Influence of the pH on the pocket field of cytochrome c type proteins

We performed hole burning Stark effect experiments on a cytochrome c type protein. From the behavior of the holes in an external electric field conclusions on local electric fields in the heme pocket can be drawn. We varied the pH value in order to study the influence of different groups on the pocket field: (a) titratable amino acid residues, (b) the porphyrin side chains (especially the propionates) and (c) the two axial ligands His18 and Met80. Surprisingly, a variation of the pH between 4.1 and 12.7 has no influence on the Stark effect. However, at a pH-level < 4.1, the Stark pattern changes. From our results, we concluded that the main influence is due to His18.