Initial characterization of the ferric H175G cytochrome c peroxidase cavity mutant using magnetic circular dichroism spectroscopy: phosphate from the buffer as an axial ligand

Abstract

Electronic absorption and magnetic circular dichroism (MCD) data are reported for the H175G cavity mutant of yeast cytochrome c peroxidase (H175G CCP) in its ligand-free ferric state at 4 °C under slightly acidic conditions (pH 5.9). Initial analysis of the MCD spectrum of this state suggested a five-coordinate structure with ligation by an aspartate or glutamate. However, examination of the crystal structure of wild-type CCP revealed no plausible carboxylate-containing amino-acid residues close enough to the heme iron to serve as the ligand. An alternative interpretation of the MCD data is that the phosphate ion from the buffer is bound to the ferric heme iron. A phosphate group is similar in electronic character to a carboxylate and could give an MCD signal similar to that of a carboxylate-bound heme. Phosphate coordination to the ferric heme iron, albeit trans to imidazole, has recently been seen in a 2.0-Å-resolution crystal structure of imidazole-bound ferric H175G CCP in phosphate buffer [Biochemistry 40 (5) (2001) 1265]. To investigate the validity of phosphate ligation, the pH 5.9 species in the 100 mM phosphate buffer was exchanged into the pH 5.9, 100 mM MES (2-[ N-morpholino]ethanesulfonic acid) buffer. The MCD spectrum of the species in the MES buffer was spectrally distinct from that in the phosphate buffer, indicating that the original spectrum depends on the presence of a phosphate ion. We conclude that in the phosphate buffer, the exogenous ligand-free ferric state of the H175G CCP cavity mutant is actually coordinated by a phosphate ion from the buffer.