Peroxidase Activity of Myoglobin Is Enhanced by Chemical Mutation of Heme-Propionates

Peroxidase activity of a myoglobin reconstituted with a chemically modified heme 1 is reported. The heme 1 bearing a total of eight carboxylates bound to the terminal of propionate side chains is incorporated into apomyoglobin from horse heart to obtain a new reconstituted myoglobin, rMb( 1 ), with a unique binding domain structure. The UV−vis, CD, and NMR spectra of rMb( 1 ) are comparable with those of native myoglobin, nMb. The mixing of rMb( 1 ) with hydrogen peroxide yields a peroxidase compound II-like species, rMb( 1 )-II, since the spectrum of rMb( 1 )-II is identical with that observed for nMb. Stoichiometric oxidation of several small molecules by rMb( 1 )-II, demonstrates the significant reactivity. (i) The oxidation of cationic substrate such as [Ru(NH 3 ) 6 ] 2+ by rMb( 1 )-II is faster than that observed for oxoferryl species of nMb, nMb-II. (ii) Anionic substrates such as ferrocyanide are unsuitable for the oxidation by rMb( 1 )-II. (iii) Oxidations of catechol, hydroquinone, and guaiacol are dramatically enhanced by rMb( 1 )-II (14−32-fold) compared to those observed for nMb-II. Thus, the chemical modification of heme-propionates can alter substrate specificity. Steady-state kinetic measurements indicate that both the reactivity and substrate affinity toward guaiacol oxidation by rMb( 1 ) are improved, so that the specificity, k cat / K m, is 13-fold higher than that in nMb. This result strongly suggests that the artificially modified heme-propionates may increase the accessibility of neutral aromatic substrates to the heme active site. The present work demonstrates that the chemical mutation of prosthetic group is a new strategy to create proteins with engineered function.