Effect of pH on axial ligand coordination of cytochrome c '' from Methylophilus methylotrophus and horse heart cytochrome c

The effect of protons on the axial ligand coordination and on structural as pects of the protein moiety of cytochrome c " from Methylophilus methylotro phus, an obligate methylotroph, has been investigated down to very low pH ( i.e., 0.3). The unusual resistance of this cytochrome to very low pH values has been exploited to carry out this study in comparison with horse heart cytochrome c. The experiments were undertaken at a constant phosphate conce ntration to minimize the variation of ionic strength with pH, The pH-linked effects have been monitored at 23 degrees C in the oxidized forms of both cytochromes by following the variations in the electronic absorption, circu lar dichroism and resonance Raman spectra, This approach has enabled the co nformational changes of the heme surroundings to be monitored and compared with the concomitant overall structural rearrangements of the molecule. The results indicate that horse heart cytochrome c undergoes a first conformat ional change at around pH 2.0. This event is possibly related to the cleava ge of the Fe-Met80 bond and a likely coordination of a H2O molecule as a si xth axial ligand, Conversely, in cytochrome c " from M. methylotrophus, a v ariation of the axial ligand coordination occurs at a pH that is about 1 un it lower. Further, it appears that a concerted cleavage of both His ligands takes place, suggesting indeed that the different axial ligands present in horse heart cytochrome c (Met/His) and in cytochrome c " from M. methylotr ophus (His/His) affect the heme conformational changes.