Subunit-subunit interactions play a key role in the heme-degradation reaction of HutZ from Vibrio cholerae
Subunit-subunit interactions play a key role in the heme-degradation reaction of HutZ from Vibrio cholerae
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亚基-亚基相互作用在霍乱弧菌 HutZ 的血红素降解反应中发挥关键作用
DOI：
10.1039/c9dt00604d
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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;Ota Kazuki;Sekine Yukari;Dojun Nobuhiko;Ishimori Koichiro
来自霍乱弧菌的二聚体蛋白HutZ是催化血红素的氧依赖性降解的蛋白质。有趣的是，HutZ的抗坏血酸支持的血红素降解活性取决于pH：在pH 8.0时，HutZ降解不到10%的血红素，但在pH 6.0时，几乎90%的血红素被降解。在这里，我们研究了pH依赖的构象变化，在HutZ使用荧光光谱。Trp 109估计位于血红素的大约21处，并且存在于含有血红素轴向配体的不同亚基中。因此，我们假设血红素和Trp 109之间的距离反映了亚基-亚基取向的变化。基于Trp 109到血红素的共振能量转移，我们估计血红素和Trp 109之间的距离在pH 8.0时约为17 μ m，而在pH 6.0时距离增加不到2 μ m。我们推测，这种变化导致电子捐赠从近端组氨酸的减少，从而导致血红素降解活性的增强。为了证实这种情况下，我们突变的Ala 31，位于二聚体接口，缬氨酸改变通过亚基-亚基相互作用的距离。A31 V突变体的血红素和Trp 109之间的距离延长至24-27 nm。虽然共振拉曼光谱和还原速率的血红素表明，这种突变导致减少电子捐赠从血红素轴向配体，抗坏血酸支持血红素降解活性没有观察到。根据我们的研究结果，可以提出，两个原聚体的相对定位是重要的，在确定血红素降解率由HutZ。
HutZ, a dimeric protein, from Vibrio cholerae is a protein that catalyzes the oxygen-dependent degradation of heme. Interestingly, the ascorbic acid-supported heme-degradation activity of HutZ depends on pH: less than 10% of heme is degraded by HutZ at pH 8.0, but nearly 90% of heme is degraded at pH 6.0. We examined here pH-dependent conformational changes in HutZ using fluorescence spectroscopy. Trp109 is estimated to be located approximately 21 Å from heme and is present in a different subunit containing a heme axial ligand. Thus, we postulated that the distance between heme and Trp109 reflects subunit–subunit orientational changes. On the basis of resonance energy transfer from Trp109 to heme, we estimated the distance between heme and Trp109 to be approximately 17 Å at pH 8.0, while the distance increased by less than 2 Å at pH 6.0. We presumed that such changes led to a decrease in electron donation from the proximal histidine, resulting in enhancement of the heme-degradation activity. To confirm this scenario, we mutated Ala31, located at the dimer interface, to valine to alter the distance through the subunit–subunit interaction. The distance between heme and Trp109 for the A31V mutant was elongated to 24–27 Å. Although resonance Raman spectra and reduction rate of heme suggested that this mutation resulted in diminished electron donation from the heme axial ligand, ascorbic acid-supported heme-degradation activity was not observed. Based on our findings, it can be proposed that the relative positioning of two protomers is important in determining the heme degradation rate by HutZ.