Qian W, Sun Y L, Wang Y H, Zhuang J H, Xie Y, Huang Z X
Department of Chemistry, Fudan University, Shanghai, China.
Biochemistry. 1998 Oct 6;37(40):14137-50. doi: 10.1021/bi9805036.
To characterize the roles played by Glu44 and Glu56 of cytochrome b5 in the formation of the electrostatic complex between cytochrome c and cytochrome b5, the Glu44, Glu56, or both sites were changed to alanine by site-directed mutagenesis. The influence of these two residues on the protein stability was probed by investigating the kinetic behaviors of protein denaturation in urea or upon heating and the heme-transfer reactions between apo-myoglobin and the variants of cytochrome b5. It has been found that when the Glu44 and/or Glu56 are mutated to alanine, the protein stability increases slightly due to the fact that the hydrophilic residue is changed to a hydrophobic one, resulting in the two pairs of helices surrounding the heme taking a more compact conformation. The difference in voltammetric behavior of cytochrome c, cytochrome b5, and its three mutants, Cyt b5 E44A, E56A, and E44/56A, alone and in 1:1 protein complexes demonstrates that both Glu44 and Glu56 of cytochrome b5 take part in the electrostatic interaction with cytochrome c. The entropy changes, DeltaS degreesrc and enthalpy changes, DeltaH degrees, derived from the temperature dependence of the formal reduction potentials of each protein in different protein systems suggest that, because of the mutual interaction with cytochrome c, cytochrome b5 mutants, especially the E44A-containing mutants, in the protein complexes suffer greater conformational changes upon reduction than that of the wild type. The variation of these thermodynamic parameters indicates that the strength of mutual interactions between cytochrome c and cytochrome b5 or its mutants has the following order: Cyt c/Cyt b5 > Cyt c/Cyt b5 E56A > Cyt c/Cyt b5 E44A > Cyt c/Cyt b5 E44/56A.
为了表征细胞色素b5的Glu44和Glu56在细胞色素c与细胞色素b5之间静电复合物形成过程中所起的作用，通过定点诱变将Glu44、Glu56或这两个位点都替换为丙氨酸。通过研究蛋白质在尿素中或加热时的变性动力学行为以及脱辅基肌红蛋白与细胞色素b5变体之间的血红素转移反应，探究了这两个残基对蛋白质稳定性的影响。已发现，当Glu44和/或Glu56突变为丙氨酸时，由于亲水性残基变为疏水性残基，蛋白质稳定性略有增加，导致围绕血红素的两对螺旋采取更紧密的构象。细胞色素c、细胞色素b5及其三个突变体Cyt b5 E44A、E56A和E44/56A单独以及在1:1蛋白质复合物中的伏安行为差异表明，细胞色素b5的Glu44和Glu·56都参与了与细胞色素c的静电相互作用。从不同蛋白质系统中每种蛋白质的形式还原电位对温度的依赖性得出的熵变ΔS°rc和焓变ΔH°表明，由于与细胞色素c的相互作用，蛋白质复合物中的细胞色素b5突变体，尤其是含E44A的突变体，在还原时比野生型经历更大的构象变化。这些热力学参数的变化表明，细胞色素c与细胞色素b5或其突变体之间相互作用的强度具有以下顺序：Cyt c/Cyt b5 > Cyt c/Cyt b5 E56A > Cyt c/Cyt b5 E44A > Cyt c/Cyt b5 E44/56A。
