Mechanism of heme degradation by heme oxygenase

(1)To identify the axial heme ligand of heme oxygenase-2(HO-2)，we prepared His45to Ala(H45A)mutant。H45A was completely devoid of the heme dedradation activity。A5-coordinate-type ferrous NO EPR spectrum was observed for the heme-H45A complex.These indicate that His45is the proximal ligand of heme oxygenase-2.(2)The O-I D22-D2 and CO reactions with the heme，hydroxyheme，and verdoheme complexes of HO were studied。The O I D 22 D 2 affinities for heme and hydroxyheme are very high，but the CO affinities are only 1-6-fold higher than the O I D 22 I D 2 affinities.Thus，HO discriminates much more strongly against CO binding than myoglobin.(3)On the basis of Raman spectra of O I D22 ii D2-bound form of the heme-HO complex，a highly bent Fe-O-O geometry has been proposed。However，the interaction of bound oxygen with the distal amino acid residue has not been identified.To clarify this，we have carried out EPR measurements of the cobalt(II)porphyrin HO complex and revealed that the bound-O I D22ii D2 formes hydrogen-bond interactions with distal amino acid residue。(4)We investigated the mechanism of the conversion of hydroxyhemin to verdoheme and confirmed that our previous conclusion that this step requires one reducing equivalent along with molecular oxygen。(5)We analyzed the first step of the heme degradation。We found that the molecular oxygen bound to heme-HO complex is stabilized by an H-bond and that hydroperoxy-HO genarated by cryoreduction catalyzes the formation of hydroxyheme.(6)We established the expression system of bacterial HO(Hmu O)。Hmu O binds hemin stoichiometrically to form a hexacoordinate high spin hemin-Hmu O complex.When ascorbic acid is used as the electron donor，Hmu O converted hemin to biliverdin with hydroxyhemin and verdoheme as intermediates.Other enzymatic and protein-chemical properties closely resembled those of mammalian HOs.

(1)To identify the axial heme ligand of heme oxygenase-2 (HO-2), we prepared His45 to Ala (H45A) mutant. H45A was completely devoid of the heme dedradation activity. A 5-coordinate-type ferrous NO EPR spectrum was observed for the heme-H45A complex. These indicate that His 45 is the proximal ligand of heme oxygenase-2. (2)The OィイD22ィエD2 and CO reactions with the heme, hydroxyheme, and verdoheme complexes of HO were studied. The OィイD22ィエD2 affinities for heme and hydroxyheme are very high, but the CO affinities are only 1-6-fold higher than the OィイD22ィエD2 affinities. Thus, HO discriminates much more strongly against CO binding than myoglobin. (3)On the basis of Raman spectra of OィイD22ィエD2-bound form of the heme-HO complex, a highly bent Fe-O-O geometry has been proposed. However, the interaction of bound oxygen with the distal amino acid residue has not been identified.To clarify this, we have carried out EPR measurements of the cobalt(II) porphyrin HO complex and revealed that the bound-OィイD22ィエD2 formes hydrogen-bond interactions with distal amino acid residue. (4)We investigated the mechanism of the conversion of hydroxyhemin to verdoheme and confirmed that our previous conclusion that this step requires one reducing equivalent along with molecular oxygen. (5)We analyzed the first step of the heme degradation. We found that the molecular oxygen bound to heme-HO complex is stabilized by an H-bond and that hydroperoxy-HO genarated by cryoreduction catalyzes the formation of hydroxyheme. (6)We established the expression system of bacterial HO (Hmu O). Hmu O binds hemin stoichiometrically to form a hexacoordinate high spin hemin-Hmu O complex. When ascorbic acid is used as the electron donor, Hmu O converted hemin to biliverdin with hydroxyhemin and verdoheme as intermediates. Other enzymatic and protein-chemical properties closely resembled those of mammalian HOs.