Axial heme ligation in the cytochrome bc(1) complexes of mitochondrial and photosynthetic membranes. A near-infrared magnetic circular dichroism and electron paramagnetic resonance study

The combination of EPR and low-temperature near-IR magnetic circular dichroism spectroscopies have been used to investigate the axial ligation of the cytochromes in the cytochrome bc(1) complexes from bovine heart mitochondria, Rhodobacter capsulatus, Rhodobacter sphaeroides, and Rhodospirillum rubrum. and the purified cytochromes c(1) from bovine heart mitochondria, Rb, capsulatus and Rb. sphaeroides, The possibility of axial ligation of cytochrome c, by the amino terminus of the polypeptide was also assessed by acetylating the N-terminus of Rb. capsulatus cytochrome c(1) and comparing the properties of the acetylated and unmodified samples. The results are consistent with bis-histidine axial ligation for the high- and low-potential b-type cytochromes and histidine/methionine axial ligation for the c(1)-type cytochrome in the intact cytochrome bc(1) complexes. Purified samples of cytochrome c(1) an mixtures of two forms, one with histidine/methionine and the other with bis-histidine axial ligation, The form with his-histidine axial ligation is also assembled in the M183L mutant of the Rb. capsulatus cyt bc(1) complex in which the methionine residue coordinating cyt c(1) is replaced by a leucine. The bis-histidine form appears to be an artifact of dissociation of cytochrome c(1) from the cytochrome bc(1) complex and is greatly enhanced particularly in the bacterial cytochromes c(1) by sample handling and the addition of 50% (v/v) ethylene glycol or glycerol.