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(IV)OH) 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 DeltaE(Q) = 2.17 mm/s and DeltaE(Q) = 1.05 mm/s, respectively. For P450cam, they are DeltaE(Q) = 1.84 mm/s and DeltaE(Q) = 0.66 mm/s, respectively. The experimentally determined quadrupole splittings (P450(BM3), DeltaE(Q) = 2.16 mm/s; P450cam, DeltaE(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.
Behan
Rachel K
RK
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Hoffart
Lee M
LM
Stone
Kari L
KL
Krebs
Carsten
C
Green
Michael T
MT
D015502:Absorptiometry, Photon; D000818:Animals; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D006420:Hemeproteins / Q000737:chemistry*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007501:Iron / Q000737:chemistry*; D008024:Ligands; D008958:Models, Molecular; D015204:Spectroscopy, Mossbauer
D006420:Hemeproteins; D008024:Ligands; C066317:ferryl iron; D003577:Cytochrome P-450 Enzyme System; D007501:Iron