title: Active site topologies of bacterial cytochromes P450101 (P450cam), P450108 (P450terp), and P450102 (P450BM-3). In situ rearrangement of their phenyl-iron complexes.
abstract: The reactions of cytochromes P450101 (P450cam), P450108 (P450terp), and P450102 (P450BM-3) with phenyldiazene result in the formation of phenyl-iron complexes with absorption maxima at 474-478 nm. Treatment of the cytochrome P450 complexes with K3Fe(CN)6 decreases the 474-478 nm absorbance and shifts the phenyl group from the iron to the porphyrin nitrogens. Acidification and extraction of the prosthetic group from each of the ferricyanide-treated enzymes yields a different mixture of the four possible N-phenylprotoporphyrin IX regioisomers. The ratios of the regioisomers with the phenyl ring on pyrrole rings B, A, C, and D (in order of elution from the high performance liquid chromatography column) are, respectively: cytochrome P450cam, 0:0:1:4; P450terp, 0:0:0:1; and P450BM-3, 2:10:2:1. The isomer ratio for recombinant cytochrome P450BM-3 without the cytochrome P450 reductase domain (2:9:2:1) shows that the reductase domain does not detectably perturb the active site topology of cytochrome P450BM-3. Potassium ions modulate the intensity of the spectrum of the phenyl-iron complex of cytochrome P450cam, but do not alter the N-phenyl isomer ratio. Computer graphics analysis of the crystal structure of the cytochrome P450cam phenyl-iron complex indicates that the active site of cytochrome P450cam is open above pyrrole ring D and, to a small extent, pyrrole ring C, in complete agreement with the observed N-phenylprotoporphyrin IX regioisomer pattern. The regioisomer ratios indicate that the active site of cytochrome P450terp is only open above pyrrole ring D, whereas that of cytochrome P450BM-3 is open to some extent above all the pyrrole rings but particularly above pyrrole ring A. The bacterial enzymes thus have topologies distinct from each other and from those of the mammalian enzymes so far investigated, which have active sites that are open to a comparable extent above pyrrole rings A and D.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446.
authors.forename: S F
authors.initials: SF
authors.lastname: Tuck
authors.forename: J A
authors.initials: JA
authors.lastname: Peterson
authors.forename: P R
authors.initials: PR
authors.lastname: Ortiz de Montellano
mesh_terms: D001426:Bacterial Proteins; D001665:Binding Sites; D019475:Camphor 5-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005292:Ferricyanides / Q000378:metabolism; D007097:Imines / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008958:Models, Molecular; D009251:NADPH-Ferrihemoprotein Reductase; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry
chemical_list: D001426:Bacterial Proteins; D005292:Ferricyanides; D007097:Imines; D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; C011981:phenyldiazene; D006899:Mixed Function Oxygenases; C073494:terpineol hydroxylase; D019475:Camphor 5-Monooxygenase; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; C028033:potassium ferricyanide