Role of His63 in HutZ from Vibrio cholerae in the heme degradation reaction and heme binding
Role of His63 in HutZ from Vibrio cholerae in the heme degradation reaction and heme binding
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霍乱弧菌 HutZ 中 His63 在血红素降解反应和血红素结合中的作用
DOI：
10.1039/c9dt00926d
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发表时间：
2019
影响因子：
4
通讯作者：
Ishimori Koichiro
中科院分区：
文献类型：
--
作者：
Uchida Takeshi;Dojun Nobuhiko;Sekine Yukari;Ishimori Koichiro
来自霍乱弧菌的HutZ是一种二聚体酶，其通过与哺乳动物血红素加氧酶类似的催化机制催化血红素的氧依赖性降解。然而，与血红素加氧酶不同，HutZ以1：1的比例氧化血红素的β-或δ-内消旋位置，血红素加氧酶仅在α-内消旋位置引发血红素的降解。 His 63是与血红素7-丙酸酯基团潜在地形成氢键的残基。为了建立His 63在HutZ和血红素结合的血红素降解的区域选择性中的作用，我们构建了His 63的突变体。有趣的是，H63 L突变体保留了与野生型HutZ相当水平的β-或δ-区域选择性。抗坏血酸辅助血红素降解的HutZ是pH值依赖性的，在pH 6.0，但不高于pH 8.0的活性。与野生型蛋白质相比，H63 L突变体是无活性的，即使在pH 6.0下，与pH 8.0下的血红素结合亲和力相比，对血红素的亲和力显著降低，如Asp 132至瓦尔的突变体所观察到的，Asp 132至Val的突变体位于血红素轴向配体His 170的氢键距离内，但在不同的原聚体中。此外，血红素和Trp 109之间的距离从野生型HutZ的16-18 μ m增加到H63 L突变体的24-28 μ m，这表明突变改变了原聚体的方向，因为Trp 109位于血红素轴向配体的另一个亚基中。我们的研究结果共同表明，His 63定位在血红素附近不有助于血红素降解的区域选择性，但在维持HutZ的亚基的取向，以发挥血红素降解的功能中起着关键作用。
HutZ from Vibrio cholerae is a dimeric enzyme that catalyzes oxygen-dependent degradation of heme via a similar catalytic mechanism to mammalian heme oxygenase. However, HutZ oxidizes the β- or δ-meso position of heme at a ∼1 : 1 ratio distinct from heme oxygenase, which initiates the degradation of heme solely at the α-meso position. His63 is a residue that potentially forms hydrogen bond with the heme 7-propionate group. To establish the role of His63 in regioselectivity of heme degradation by HutZ and heme binding, we constructed mutants of His63. Interestingly, the H63L mutant retained a comparable level of β- or δ-regioselectivity as wild-type HutZ. Ascorbic acid-assisted heme degradation by HutZ is pH-dependent, showing activity at pH 6.0 but not above pH 8.0. Compared to the wild-type protein, the H63L mutant was inactive, even at pH 6.0, and affinity for heme was significantly decreased in contrast with a comparable heme binding affinity at pH 8.0, as observed for the mutant of Asp132 to Val, which is located within hydrogen bonding distance of the heme axial ligand His170, but in a different protomer. In addition, the distance between heme and Trp109 increased from 16–18 Å for wild-type HutZ to 24–28 Å for the H63L mutant, indicating that protomer orientation is altered by the mutation, since Trp109 is in another subunit of the heme axial ligand. Our results collectively suggest that His63 positioned near heme does not contribute to regioselectivity of heme degradation but plays a key role in maintaining the orientation of subunits for HutZ to function of heme degradation.