Molucular mechanisms of oxygen activation at the three steps in heme oxygenase reaction

（1）测量了血红素加氧酶（HO）的α-羟基血红素和绿血红素复合物的共振拉曼光谱。我们发现，铁的α-羟基血红素和亚铁绿血红素配合物显示出非典型的拉曼模式，这被解释为卟啉共轭π电子系统的对称性降低的结果。(2)To为了鉴定HO-2的轴向血红素配体，我们制备了His 45到Ala（H45 A）和Hisi 52到Ala（Hi52 A）突变体。H45 A完全没有血红素降解活性。血红素-H45 A复合物的EPR谱为5配位型亚铁NO EPR谱，而H152 A突变体的光谱和酶性质与野生型完全相同。HO-1的His 132对血红素的降解也不重要。（3）研究了HO的血红素、羟基血红素和绿血红素配合物与O_2和CO的反应。O_2对血红素和羟基血红素的亲和力非常高，但CO亲和力仅比O_2亲和力高1-6倍。因此，HO比肌红蛋白更强烈地歧视CO结合。绿血红素络合物的CO亲和力比血红素络合物的CO亲和力弱约10，000倍。(4)On根据血红素-HO络合物O_2结合态的拉曼光谱，提出了一种高度弯曲的Fe-O-O构型。然而，结合氧与远端氨基酸残基的相互作用尚未确定。为了阐明这一点，我们对钴卟啉HO配合物进行了EPR测量，发现结合的-O_2与远端氨基酸残基形成氢键相互作用。

(1)The resonance Raman spectra for alpha-hydroxyheme and verdoheme complexes of heme oxygenase (HO) was measured. We found that the ferric alpha-hydroxyheme and ferrous verdoheme complexes showed atypical Raman patterns, which are interpreted as the result of the symmetry lowering of the porphyrin-conjugating pi-electron system. (2)To identify the axial heme ligand of HO-2, we prepared His45 to Ala (H45A) and Hisi 52 to Ala (Hi 52A) mutants. H45A was completely devoid of the heme dedradation activity. A 5-coordinate-type ferrous NO EPR spectrum was observed for the heme-H45A complex, On the contrary, H152A mutant exhibited spectroscopic and enzymatic properties identical to those of wild-type. His132 of HO-1 was also not important for the heme degradation. (3)The O_2 and CO reactions with the heme, hydroxyheme, and verdoheme complexes of HO were studied. The 02 affinities for heme and hydroxyheme are very high, but the CO affinities are only 1-6-fold higher than the O_2 affinities. Thus, HO discriminates much more strongly against CO binding than myoglobin. The CO affinities of the verdoheme complexes are about 10,000 times weaker than those of the heme complex. (4)On the basis of Raman spectra of O_2-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_2 forms hydrogen-bond interactions with distal amino acid residue.