KINETIC MECHANISM OF CYTOCHROME-C FOLDING - INVOLVEMENT OF THE HEME AND ITS LIGANDS
KINETIC MECHANISM OF CYTOCHROME-C FOLDING - INVOLVEMENT OF THE HEME AND ITS LIGANDS
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DOI：
10.1021/bi00188a023
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发表时间：
1994-06-07
期刊：
影响因子：
2.9
通讯作者：
RODER, H
中科院分区：
文献类型：
--
作者：
ELOVE, GA;BHUYAN, AK;RODER, H
共价连接的血红素及其轴向配体不仅对细胞色素c的结构和功能至关重要，而且在折叠过程中也起着重要作用。在典型的变性条件下（pH约为7的高浓度盐酸胍或尿素），轴向配体之一的组氨酸18仍然与氧化态血红素铁结合，但第二个配体甲硫氨酸80被一个非天然的组氨酸配体（马细胞色素c中的组氨酸26或组氨酸33）所取代。利用淬灭流动和核磁共振方法，测量了盐酸胍变性的马细胞色素c中几个单个酰胺质子的氢交换速率。观察到一个高度受保护（140倍）的主链酰胺，即组氨酸18的酰胺，表明存在一个持久的氢键，这与未折叠状态下组氨酸18侧链与血红素的配位一致。在4.5 M盐酸胍中，将氧化态细胞色素c从pH 7.8快速酸化至4.6或更低时，血红素吸光度的变化呈现两个动力学阶段，速率分别为110和25 s⁻¹，这归因于未折叠状态下非天然组氨酸配体从血红素上的解离。通过停流方法研究了在各种初始和最终条件下从盐酸胍变性的细胞色素c的折叠动力学，使用色氨酸荧光作为构象探针，索雷特吸光度作为血红素配位状态的探针。一个快速动力学阶段（80 s⁻¹）伴随着荧光的大幅下降和较小的吸光度变化，这与早期脉冲NH交换测量中检测到的具有相互作用的链末端的部分折叠中间体的形成相一致[罗德，H.，埃洛夫，G. A.，英格伦德，S. W.（1988）《自然》335，700]。在中性pH下，一个中间动力学阶段（1.8 s⁻¹）占吸光度变化的78%和荧光变化的47%。相比之下，在pH 5时的折叠动力学以快速阶段为主，中间阶段的幅度降低到约10%。pH依赖的幅度变化显示出滴定行为，表观pK约为5.7，这与单个组氨酸残基的质子化一致。中间阶段也可通过添加200 mM咪唑来抑制。由于这两种条件都会干扰组氨酸配位，所以中间动力学阶段归因于存在一个非天然的组氨酸配体（组氨酸26或组氨酸33），它可能被困在一个部分折叠的中间体中。为了在不受血红素配位事件干扰的情况下研究氢键结构的形成，利用淬灭流动和二维核磁共振方法测量了在pH 5时折叠反应过程中对NH交换的保护时间进程。与在较高pH下的早期结果相反，在快速折叠阶段所有酰胺质子都已经获得了广泛的保护，这表明是一个更协同的结构转变。除了N - 和C - 末端酰胺质子外，所有质子在100毫秒时间尺度上都表现出一个较小的保护阶段，这表明在较低pH下也发现了N - 和C - 末端螺旋的一些优先相互作用。提出了一个动力学机制，该机制解释了在各种条件下细胞色素c折叠过程中观察到的大部分结构和动力学数据。该模型预测，具有不同轴向配体的未折叠分子的不同群体产生了多个平行的折叠途径，正如先前观察到的[埃洛夫，G. A.，罗德，H.（1991）《美国化学会专题论文集》470，50]。
The covalently attached heme and its axial ligands not only are essential for the structure and function Of cytochrome c but they also play an important role in the folding process. Under typical denaturing conditions (concentrated guanidine hydrochloride or urea near pH 7), one of the axial ligands, His 18, remains bound to the oxidized heme iron, but the second ligand, Met 80, is replaced by a non-native histidine ligand (His 26 or His 33 in horse cytochrome c). Using quenched-flow and NMR methods, hydrogen exchange rates were measured for several individual amide protons in guanidine-denatured horse cytochrome c. The observation of a single highly protected (140-fold) backbone amide, that of His 18, suggests the presence of a persistent H-bond consistent with heme ligation of the His 18 side chain in the unfolded state. Heme absorbance changes induced by rapid acidification of oxidized cytochrome c in 4.5 M guanidine hydrochloride from pH 7.8 to 4.6 or below exhibit two kinetic phases with rates of 110 and 25 s(-1), attributed to the dissociation of non-native histidine ligands from the heme in the unfolded state. The kinetics of folding from guanidine-denatured cytochrome c under a variety of initial and final conditions was investigated by stopped-flow methods, using tryptophan fluorescence as a conformational probe and Soret absorbance as a probe for the ligation state of the heme. A fast kinetic phase (80 s(-1)) accompanied by a major decrease in fluorescence and a minor absorbance change coincides with the formation of a partially folded intermediate with interacting chain termini detected in earlier pulsed NH exchange measurements [Roder, H., Elove, G. A., and Englander, S. W. (1988) Nature 335 700]. At neutral pH, an intermediate kinetic phase (1.8 s(-1)) accounts for 78% of the absorbance change and 47% of the fluorescence change. In contrast, the folding kinetics at pH 5 is dominated by the fast phase, and the amplitude of the intermediate phase is reduced to similar to 10%. The pH-dependent amplitude changes show titration behavior with an apparent pK of similar to 5.7, consistent with the protonation of a single histidine residue. The intermediate phase can also be suppressed by the addition of 200 mM imidazole. Since both of these conditions interfere with histidine ligation, the intermediate kinetic phase is attributed to the presence of a non-native histidine ligand (His 26 or His 33) that can become trapped in a partially folded intermediate. In order to investigate the formation of H-bonded structure without interference from heme ligation events, quenched-flow and two-dimensional NMR methods were used to measure the time course of protection against NH exchange during the folding reaction at pH 5. In contrast to earlier results at higher pH, all amide protons had already acquired extensive protection during the fast folding phase, indicating a more cooperative structural transition, All except the N- and C-terminal amide protons exhibit a minor protection phase on the 100-ms time scale, suggesting that some preferential interaction of N- and C-terminal helices is also found at lower pH. A kinetic mechanism is presented that accounts for most of the observed structural and kinetic data on the cytochrome c folding process under various conditions. The model predicts that distinct populations of unfolded molecules with alternative axial ligands give rise to multiple parallel folding pathways, as previously observed [Elove, G. A., and Roder, H. (1991) ACS Symp. Ser. 470, 50].