Evidence for basic ferryls in cytochromes P450

Using a combination of Mossbauer spectroscopy and density functional calculations, we have determined that the ferryl forms of P450(BM3) and P450cam are protonated at physiological pH. Density functional calculations were performed on large active-site models of these enzymes to determine the theoretical Mossbauer parameters for the ferryl and protonated ferryl ((FeOH)-O-IV) species. These calculations revealed a significant enlargement of the quadrupole splitting parameter upon protonation of the ferryl unit. The calculated quadrupole splittings for the protonated and unprotonated ferryl forms of P450(BM3) are Delta E-Q = 2.17 mm/s and Delta E-Q = 1.05 mm/s, respectively. For P450cam, they are Delta E-Q = 1.84 mm/s and Delta E-Q = 0.66 mm/s, respectively. The experimentally determined quadrupole splittings (P450(BM3), Delta E-Q = 2.16 mm/s; P450cam, Delta E-Q = 2.06 mm/s) are in good agreement with the values calculated for the protonated forms of the enzymes. Our results suggest that basic ferryls are a natural consequence of thiolate-ligated hemes.