Precise design of artificial cofactors for enhancing peroxidase activity of myoglobin: myoglobin mutant H64D reconstituted with a "single-winged cofactor" is equivalent to native horseradish peroxidase in oxidation activity.

H64D myoglobin mutant was reconstituted with two different types of synthetic hemes that have aromatic rings and a carboxylate-based cluster attached to the terminus of one or both of the heme-propionate moieties, thereby forming a "single-winged cofactor" and "double-winged cofactor," respectively. The reconstituted mutant myoglobins have smaller K(m) values with respect to 2-methoxyphenol oxidation activity relative to the parent mutant with native heme. This suggests that the attached moiety functions as a substrate-binding domain. However, the k(cat) value of the mutant myoglobin with the double-winged cofactor is much lower than that of the mutant with the native heme. In contrast, the mutant reconstituted with the single-winged cofactor has a larger k(cat) value, thereby resulting in overall catalytic activity that is essentially equivalent to that of the native horseradish peroxidase. Enhanced peroxygenase activity was also observed for the mutant myoglobin with the single-winged cofactor, thus indicating that introduction of an artificial substrate-binding domain at only one of the heme propionates in the H64D mutant is the optimal engineering strategy for improving the peroxidase activity of myoglobin.