Correlations between catalytic function and the number and orientation of active site His residues in binuclear non-heme iron enzymes suggest that they play an important role in functional tuning. De novo four-helix bundle proteins such as DFsc are uniquely suited for studying these structure-function relationships due to their simplicity, stability, and ease of mutation. The recent alteration of function from oxidation to N-hydroxylation in DFsc through four mutations (one active site His and three supporting mutations) demonstrates the feasibility of using this scaffold for investigations into the geometric and electronic factors that influence catalytic tuning of di-iron active sites. To complement the existing 2-His/4-carboxylate and 3-His/4-carboxylate variants of DFsc, we have rationally redesigned the DFsc active site to mimic those found in nature that contain additional carboxylates (rubrerythrins, symerythrin) or additional histidines (FprA, MIOX).  The secondary structure, stability, and metal-binding capacity of these new proteins have been characterized.  In addition, their ability to catalyze a range of O2- and H2O2-dependent reactions has been investigated.  As the active sites of the proteins in this series differ stepwise by either a single His or carboxylate residue, comparisons of the spectroscopic and geometric properties of these structurally-similar but functionally distinct proteins provide insight into the roles of charge and coordination number in tuning catalytic activity.

Using de novo protein models to understand functional tuning in binuclear non-heme iron enzymes

248th National Meeting (2014)

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