Oxygenated cytochrome P-450cam: evidence against axial histidine ligation of iron.

Because of their unusual spectral and catalytic properties, the cytochrome P-4.50 enzymes have been intensely investigated [ 11. A major objective has been to identify the non-porphyrin ligand(s) to the central iron in states l-5 of the P-450 reaction cycle (see Scheme 1). Comparative physical properties of synthetic porphyrin complexes with various axial ligands, myoglobin complexes with axial histidine ligation, and P450 reaction states 1,2 and 5 have provided convincing evidence for cysteinate ligation in the latter [2-91. Synthetic systems whose properties mimic those of reaction state 4 have not been prepared, making its axial ligand more difficult to ascertain. The identity of this ligand may be crucial for understanding the dioxygen activation step (Scheme 1,4 + 1). Magnetic circular dichroism (MCD) spectroscopy has been particularly useful in studying the P-450 class of enzymes [5,8,10-171 because of its frequent ability to distinguish between porphyrin chromophores with different axial ligands. We have therefore obtained the MCD spectra of oxy-P-450, and of oxymyoglobin, the latter being a heme protein with known axial ligation by histidine. Substantial differences are seen, strongly suggesting the presence of an axial ligand in oxy-P-450_ other than neutral histidine.