Department of Chemistry, University of Rochester, Rochester, NY, 14627-0216, USA.
Department of Chemistry and Biochemistry, Messiah College, Mechanicsburg, PA, 17055, USA.
J Biol Inorg Chem. 2018 Oct;23(7):1073-1083. doi: 10.1007/s00775-018-1603-3. Epub 2018 Aug 24.
Heme c is characterized by its covalent attachment to a polypeptide. The attachment is typically to a CXXCH motif in which the two Cys form thioether bonds with the heme, "X" can be any amino acid other than Cys, and the His serves as a heme axial ligand. Some cytochromes c, however, contain heme attachment motifs with three or four intervening residues in a CXCH or CXCH motif. Here, the impacts of these variations in the heme attachment motif on heme ruffling and electronic structure are investigated by spectroscopically characterizing CXCH and CXCH variants of Hydrogenobacter thermophilus cytochrome c. In addition, a novel CXCH variant is studied. H and C NMR, EPR, and resonance Raman spectra of the protein variants are analyzed to deduce the extent of ruffling using previously reported relationships between these spectral data and heme ruffling. In addition, the reduction potentials of these protein variants are measured using protein film voltammetry. The CXCH and CXCH variants are found to have enhanced heme ruffling and lower reduction potentials. Implications of these results for the use of these noncanonical motifs in nature, and for the engineering of novel heme peptide structures, are discussed.
亚铁血红素 c 通过其与多肽的共价连接来表征。这种连接通常是与CXXCH 基序结合，其中两个半胱氨酸与亚铁血红素形成硫醚键，“X”可以是除半胱氨酸以外的任何氨基酸，而组氨酸则作为亚铁血红素轴向配体。然而，一些细胞色素 c 含有亚铁血红素附着基序，其中在 CXCH 或 CXCH 基序中有三个或四个插入残基。在这里，通过光谱学方法对热氢杆菌细胞色素 c 的 CXCH 和 CXCH 变体进行表征，研究了亚铁血红素附着基序中的这些变化对亚铁血红素起皱和电子结构的影响。此外，还研究了一种新型的 CXCH 变体。对蛋白变体的 H 和 C NMR、EPR 和共振拉曼光谱进行了分析，使用先前报道的这些光谱数据与亚铁血红素起皱之间的关系来推断起皱的程度。此外，还使用蛋白膜伏安法测量了这些蛋白变体的还原电势。发现 CXCH 和 CXCH 变体具有增强的亚铁血红素起皱和更低的还原电势。这些结果对自然中使用这些非典型基序以及对新型亚铁血红素肽结构的工程设计具有重要意义。
