Evidence for Water Binding to the Fe Center in Cytochrome P450cam Obtained by 17O Electron Spin-Echo Envelope Modulation Spectroscopy

Orientation selective and multiple frequency ESEEM experiments on substrate free cyctochrome P450cam (CP450cam) with {sup 17}O-enriched water are reported. The {sup 17}O ESEEM frequencies were obtained from Fourier transformation of the ratio of ESEEM waveforms of CP450cam with enriched water and CP450cam with non-enriched water. Numerical simulations were carried out to determine the isotropic and the anisotropic hyperfine interactions and the quadrupole interaction of the {sup 17}O. From the magnitude (e{sup 2}qQ/h = 6.6 MHz) and asymmetry ({eta} = 0.95) of the {sup 17}O quadrupole interaction, we conclude that the distal axial ligand of Fe{sup 3+} in the CP450 heme is a water molecule. Moreover, from the orientation of the {sup 17}O quadrupole tensor, the orientation of the water molecule was found to be confined (within {+-}10{degree}) with respect to the Fe-N directions within the heme plane. Two possible sets of isotropic and anisotropic components of the {sup 17}O hyperfine interaction, ({+-}2.6, {+-}10.3 MHz) and ({+-}0.4, {+-}1.8 MHz), were found to satisfactorily reproduce the experimental results. Both sets indicate very small {sup 17}O hyperfine couplings which is consistent with the unpaired electron residing predominately in the d{sub yz} orbital of the Fe{sup 3+}. Ligand field models for each set are more » presented. 50 refs., 11 figs., 1 tab. « less