Cytoplasmic Heme-Binding Protein (HutX) from Vibrio cholerae Is an Intracellular Heme Transport Protein for the Heme-Degrading Enzyme, HutZ
Cytoplasmic Heme-Binding Protein (HutX) from Vibrio cholerae Is an Intracellular Heme Transport Protein for the Heme-Degrading Enzyme, HutZ
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DOI：
10.1021/acs.biochem.5b01273
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发表时间：
2016-02-16
期刊：
影响因子：
2.9
通讯作者：
Uchida, Takeshi
中科院分区：
文献类型：
--
作者：
Sekine, Yukari;Tanzawa, Takehito;Uchida, Takeshi
HutZ是来自霍乱弧菌的胞质血红素结合蛋白。尽管我们先前已经将HutZ鉴定为血红素降解酶[Uchida，T.，等人（2012）Chem.Commun. 48，6741-6743]，HutZ的血红素转运蛋白仍然未知。为了鉴定HutZ的血红素转运蛋白，我们专注于血红素利用操纵子hutWXZ。为此，我们在大肠杆菌中构建了HutX的表达系统，并将其纯化至均一。吸收光谱分析表明，HutX与血红素结合的化学计量比为1：1，解离常数为7.4 nM。HutX的晶体结构显示出与来自大肠杆菌的同源蛋白ChuX相似的折叠。coli O157：H7。HutX和ChuX的结构比较以及血红素-HutX的共振拉曼光谱表明铁血红素的轴向配体是Tyr 90。与HutX结合的血红素以8.3 × 10（-2）和1.5 × 10（-2）s（-1）的双相解离动力学转移到HutZ，该值明显大于从HutX转移到脱辅基肌红蛋白的值。表面等离子体共振实验证实，HutX与HutZ相互作用的解离常数类似于400 μ M。这些结果表明，血红素从HutX转移到HutZ通过特定的蛋白质蛋白质相互作用。因此，我们可以得出结论，HutX是HutZ的胞质血红素转运蛋白。
HutZ is a cytoplasmic heme-binding protein from Vibrio cholerae. Although we have previously identified HutZ as a heme-degrading enzyme [Uchida, T., et al. (2012) Chem. Commun. 48, 6741-6743], the heme transport protein for HutZ remained unknown. To identify the heme transport protein for HutZ, we focused on the heme utilization operon, hutWXZ. To this end, we constructed an expression system for HutX in Escherichia coli and purified it to homogeneity. An absorption spectral analysis demonstrated that HutX binds heme with a 1:1 stoichiometry and a dissociation constant of 7.4 nM. The crystal structure of HutX displays a fold similar to that of the homologous protein, ChuX, from E. coli O157:H7. A structural comparison of HutX and ChuX, and resonance Raman spectra of heme-HutX, suggest that the axial ligand of the ferric heme is Tyr90. The heme bound to HutX is transferred to HutZ with biphasic dissociation kinetics of 8.3 x 10(-2) and 1.5 x 10(-2) s(-1), values distinctly larger than those for transfer from HutX to apomyoglobin. Surface plasmon resonance experiments confirmed that HutX interacts with HutZ with a dissociation constant of similar to 400 mu M. These results suggest that heme is transferred from HutX to HutZ via a specific protein protein interaction. Therefore, we can conclude that HutX is a cytoplasmic heme transport protein for HutZ.