Functions of fluctuation in the heme-binding loops of cytochrome b5 revealed in the process of heme incorporation
Functions of fluctuation in the heme-binding loops of cytochrome b5 revealed in the process of heme incorporation
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DOI：
10.1021/bi9922289
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发表时间：
2000-05-23
期刊：
影响因子：
2.9
通讯作者：
Morishima, I
中科院分区：
文献类型：
--
作者：
Ihara, M;Takahashi, S;Morishima, I
细胞色素B（5）（cyt B（5））利用位于两个血红素结合环中心的两个轴向组氨酸His 63和His 39来固定血红素。先前对细胞色素B（5）的apo形式（apocyt B（5））的NMR研究揭示，与包括His 39的其它环相比，包括His 63的环表现出更大的波动[Falzone，C. J.，迈耶，M。R.，Whiteman，E. L.，摩尔角D、和Lecomte，J.T.（1996）Biochemistry 35，6519-6526]。为了理解这种波动的意义，我们使用两种细胞色素B（5）的突变体（其中一个轴向组氨酸被亮氨酸取代）比较了两个环的血红素结合和解离速率。结果表明，波动环具有显着较慢的血红素解离速率和较快的血红素缔合速率比其他环。为了进一步验证波动环的重要性，使用光学吸收和CD光谱研究了野生型脱细胞B（5）的血红素缔合过程。结果表明，该过程通过两个途径进行，并且主导途径涉及位于波动环中的His 63的初始协调。尿素浓度依赖性的速率常数表明，折叠的波动回路与His 63的协调。有人建议，波动使环有一个更大的血红素结合构象的血红素环接触。这种起伏的血红素结合环可能有助于血红素结合蛋白的人工设计。
Cytochrome b(5) (cyt b(5)) holds heme using two axial histidines, His63 and His39, that are located in the centers of the two heme-binding loops. The previous NMR study on the apo form of cyt b(5) (apocyt b(5)) revealed that the loop including His63 exhibits a larger fluctuation compared to the other loop including His39 [Falzone, C. J., Mayer, M. R., Whiteman, E. L., Moore, C. D., and Lecomte, J. T. (1996) Biochemistry 35, 6519-6526]. To understand the significance of the fluctuation, the heme association and dissociation rates of the two loops were compared using two mutants of cyt b(5) in which one of the axial histidines was replaced with leucine. It was demonstrated that the fluctuating loop possesses a significantly slower heme dissociation rate and a faster heme association rate than the other loop. To further verify the importance of the fluctuating loop, the heme association process of wild-type apocyt b(5) was investigated using optical absorption and CD spectroscopies. It was indicated that the process proceeds through the two pathways, and that the dominant pathway involves the initial coordination of His63 located in the fluctuating loop. The urea concentration dependency of the rate constants revealed that the folding of the fluctuating loop is associated with the coordination of His63. It was suggested that the fluctuation enables the loop to have a larger heme-loop contact in the heme-bound conformation. The fluctuating heme-binding loops might be useful for the artificial design of heme-binding proteins.