Allen James W A, Ferguson Stuart J
Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
Biochem J. 2003 Nov 1;375(Pt 3):721-8. doi: 10.1042/BJ20030752.
Cytochromes c are typically characterized by the covalent attachment of haem to polypeptide through two thioether bonds with the cysteine residues of a Cys-Xaa-Xaa-Cys-His peptide motif. In many Gram-negative bacteria, the haem is attached to the polypeptide by the periplasmically functioning cytochrome c maturation (Ccm) proteins. Exceptionally, Hydrogenobacter thermophilus cytochrome c552 can be expressed as a stable holocytochrome both in the cytoplasm of Escherichia coli in an apparently uncatalysed reaction and also in the periplasm in a Ccm-mediated reaction. In the present study we show that a Met60-->Ala variant of c552, which does not have the usual distal methionine ligand to the haem iron of the mature cytochrome, can be made in the periplasm by the Ccm system. However, no holocytochrome could be detected when this variant was expressed cytoplasmically. These data highlight differences between the two modes of cytochrome c assembly. In addition, we report investigations of haem attachment to cytochromes altered to have the special Cys-Trp-Ser-Cys-Lys haem-binding motif, and Cys-Trp-Ser-Cys-His and Cys-Trp-Ala-Cys-His analogues, of the active-site haem of nitrite reductase NrfA.
细胞色素c的典型特征是通过两个硫醚键将血红素与多肽共价连接，这两个硫醚键与Cys-Xaa-Xaa-Cys-His肽基序的半胱氨酸残基相连。在许多革兰氏阴性细菌中，血红素通过周质中起作用的细胞色素c成熟（Ccm）蛋白与多肽相连。例外的是，嗜热栖热菌细胞色素c552在大肠杆菌细胞质中以明显无催化的反应形式，以及在周质中以Ccm介导的反应形式，都能被表达为稳定的全细胞色素。在本研究中，我们表明c552的Met60→Ala变体，该变体在成熟细胞色素的血红素铁上没有通常的远端甲硫氨酸配体，可以通过Ccm系统在周质中产生。然而，当这种变体在细胞质中表达时，未检测到全细胞色素。这些数据突出了细胞色素c组装的两种模式之间的差异。此外，我们报告了对血红素与细胞色素结合的研究，这些细胞色素经过改造后具有亚硝酸盐还原酶NrfA活性位点血红素的特殊Cys-Trp-Ser-Cys-Lys血红素结合基序，以及Cys-Trp-Ser-Cys-His和Cys-Trp-Ala-Cys-His类似物。
