EVIDENCE FOR WATER BINDING TO THE FE CENTER IN CYTOCHROME P450CAM OBTAINED BY O-17 ELECTRON-SPIN ECHO ENVELOPE MODULATION SPECTROSCOPY

Orientation selective and multiple frequency ESEEM experiments on subs trate free cytochrome P450cam (CP450cam) with O-17-enriched water are reported. The O-17 ESEEM frequencies were obtained from Fourier transf ormation of the ratio of ESEEM waveforms of CP450cam with enriched wat er and CP450cam with nonenriched water. Numerical simulations were car ried out to determine the isotropic and the anisotropic hyperfine inte ractions and the quadrupole interaction of the O-17. From the magnitud e (e(2)-qQ/h = 6.6 MHz) and asymmetry (eta = 0.95) of the O-17 quadrup ole interaction, we conclude that the distal axial ligand of Fe3+ in t he CP450 heme is a water molecule. Moreover, from the orientation of t he O-17 quadrupole tenser, the orientation of the water molecule was f ound to be confined (within +/-10 degrees) with respect to the Fe-N di rections within the heme plane. Two possible sets of isotropic and ani sotropic components of the O-17 hyperfine interaction, (+/-2.6, +/-0.3 MHz) and (+/-0.4, +/-1.8 MHz), were found to satisfactorily reproduce the experimental results. Both sets indicate very small O-17 hyperfin e couplings which is consistent with the unpaired electron residing pr edominately in the d(yz) orbital of the Fe3+. Ligand field models for each set are presented.