| Abstract
| - The de novo protein DF1 is a minimal model for diiron and dimanganese metalloproteins, suchas soluble methane monooxygenase. DF1 is a homodimeric four-helix bundle whose dinuclear center isformed by two bridging Glu side chains, two chelating Glu side chains, and two monodentate His ligands.Here, we report the di-Mn(II) and di-Co(II) derivatives of variants of this protein. Together with previouslysolved structures, 23 crystallographically independent four-helix bundle structures of DF1 variants havebeen determined, which differ in the bound metal ions and size of the active site cavity. For the di-Mn(II)derivatives, as the size of the cavity increases, the number and polarity of exogenous ligands increases.This collection of structures was analyzed to determine the relationship between protein conformation andthe geometry of the active site. The primary mode of backbone movement involves a coordinated tiltingand sliding of the first helix in the helix−loop−helix motif. Sliding depends on crystal-packing forces, thesteric bulk of a critical residue that determines the dimensions of the active site access cavity, and theintermetal distance. Additionally, a torsional motion of the bridging carboxylates modulates the intermetaldistance. This analysis provides a critical evaluation of how conformation, flexibility, and active siteaccessibility affect the geometry and ligand-binding properties of a metal center. The geometric parametersdefining the DF structures were compared to natural diiron proteins; DF proteins have a restricted activesite cavity, which may have implications for substrate recognition and chemical stability.
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