Gray K A, Davidson E, Daldal F
Department of Biology, University of Pennsylvania, Philadelphia 19104-6018.
Biochemistry. 1992 Dec 1;31(47):11864-73. doi: 10.1021/bi00162a027.
Site-directed mutagenesis was used to investigate which of the highly conserved methionine residues (M183 and M205) provides the sixth axial ligand to the heme Fe in the cyt c1 subunit of the bc1 complex from the bacterium Rhodobacter capsulatus. These residues were changed to leucine (cM183L) and valine (cM205V). Two additional mutants were constructed, 1 in which a stop codon was inserted at M205 (cM205*) and the second in which 127 amino acids were deleted between the signal sequence and the putative C-terminal transmembrane alpha-helix (c delta SfuI). Only cM205V grew photosynthetically, and membranes isolated from this strain catalyzed quinol-dependent reduction of cyt c in amounts similar to that in a wild-type strain. Even though cM183L could not grow photosynthetically, it contained all the appropriate polypeptides and cofactors of the bc1 complex, as shown by SDS-PAGE and optical difference spectroscopy of intact membrane particles. Neither of the two deletion mutants contained a stable complex. Flash absorption spectroscopy using chromatophores showed no cytochrome c rereduction after oxidation by the reaction center in cM183L. The bc1 complex from each strain was isolated and characterized. Oxidation reduction midpoint potential titrations revealed that cyt c1 from cM183L had a dramatically shifted Em value (delta Em = -390 mV) compared with wild type and cM205V. While the optical absorption spectrum of cyt c1 from cM183L suggested that the c-type heme was low-spin, nonetheless it was able to react with the exogenous ligand carbon monoxide. The overall data support that M183, and not M205, is the sixth ligand to the heme Fe of cyt c1 of the bc1 complex.
定点诱变用于研究来自荚膜红细菌的bc1复合物的细胞色素c1亚基中高度保守的甲硫氨酸残基（M183和M205）哪一个为血红素铁提供第六个轴向配体。这些残基被替换为亮氨酸（cM183L）和缬氨酸（cM205V）。构建了另外两个突变体，一个是在M205处插入一个终止密码子（cM205*），另一个是在信号序列和假定的C端跨膜α螺旋之间缺失127个氨基酸（cΔSfuI）。只有cM205V能够进行光合生长，从该菌株分离的膜催化的依赖喹啉的细胞色素c还原量与野生型菌株相似。尽管cM183L不能进行光合生长，但通过SDS-PAGE和完整膜颗粒的光学差分光谱显示，它含有bc1复合物的所有合适的多肽和辅因子。两个缺失突变体都不包含稳定的复合物。使用载色体的闪光吸收光谱显示，在cM183L中，反应中心氧化后细胞色素c没有再还原。分离并表征了每个菌株的bc1复合物。氧化还原中点电位滴定显示，与野生型和cM205V相比，cM183L的细胞色素c1的Em值发生了显著变化（ΔEm = -390 mV）。虽然cM183L的细胞色素c1的光吸收光谱表明c型血红素是低自旋的，但它仍然能够与外源性配体一氧化碳反应。总体数据支持M183而非M205是bc1复合物细胞色素c1血红素铁的第六个配体。
