Mechanism of heme degradation by heme oxygenase

（1）To identify the axial heme ligand of heme oxygenase-2（HO-2）， we prepared His45 to Ala （H45A） mutant。H45 A完全违背了Heme分流活动的规定。A5-coordinate型ferrous NO EPR谱被观察到用于Heme-H45 A复合体。这些指标表明，他的45是血红素氧合酶的正分子配体-2。（2）The OイイD22イエD2 and CO reactions with the heme， hydroxyheme， and verdoheme complexes of HO were studied。The O-I D22 YE D2 Affinities for heme and hydroxyheme非常高，但CO Affinities are only 1- 6-fold higher than the O-I D22 YE D2 Affinities。因此， HO发现更多的是对CO绑定比myoglobin更强。（3）在O-Y-D22-D2-bound形式的基础上，一个高度弯曲的Fe-O-O几何图形已经提出。However，与distal amino acid residue的绑定氧的相互作用尚未被识别。为了澄清这一点，我们已经提出了钴的EPR测量（II）卟啉HO复合体，并揭示了与distal amino acid residue的绑定O型D22型氢键相互作用。（4）我们研究了氢氧素转化为verdoheme的机制，并确认了我们先前的结合，这一步需要一个减少分子氧的相等性。（5）我们分析了对Heme降级的第一步。我们发现了分子氧结合到Heme-HO复合体是由H-邦德和由低温催化生成的羟基-HO复合体的形成。（6）We established the expression system of bacterial HO （Hmu O）。Hmu O binds Hemin stoichiometrically形成了一个高自旋Hemin-Hmu O复合体。当鞘酸被用作电子捐赠者时， Hmu O将血红素转化为含有羟基血红素和苯多烯的双氧嘧啶。其他酶和蛋白质化学性质封闭地重新组装了哺乳动物的那些。

(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.