Production and characterisation of Met80X mutants of yeast iso-1-cytochrome c:: spectral, photochemical and binding studies on the ferrous derivatives
Production and characterisation of Met80X mutants of yeast iso-1-cytochrome c:: spectral, photochemical and binding studies on the ferrous derivatives
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DOI：
10.1016/s0301-4622(02)00085-6
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发表时间：
2002-07-10
影响因子：
3.8
通讯作者：
Wilson, MT
中科院分区：
文献类型：
--
作者：
Silkstone, G;Stanway, G;Wilson, MT
酵母异-1-细胞色素c的铁配体Met 80已突变为不能与铁结合的残基。表达并纯化了Met 80 Ala、Ser、Asp、Glu等蛋白。所有突变蛋白质都表现出明确的pH依赖性光谱跃迁，这些光谱跃迁报告了在高pH下驱动血红素低自旋的内在配体（可能是赖氨酸的ε-NH 2的氮）的结合。pK值依赖于突变体。所有的突变体蛋白质结合外在配体，如CO，在其亚铁态，我们报告的表观量子产率（φ）CO光解。phi值的范围从Met 80 Ala的0.004到Met 80 Asp的0.04。我们还报告了结合固有赖氨酸残基的速率常数值。这个常数的值，为phi和为pK值的细胞色素结构的刚性方面进行了讨论。我们还表明，突变体蛋白结合细胞色素c氧化酶，无论是在铁和亚铁状态具有高亲和力。这些蛋白质作为光激活的电子供体的电子转移的研究的潜力进行了讨论。(C)2002 Elsevier Science B. V.保留所有权利。
The iron ligand, Met80, of yeast iso-1-cytochrome c has been mutated to residues that are unable to bind to the iron. The resultant proteins, Met80Ala, Ser, Asp, Glu, have been expressed and purified. All mutant proteins exhibit well defined pH dependent spectral transitions that report the binding, at high pH, of an intrinsic ligand (probably the nitrogen of an epsilon-NH2 of a lysine) that drives the heme low-spin. The pK values are mutant dependent. All the mutant proteins bind extrinsic ligands, such as CO, in their ferrous states and we report the apparent quantum yield (phi) for CO photo-dissociation. The values of phi range from 0.004 for Met80Ala to 0.04 for Met80Asp. We also report values for the rate constant for binding the intrinsic lysine residue. The values for this constant, for phi and for the pK values are discussed in terms of the rigidity of the cytochrome structure. We also show that the mutant proteins bind with high affinity to cytochrome c oxidase, both in the ferric and ferrous states. The potential of these proteins to act as light activated electron donors for the study of electron transfer is discussed. (C) 2002 Elsevier Science B.V. All rights reserved.