NONCOVALENT BINDING OF HEME INDUCES A COMPACT APOCYTOCHROME-C STRUCTURE
NONCOVALENT BINDING OF HEME INDUCES A COMPACT APOCYTOCHROME-C STRUCTURE
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DOI：
10.1021/bi00189a043
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发表时间：
1994-06-14
期刊：
影响因子：
2.9
通讯作者：
CAMPBELL, GA
中科院分区：
文献类型：
--
作者：
DUMONT, ME;CORIN, AF;CAMPBELL, GA
线粒体全细胞色素c含有血红素，血红素在细胞色素c血红素裂解酶催化的反应中共价连接到蛋白质上。在没有血红素的情况下，脱辅基细胞色素c，即全细胞色素c的前体，被展开。我们发现，纯化脱辅基细胞色素c非共价结合血红素。结合伴随着血红素的光吸收光谱的变化和蛋白质的色氨酸荧光的淬灭。脱辅基细胞色素c对血红素的亲和力，以及结合的化学计量，似乎取决于是否存在氰化物和血红素的氧化态。在还原条件下，在氰化物存在下，缔合似乎是1：1，结合常数约为10（7）M（-1）。在氧化条件下，每个脱辅基细胞色素c分子可能结合有多个血红素。在与血红素结合后，脱辅基细胞色素c在凝胶过滤色谱和速度梯度超离心中表现出与全细胞色素c相似的迁移率，表明血红素-蛋白质复合物采用了几乎与全细胞色素c一样紧凑的结构。脱辅基细胞色素c的圆二色性光谱的变化与结合血红素的蛋白质的α-螺旋含量的增加是一致的。非共价血红素-脱辅基细胞色素c复合物的紧凑结构可能代表细胞色素c从头折叠的中间体。
Mitochondrial holocytochrome c contains heme that is covalently attached to the protein in a reaction catalyzed by the enzyme cytochrome c heme lyase. In the absence of heme, apocytochrome c, the precursor to holocytochrome c, is unfolded. We find that purified apocytochrome c binds noncovalently to heme. Binding is accompanied by changes in the optical absorption spectrum of heme and by quenching of the tryptophan fluorescence of the protein. The affinity of apocytochrome c for heme, as well as the stoichiometry of binding, appears to depend on whether or not cyanide is present and on the oxidation state of heme. Under reducing conditions, in the presence of cyanide, the association appears to be 1:1, with a binding constant of about 10(7) M(-1). Under oxidized conditions, there may be multiple hemes bound per molecule of apocytochrome c. Upon binding to heme, apocytochrome c exhibits a mobility similar to that of holocytochrome c in gel filtration chromatography and velocity gradient ultracentrifugation, indicating that the heme-protein complex adopts a structure that is almost as compact as that of holocytochrome c. Changes in the circular dichroism spectrum of apocytochrome c are consistent with an increase in the alpha-helical content of the protein on binding heme. The compact structure of the noncovalent heme-apocytochrome c complex may represent an intermediate in the de novo folding of cytochrome c.