Unfolding the unique c-type heme protein, Chlamydomonas reinhardtii cytochrome f

Ali Sabahi, Pernilla Wittung-Stafshede
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引用次数: 7

Abstract

We have studied the unfolding reaction of cytochrome f from the green alga Chlamydomonas reinhardtii. Cytochrome f is different from all other c-type heme proteins in that it is a large, two-domain protein with predominantly β-sheet structure. Moreover, the sixth axial ligand to the heme-iron is unique in cytochrome f: it is provided by the N-terminal α-amino group. Unfolding of oxidized and reduced cytochrome f by guanidine hydrochloride (GuHCl) was monitored by far-UV circular dichroism (CD), Soret absorption, and tyrosine emission: the same unfolding curves were obtained regardless of method. Neither oxidized nor reduced unfolded cytochrome f can be refolded at neutral pH. At pH 3.5 refolding takes place (upon dilution to lower denaturant concentrations or by electron injection to the unfolded, oxidized form), although the reaction is extremely slow. Reduced cytochrome f appears much more resistant towards denaturant perturbation than the oxidized form (in pH range 7–3.5). The heme in unfolded cytochrome f remains low-spin to pH 4 but turns high-spin at pH 3.5 (presumably due to protonation of the N-terminal amino group). Our results suggest that the unfolding process for cytochrome f is complex, involving kinetically trapped intermediates not resolvable by spectroscopy.

揭示独特的c型血红素蛋白,莱茵衣藻细胞色素f
研究了莱茵衣藻细胞色素的展开反应。细胞色素f不同于所有其他c型血红素蛋白,因为它是一个大的,双结构域蛋白,主要具有β-片结构。此外,血红素铁的第六个轴向配体在细胞色素f中是独特的:它由n端α-氨基提供。采用远紫外圆二色性(CD)、Soret吸收和酪氨酸发射监测盐酸胍(GuHCl)对氧化和还原细胞色素f的展开,无论采用何种方法,均可获得相同的展开曲线。在中性pH下,氧化的和还原的未折叠细胞色素f都不能再折叠。在pH为3.5时,虽然反应非常缓慢,但会发生再折叠(稀释至较低的变性剂浓度或通过电子注入未折叠的氧化形式)。还原后的细胞色素f比氧化后的细胞色素f (pH值范围7-3.5)更能抵抗变性剂的扰动。未折叠细胞色素f中的血红素在pH值为4时保持低自旋,但在pH值为3.5时变为高自旋(可能是由于n端氨基的质子化作用)。我们的结果表明,细胞色素f的展开过程是复杂的,涉及动力学捕获的中间体,不能通过光谱来分辨。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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