The Catalytic Pathway of Cytochrome P450cam at Atomic Resolution

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. Molecular replacement with the coordinates of oxidized P450cam complexed with camphor (PDB code 2CPP) as search model was used to determine the ferric P450.cam structure in a monoclinic crystal form that contains two molecules per asymmetric unit with an intermolecular K + ion binding site. The electron densities of the active sites of the two molecules in the asymmetric unit are not the same but in different crystals of the same complex corresponding molecules have similar electron densities suggesting that the crystal lattice contacts influence the dynamics of the molecules differently. Indeed the crystallographic environments of the two molecules differ especially in the vicinity of the intramolecular K + binding site which is connected through Tyr 96 directly to the camphor binding site. In one molecule this K + site is close to a neighboring molecule in the lattice. In general for the experiments reported here similar electron density features are seen in molecules 1 and 2 but mixtures seem to be present in molecule 2 that could not be modeled satisfactorily. It is possible that the two molecules represent slightly different stages of the reaction or different mixtures of species.

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The literature of cytochrome P450 is so vast and so many people have made important contributions that it is impossible in the space allowed to give even a representative set of references. We have tried to cite review articles whenever possible to provide links to the original papers. We apologize to those we have been unable to include. We gratefully acknowledge G. Holtermann for designing the pressure cell; M. Davies N. C. Gerber E. J. Mueller and M. Vidakovic for supplying us with protein; C. Jung G. Rosenbaum and K. Scheffzek for discussions; and R. S. Goody K. C. Holmes and W. Kabsch for continuous support and encouragement. P. Ortiz de Montellano and J. A. Peterson provided useful advice. The referees made a number of very helpful suggestions that greatly improved the manuscript. We thank the Alexander von Humboldt Stiftung the Human Frontiers Science Project and the Richard und Anne Liese Leyendecker Stiftung for generous support and acknowledge the European Community biotech grant BIO2-CT942060 to I.S. NIH grants GM31756 and GM33775 to S.G.S and GM26788 to G.A.P. and D.R. Beamline X12C is supported by the U.S. Department of Energy Offices of Health and Environmental Research and of Basic Energy Sciences NIH and NSF. The coordinates have been submitted to the PDB: 1d26 1d24 1d28 and 1d29 for the ferric reduced oxy and oxyferryl complex respectively.