IDENTIFICATION OF HISTIDINE-25 AS THE HEME LIGAND IN HUMAN LIVER HEME OXYGENASE
IDENTIFICATION OF HISTIDINE-25 AS THE HEME LIGAND IN HUMAN LIVER HEME OXYGENASE
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DOI：
10.1021/bi00250a026
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发表时间：
1994-11-22
期刊：
影响因子：
2.9
通讯作者：
DEMONTELLANO, PRO
中科院分区：
文献类型：
--
作者：
SUN, J;LOEHR, TM;DEMONTELLANO, PRO
据报道，对人肝血红素加氧酶 (HO) 的 His25Ala 突变体及其与血红素的复合物进行了电子和共振拉曼光谱研究。在酶.底物复合物的氧化（三价铁）形式中，血红素处于高自旋、五配位状态。这与野生型酶中的相同复合物不同，在野生型酶中，血红素是六配位的，在类似水合肌红蛋白的环境中与近端组氨酸和水分子连接。 H25A 血红素加氧酶突变体复合物的还原（亚铁）形式失去了非常显着的共振拉曼带，类似于野生型复合物中所见的 217 cm(-1)，该共振拉曼带已被明确指定为近端 Fe-N(His) 振动频率 [Sun et al. 2017]。 (1993) 生物化学 32, 14151;高桥等人。 (1993) 生物化学 33, 1010]。突变蛋白光谱中缺乏该条带，明确表明 His 25 是血红素底物的近端配体。此外，这种亚铁血红素-H25A H2O复合物作为五配位高自旋物质和四配位中间自旋物质之间的平衡混合物存在。尽管 H25A 突变蛋白没有表现出血红素加氧酶活性，但血红素能够结合一氧化碳。对 H25A HO 复合物的 CO 加合物的研究表明，nu(CO) 和 nu(Fe-CO) 频率分别为 1960 cm(-1) 和 529 cm(-1)，这是血红素上具有弱近端配体的疏水性一氧化碳结合位点的特征。此外，这些频率表明加合物中CO反式的配体不可能是组氨酸，这一结果与具有点突变的酶中不存在His 25配体完全一致。血红素-H25A HO 的三价铁和二价铁形式的第五配体被排除为来自另一种组氨酸或酪氨酸。然而，目前尚不清楚弱配体的确切身份以及附着位点（相对于野生型酶的近端与远端）。
Electronic and resonance Raman spectroscopic studies are reported for the His25Ala mutant of human liver heme oxygenase (HO) and its complex with heme. In the oxidized (ferric) form of the enzyme.substrate complex, the heme is shown to be in a high-spin, five-coordinate state. This is distinct from the same complex in the wild-type enzyme in which the heme is six-coordinate, ligated to a proximal histidine and a water molecule in an environment reminiscent of aquometmyoglobin. The reduced (ferrous) form of the complex of the H25A heme oxygenase mutant has lost the very prominent resonance Raman band at similar to 217 cm(-1) seen in the wild-type complex that has been unambiguously assigned to the proximal Fe-N(His) vibrational frequency [Sun et al. (1993) Biochemistry 32, 14151; Takahashi et al. (1993) Biochemistry 33, 1010]. The absence of this band in the spectrum of the mutant protein definitively identifies His 25 as the proximal ligand of the heme substrate. Furthermore, this ferrous heme-H25A HO complex exists as an equilibrium mixture between a five-coordinate, high-spin species and a four-coordinate, intermediate-spin species. Although the H25A mutant protein shows no heme oxygenase activity, the heme is competent to bind carbon monoxide. Studies of the CO adduct of the H25A HO complex show nu(CO) and nu(Fe-CO) frequencies at 1960 and 529 cm(-1), respectively, that are characteristic of a hydrophobic carbon monoxide binding site on a heme with a weak proximal ligand. Furthermore, these frequencies indicate that the ligand trans to the CO in the adduct cannot be a histidine, a result that is entirely consistent with the absence of the His 25 ligand in the enzyme with the point mutation. The fifth ligand in the ferric and ferrous forms of the heme-H25A HO is ruled out as being from another histidine or a tyrosine. However, the exact identity of the weak ligand as well as the site of attachment (proximal versus distal, relative to the wild type enzyme) are not known at the present time.