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 apomyoglobi n from horse heart to obtain a new reconstituted myoglobin, rMb(l), with a unique binding domain structure. The UV-vis, CD, and NMR spectra of rMb(l) are comparable with those of native myoglobin, nMb. The mixing of rMb(l) wi th hydrogen peroxide yields a peroxidase compound II-like species, rMb(l)-I I, since the spectrum of rMb(1)-II is identical with that observed for nMb. Stoichiometric oxidation of several small molecules by rMb(1)-II, demonstr ates the significant reactivity. (i) The oxidation of cationic substrate su ch as [Ru(NH3)(6)](2+) by rMb(1)-II is faster than that observed far oxofer ryl species of nMb, nMb-II. (ii) Anionic substrates such as ferrocyanide ar e unsuitable for the oxidation by rMb(1)-II. (iii) Oxidations of catechol, hydroquinone, and guaiacol are dramatically enhanced by rMb(l)-II(14-32- fo ld) compared to those observed for nMb-II. Thus, the chemical modification of heme-propionates can alter substrate specificity. Steady-state kinetic m easurements 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 tha t the artificially modified heme-propionates may increase the accessibility of neutral aromatic substrates to the heme active site. The present work d emonstrates that the chemical mutation of prosthetic group is a new strateg y to create proteins with engineered function.