DETERMINANTS OF THE SPIN-STATE OF THE RESTING STATE OF CYTOCHROME-P450CAM

The origin of the low-spin ground state of the substrate-free ferric resting state of cytochrome P450cam has been investigated with use of the combined techniques of restricted open-shell Hartree-Fock INDO/S calculations together with molecular dynamics. The presence of water as the heme-iron ligand, while resulting in a small energy separation of the S = 5/2 and 1/2 ferric heme spin states, is by itself insufficient to explain the experimentally observed low-spin resting state. However, the inclusion of the electrostatic field of the protein in the INDO/S Hamiltonian, using the optimized X-ray coordinates and electrostatic potential-derived partial charges found in AMBER, results in a low-spin resting state of cytochrome P-450cam. Molecular dynamic simulations of the optimized X-ray structure further support the role of the protein in modulating the spin state equilibrium. The dynamic motion of the heme unit is not sufficient to account for the predominance of the low-spin state, while the dynamic effect of the field of the protein favors the low-spin state. By contrast, in the camphor-bound cytochrome P450cam, the field of the protein does not reverse the high-spin state found for the optimized X-ray structure of the heme unit. These results have revealed a heretofore unidentified role of the protein in modulating spin equilibrium, a property of the ferric heme unit that is central to the maintenance of the enzymatic function of the cytochrome P450s.