X-ray absorption near edge studies of cytochrome P-450-CAM, chloroperoxidase, and myoglobin. Direct evidence for the electron releasing character of a cysteine thiolate proximal ligand.
The low spin ferric and low and high spin ferrous forms of myoglobin, bacterial cytochrome P-450-CAM, and chloroperoxidase have been examined by Fe-K x-ray absorption edge spectroscopy. The positions of the absorption edge and the shapes of preedge and edge regions of imidazole adducts of ferric P-450-CAM and chloroperoxidase are essentially the same when compared with thiolate-ligated ferric myoglobin. As these three protein derivatives all have six-coordinate, low spin, ferric hemes with axial imidazole and thiolate ligands, the superposition of x-ray absorption edge spectral properties demonstrates that the protein environment does not effect the spectra, provided one compares heme iron centers with identical coordination numbers, spin and oxidation states, and ligand sets. In contrast, a 0.96 eV difference is observed in the energy of the absorption edge for imidazole- and thiolate-ligated ferric myoglobin with the latter shifted to lower energy as observed for ferrous myoglobin states. Similarly, in the low spin ferric-imidazole and ferrous-CO states, the energies of the absorption edge for chloroperoxidase and P-450-CAM are shifted in the direction of the ferrous state (to lower energy) when compared with those for analogous myoglobin derivatives. In the deoxyferrous high spin state, comparison of the edge spectra of chloroperoxidase with analogous data for cytochrome P-450-CAM suggests that the electron density at the iron is similar for these two protein states. The shifts observed in the energies of the x-ray absorption edge for the thiolate-ligated states of these proteins relative to derivatives lacking a thiolate ligand provide a direct measure of the electron releasing character of a thiolate axial ligand. These results therefore support the suggested role of the cysteinate proximal ligand of P-450 as a strong internal electron donor to promote O-O bond cleavage in the putative ferric-peroxide intermediate to generate the proposed ferryl-oxo "active oxygen" state of the reaction cycle.
Liu
H I
HI
Department of Chemistry, Stanford University, California 94305, USA.
Sono
M
M
Kadkhodayan
S
S
Hager
L P
LP
Hedman
B
B
Hodgson
K O
KO
Dawson
J H
JH
D019475:Camphor 5-Monooxygenase; D002711:Chloride Peroxidase / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D009211:Myoglobin / Q000737:chemistry*; D010084:Oxidation-Reduction; D016958:Pseudomonas putida / Q000201:enzymology; D013057:Spectrum Analysis; D014965:X-Rays
D005290:Ferric Compounds; D005296:Ferrous Compounds; D009211:Myoglobin; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D002711:Chloride Peroxidase; D019475:Camphor 5-Monooxygenase; D003545:Cysteine