Proteins that contain the heme prosthetic group are responsible for many different types of catalytic activity. Understanding the mechanisms through which a particular type of catalytic activity is favored over the others remains a significant challenge. Recently, the most common strategy for structure - function studies for a particular enzyme has involved substitution of amino acid residues by site-directed mutagenesis followed by investigations of the effect of the substitution on the catalytic activity of that system. This work describes a significant departure from this common strategy. Instead, we seek to convert a non-enzymatic hemoprotein into one that is capable of catalytic activity. In so doing, we expect to gain an understanding of the general structural requirements for particular enzymatic functions. Comparison of X-ray crystal structures of myoglobin and peroxidases reveals differences in arrangement of amino acid residues in the heme pockets. On the basis of these structural differences and the reaction mechanism of peroxidases, we have rationally designed several myoglobin mutants in order to convert myoglobin into a peroxidase-like enzyme. We have discovered that the location of the distal histidine in the active site provides a critical balance between the formation and subsequent decay of the oxo-ferryl porphyrin radical cation (compound I), a catalytic species for one- and two-electron oxidation and oxygen transfer reactions. The mutants prepared in this work have been altered in such a manner that they have permitted compound I to be observed in myoglobin for the first time. This allows us to investigate mechanistic details under single turnover conditions by use of double mixing stopped-flow spectroscopy. Furthermore, some of the mutants we have constructed might be useful as good catalysts for asymmetric oxidations. In this short review, we describe our attempts to elucidate structure-function relationships on the activation of the oxygen-oxygen bond of peroxides by hemoproteins.
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