suggest that different reaction conditions, rather than loop flexibility, might yield the desired stabilization of [Fe₄S₄] 1+ by inducing a change in the topology of the four-helix bundles.; The characterization data on the nickel-containing 63mer metallopeptides provide strong evidence that nickel(II) binds to the desired site in two of the peptides. Further evidence was gathered through nickel binding studies. The data could be analyzed for three of the 63mer-[Fe₄S₄] peptides in terms of both site-specific and stepwise binding constants. Aside from significantly weak nickel(II) binding sites, two of the metallopeptides contain 1 strong binding site per 63mer-[Fe₄S₄] unit whereas the other metallopeptide binds 0.5 Ni(II) per 63mer-[Fe₄S ₄] unit. The nature of the latter site could not be established. However, the former two sites appear to be those designed for nickel binding, demonstrating the potential utility of helix-loop-helix as scaffolds for biologically relevant bridged assemblies.