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

(1)测定了α-羟基血红素和马鞭铁血红素加氧酶(HO)络合物的共振拉曼光谱。我们发现，铁的α-羟基血红素和铁的马鞭铁络合物表现出非典型的拉曼光谱，这被解释为卟啉-共轭的电子体系对称性降低的结果。(2)为了确定HO-2的轴向血红素配基，我们制备了His45到Ala(H45A)和Hisi 52到Ala(Hi 52A)的突变体。H45A完全不具有血红素的脱水活性。血红素-H45A复合体具有5配位型亚铁电子顺磁共振波谱，而H152A突变体的光谱和酶学性质与野生型相同。HO-1的His132对血红素的降解也不重要。(3)研究了HO的亚铁血红素、羟基亚铁血红素和马鞭铁血红素络合物与O_2和CO的反应。血红素和羟基血红素的02亲和力很高，但CO亲和力只比O2亲和力高1-6倍。因此，HO对CO结合的区分能力比肌红蛋白强得多。马鞭铁血红素络合物的CO亲和力大约是血红素络合物的一万倍。(4)根据O_2-HO络合物的拉曼光谱，提出了一个高度弯曲的Fe-O-O几何构型。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(II)卟啉HO络合物进行了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.