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

• Published in: Journal of the American Chemical Society 1999-09, Vol.121 (34), p.7747-7750
• Main Authors: Hayashi, Takashi, Hitomi, Yutaka, Ando, Tsutomu, Mizutani, Tadashi, Hisaeda, Yoshio, Kitagawa, Susumu, Ogoshi, Hisanobu
• Format: Article
• Language: English
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja9841005

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(NH3)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.