HEME OXYGENASE-2 - PROPERTIES OF THE HEME COMPLEX OF THE PURIFIED TRYPTIC FRAGMENT OF RECOMBINANT HUMAN HEME OXYGENASE-2

Recombinant human microsomal heme oxygenase-2 was expressed in Escheri chia coli. Tryptic digestion of the membrane fraction, in which the wi ld-type enzyme was localized, yielded a soluble tryptic peptide of 28 kDa, which retained the ability to accept electrons from NADPH-cytochr ome P-450 reductase and the enzymatic activity for conversion of heme to biliverdin. The tryptic fragment, when purified to apparent homogen eity, bound one equivalent of heme to form a substrate-enzyme complex that had spectroscopic properties characteristic of heme proteins, suc h as myoglobin and hemoglobin. Optical absorption, Raman scattering, a nd EPR studies of the heme-tryptic fragment complex revealed that the ferric heme was six coordinate high spin at neutral pH and six coordin ate low spin at alkaline pH, with a pK(a) value of 8.5. EPR and Raman scattering studies indicated that a neutral imidazole of a histidine r esidue served as the proximal ligand in the heme-heme oxygenase-2 frag ment complex. The reaction with hydrogen peroxide converted the heme o f the heme oxygenase 2 fragment complex into a verdoheme-like intermed ate, while the reaction with m-chloroperbenzoic acid yielded a oxoferr yl species. These spectroscopic properties are similar to those obtain ed for heme oxygenase-1, and thus the catalytic mechanism of heme oxyg enase-2 appears to be similar to that of heme oxygenase-1.