The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450.

Abstract

The crystal structure of Pseudomohzdputida cytochrome P-450cam in the ferric, camphor bound form has beeg determined and partially refined to R = 0.23 at 2.6 A. The single 414 amino acid polypeptide chain (Mr = 45,000) approximates + triangular prism with a maximum dimension of -60 A and a minimum of -30 A. Twelve helical segments (A through L) account for -40% of the structure while antiparallel B pairs account for only -10%.The unexposed iron protoporphyrin IX is sandwiched between two parallel helices designated the proximal and distal helices.The heme iron atom is pentacoordinate with the axial sulfur ligand provided by Cys 357 which extends from the N- terminal end of the proximal (L) helix.A substrate molecule, 2-bornanone (camphor), is buried in an internal pocket just above the heme distal surface adjacent to the oxygen binding,site.The substrate molecule is held in place by a hydrogen bond between the side chain hydroxyl group of Tyr 96 and the camphor carbonyl oxygen atom in addition to complementary hydrophobic contacts between the camphor molecule and neighboring aliphatic and aromatic residues.The camphor is oriented such that the exosurface of C5 would contact an iron bound, "activated" oxygen atom for stereoselective hydroxylation.Cytochrome P-450 monooxygenases are a widely distributed class of B-type hemeproteins that catalyze a mixed function oxidation of hydrocarbon substrates (1).Utilizing two-electron equivalents from reduced pyridine nucleotides, the P-450 monooxygenases cleave dioxygen into water and a single oxygen atom which is inserted into a hydrocarbon bond.As a group, the P-450 monooxygenases are characterized by