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 examine d by Fe-K x-ray absorption edge spectroscopy. The positions of the abs orption edge and the shapes of preedge and edge regions of imidazole a dducts of ferric P-450-CAM and chloroperoxidase are essentially the sa me when compared with thiolate-ligated ferric myoglobin. As these thre e 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 envi ronment does not effect the spectra, provided one compares heme iron c enters 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 fer ric 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 chlorop eroxidase and P-450-CAM are shifted in the direction of the ferrous st ate (to lower energy) when compared with those for analogous myoglobin derivatives. In the deoxyferrous high spin state, comparison of the e dge spectra of chloroperoxidase with analogous data for cytochrome P-4 50-CAM suggests that the electron density at the iron is similar for t hese 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 mea sure of the electron releasing character of a thiolate axial ligand. T hese results therefore support the suggested role of the cysteinate pr oximal ligand of P-450 as a strong internal electron donor to promote O-O bond cleavage in the putative ferric-peroxide intermediate to gene rate the proposed ferryl oxo ''active oxygen'' state of the reaction c ycle.