The study of the heme degradation mechanism by heme oxygenase based on ESR and NMR spectroscopied and crystal structures

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Rudi Schimd and his coworkers in the middle of 1960s discovered heme oxygenase, an enzyme that is in charge of the physiological degradation of heme. Since then, a number of researchers in the whole world have extensively devoted their efforts in the study of this enzyme. In this project, we have pursued the mechanism of the heme oxygenase reaction from the viewpoint of structural biology and obtained the following outcome. (1) We succeeded in establishing an expression system of a truncated soluble version of heme oxygenase-1, in which the C-terminal membrane-binding stretch consisting of 22 hydrophobic amino acid residues are removed. With this system we are able to routinely obtain ca. 170 mg of purified enzyme from 10-L culture (Kurume med. J. 43, 313, 1996). (2) With a home-made titration device for dioxygen and electron, we found that α -hydroxyheme can be converted to verdoheme by dioxygen in the absence of added reducing equivalents (J. Biol. Chem. 274, 18196, 1999). (3) We obtained a diffraction data with high-resolution (2.4 ^^゜__A) of heme oxygenase-1(Acta. Cryst. D54, 1017, 1998). Then, with selenomethionine mutants of heme oxygenase-1, we determined the crystal structure of rat heme oxygenase-1 (FEBS Lett. 471, 61, 2000). (4) We succeeded in preparing four verdoheme isomaers (J. Inorg. Biochem. 82, 113, 2000). (5) We reconfirmed that α -hydroxyheme can be converted to verdoheme only by dioxygen without exogenous electrons. Further we clarified the discrepancies between researchers concerning the requirement for electron are due to the preparation methods of the complex of heme oxygenase with α -hydroxyheme and also due to the reducing systems employed (Eur. J. Biochem. 269, 5231-5239, 2002). (6) We determined the crystal structures of the apo form of heme oxygenase-1 (Biochemistry 41, 7293-7300, 2002) and the azide-bound form of heme-heme oxygenase complex (J. Biol. Chem. 277, 45086-45090, 2002 )