Evidence for basic ferryls in cytochromes P450

Using a combination of Mössbauer 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 Mössbauer parameters for the ferryl and protonated ferryl (Fe^IVOH) 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 ΔE_Q = 2.17 mm/s and ΔE_Q = 1.05mm/s, respectively. For P450cam, they are ΔE_Q = 1.84 mm/s and ΔE_Q = 0.66 mm/s, respectively. The experimentally determined quadrupole splittings (P450 _BM3, ΔE_Q = 2.16 mm/s; P450cam, ΔE_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.