INTERMEDIATES IN THE REACTION PATHWAY OF CYTOCHROME P450_cam. Ilme Schlichting^*, Joel Berendzen^**, Kelvin Chu^**, Ann M. Stock⁽, Matthew Davies⁽, Ernest J. Mueller⁽, Steven Sligar⁽, Robert M. Sweet⁽, Dagmar Ringe⁽ & Gregory A. Petsko⁽. ^*Max Planck Institut für molekulare Physiologie, Abteilung Physikalische Biochemie, Rheinlanddamm 201, 44139 Dortmund and Abteilung Biophysik, Max Planck Institut für medizinische Forschung, Jahnstraße 29, 69120 Heidelberg , Germany; ^**Los Alamos National Laboratory, Biophysics Group, MS M715, Los Alamos, New Mexico 87545, USA. ⁽Center for Advanced Biotechnology and Medicine, Piscataway, NJ 008854-5638, USA; ⁽Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA; ⁽Department of Biochemistry and Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana, Illinois 61801, USA; ⁽Rosenstiel Center, Brandeis University, Waltham, Massachussetts 02254, USA

Cytochrome p450 enzymes form a family of ubiquitous heme proteins named after an absorption band at 450nm when complexed to carbon monoxide. P450 enzymes are mixed-function mono-oxygenases. They play a critical role in the synthesis and degradation of many physiologically important compounds and xenobiotics. This makes cytochrome p450s an attractive target for pharmaceutical or environmental industries. The biochemical relevance of the p450 mono-oxygenases in general is their unique ability to catalyze the hydroxylation of non-activated aliphatic or aromatic carbons. The biochemically and structurally best characterized p450 is p450_cam from Pseudomonas putida, which catalyzes the stereospecific 5-exo-hydroxylation of camphor. Although the structures of the p450_cam apoprotein and of complexes of p450 with camphor, various inhibitors and CO have been determined, the structure of the biochemically important p450.camphor.O₂ complex has not been solved ,yet, as it is unstable due to autooxidation which transforms the enzyme from the ferro to the ferric form with a rate constant of 10^-3 s^-1 at 4 (C in solution. This requires to collect the diffraction data of the relatively short-lived complex either fast or to prolong its life time. Thus, we used Laue and cryocrystallography for the crystal structure determination of intermediates occuring along the reaction coordinate of p450. The methods used, and the structures obtained will be described.