Axial heme ligation in the cytochrome bc1 complexes of mitochondrial and photosynthetic membranes. A near-infrared magnetic circular dichroism and electron paramagnetic resonance study

Abstract

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 fron bovine heart mitochondria, Rb. capsulatus and Rb. sphaeroides. The possibility of axial ligation of cytochrome c 1 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 are mixtures of two forms, one with histidine/methionine and the other with bis-histidine axial ligation. The form with bis-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.