Materials and Methods

P-450cam was crystallized according to our earlier procedure (Poulos et al., 1982). These crystals grow in the absence of camphor but with a large excess of dithiothreitol. Dithio-threitol competes with camphor in binding to the enzyme and generates electron paramagnetic resonance and absorption spectra distinct from those of the thiol-free enzymes (Sono et al., 1982). Therefore, crystals were converted to the low-spin, thiol-free form by washing extensively with 0.05 M potassium phosphate, pH 7.0, 40% ammonium sulfate, and0. 2 Mkc1. Removal of dithiothreitol appeared to depend on soak time. Crystallographic refinement with data obtained from crystals soaked for only 12-24 h indicated residual-bound dithiothreitol at relatively high occupancy (~> 60%). This was evidenced by low-temperature factors and positive F0- Fc difference density at scattering centers initially modeled as solvent oxygen atoms. A model that best accounted for F0- Fc difference density included a cluster of active site water molecules and a dithiothreitol molecule, each at about half-occupancy. Crystallographic refinement with data obtained from a single crystal washed every other day over a 2-week period gave no evidence of ordered high-occupancysulfur atoms in the substrate pocket. Furthermore, single-crystal EPR (Devaney, 1980; Devaney et al., 1980) and single-crystal polarized ab-sorption spectroscopy (Gerald C. Wagner, personal commu-nication) with crystals grown and then washed in thiol-free