DETECTION, CHARACTERIZATION, AND QUENCHING OF THE INTRINSIC FLUORESCENCE OF BOVINE HEART CYTOCHROME-C-OXIDASE
DETECTION, CHARACTERIZATION, AND QUENCHING OF THE INTRINSIC FLUORESCENCE OF BOVINE HEART CYTOCHROME-C-OXIDASE
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DOI：
10.1021/bi00356a065
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发表时间：
1986-04-22
期刊：
影响因子：
2.9
通讯作者：
ROBINSON, NC
中科院分区：
文献类型：
--
作者：
HILL, BC;HOROWITZ, PM;ROBINSON, NC
月桂基麦芽糖苷增溶的牛心细胞色素c氧化酶的固有荧光已被确定产生的色氨酸残基的氧化酶复合物。荧光强度约为N-乙酰基甜菜酰胺（NATA）的34%。该荧光水平与血红素到色氨酸的平均距离为30埃一致。大多数荧光色氨酸残基处于疏水环境中，如（1）在328 nm处的荧光发射最大值和（2）淬灭剂（Cs+、I-和丙烯酰胺）的不同有效性所示。铯是无效的浓度为0.7 M，而淬火的其他表面淬火剂，碘化物，是复杂的。低于0.2 M时，KI无效，而在0.2和0.7 M之间，发现15%的色氨酸荧光可被碘化物接近。这种模式表明，蛋白质结构的变化是由碘化物诱导的，可能与KI的离液特性有关。丙烯酰胺作为氧化酶荧光的猝灭剂是中等有效的，其Stern-Volmer常数为2 M-1，与NATA和水溶性酶醛缩酶的丙烯酰胺猝灭相比，其Stern-Volmer常数分别为12 M-1和0.3 M-1。有没有影响的色氨酸发射强度从细胞色素c氧化酶的条件下，两种蛋白质形成一个紧密的，1：1的复合物，这意味着色氨酸残基附近的细胞色素c结合位点已经淬灭的能量转移到血红素的氧化酶。用于溶解酶的月桂基麦芽糖苷浓度不影响NATA的荧光。相反，吲哚的荧光光谱的最大值移动到一个较短的波长附近的十二烷基麦芽糖苷的临界胶束浓度，表明吲哚已分配到洗涤剂胶束。吲哚在十二烷基麦芽糖苷胶束中的荧光光谱类似于细胞色素氧化酶的色氨酸荧光。这些数据支持的想法，在膜蛋白有两种可能的环境，可能会引起色氨酸的荧光特性与细胞色素c氧化酶：内部的蛋白质和蛋白质/脂质或蛋白质/去污剂界面。Cs+和I-猝灭月桂基麦芽糖苷胶束中吲哚荧光的能力远大于这些试剂作为氧化酶荧光猝灭剂的能力。这些淬灭数据表明，在细胞色素c氧化酶的荧光色氨酸残基被埋在蛋白质。因此，淬灭研究代表了一种方法，用于区分两种可能类型的疏水环境中的完整的膜蛋白。它的结论是，在细胞色素c氧化酶中的荧光双链体不对称地位于复合物中，从细胞色素c结合区，并埋在内部的蛋白质，而不是在蛋白质/脂质界面。
The intrinsic fluorescence of lauryl maltoside solubilized bovine heart cytochrome c oxidase has been determined to arise from tryptophan residues of the oxidase complex. The magnitude of the fluorescence is approximately 34% of that from n-acetyltryptophanamide (NATA). This level of fluorescence is consistent with an average heme to tryptophan distance of 30 .ANG.. The majority of the fluorescent tryptophan residues are in a hydrophobic environment as indicated by (1) the fluorescence emission maximum at 328 nm and (2) the differing effectiveness of the quenching agents: Cs+, I-, and acrylamide. Cesium was ineffective up to a concentration of 0.7 M, whereas quenching by the other surface quenching agent, iodide, was complex. Below 0.2 M, KI was ineffective whereas between 0.2 and 0.7 M 15% of the tryptophan fluorescence was found to be accessible to iodide. This pattern indicates that protein structural changes were induced by iodide and may be related to the chaotropic character of KI. Acrylamide was moderately effective as a quenching agent of the oxidase fluorescence with a Stern-Volmer constant of 2 M-1 compared with acrylamide quenching of NATA and the water-solule enzyme aldolase having Stern-Volmer constants of 12 M-1 and 0.3 M-1, respectively. There was no effect of cytochrome c on the tryptophan emission intensity from cytochrome c oxidase under conditions where the two proteins form a tight, 1:1 complex, implying that the tryptophan residues near the cytochrome c binding site are already quenched by energy transfer to the hemes of the oxidase. The lauryl maltoside concentration used to solubilize the enzyme did not affect the fluorescence of NATA. In contrast, the fluorescence spectral maximum of indole was shifted to a shorter wavelength near the critical micelle concentration of lauryl maltoside, indicating that indole had partitioned into the detergent micelle. The fluorescence spectrum of indole in a lauryl maltoside micelle resembles the tryptophan fluorescence of cytochrome oxidase. These data support the idea that in a membrane protein there are two possible environments that may give rise to the fluorescence properties of tryptophan seen with cytochrome c oxidase: the interior of the protein and the protein/lipid or protein/detergent interface. The ability of Cs+ and I- to quench the fluorescence of indole in lauryl maltoside micelles is much greater than the ability of these agents as quenchers of the oxidase fluorescence. These quenching data suggest that in cytochrome c oxidase the fluorescent tryptophan residues are buried in the protein. Thus, quenching studies represent a method for distinguishing between the two possible types of hydrophobic environments in integral membrane proteins. It is concluded that the fluorescent tryptophans in cytochrome c oxidase are asymmetrically located in the complex, removed from the cytochrome c binding region, and buried in the interior of the protein rather than at the protein/lipid interface.