title: Spectroscopic studies of isopenicillin N synthase. A mononuclear nonheme Fe2+ oxidase with metal coordination sites for small molecules and substrate.
abstract: The nonheme iron oxidase isopenicillin N synthase catalyzes the formation of two new internal bonds in the tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form the beta-lactam and thiazolidine rings of isopenicillin N. Concomitantly, O2 is reduced to 2 H2O. The recombinant enzyme from Cephalosporium acremonium (Mr = 38,400), expressed as an apoenzyme in Escherichia coli, binds 1 g atom of Fe2+/mol of enzyme to reconstitute full activity. Mössbauer spectra of the 57Fe-enriched enzyme exhibit parameters (delta = 1.30 mm/s, delta EQ = 2.70 mm/s) which unambiguously show that the active site iron is high spin Fe2+. Anaerobic binding of ACV causes a substantial decrease in the isomer shift parameter delta (delta = 1.10 mm/s, delta EQ = 3.40 mm/s) showing that the substrate perturbs the iron site and makes its coordination environment much more covalent. Nitric oxide (NO) binds to the EPR silent active site iron to give an EPR active species (g = 4.09, 3.95, 2.0; S = 3/2) similar to those of the nitrosyl complexes of many other mononuclear Fe2+-containing enzymes. The rhombicity of the EPR spectrum is increased (g = 4.22, 3.81, 1.99) by anaerobic addition of ACV suggesting that the substrate binds to or near the iron without displacing NO. Interestingly, the enzyme.ACV.NO complex displays an optical spectrum similar to that of ferric rubredoxin in which the iron has only thiol coordination. This suggests that the Fe2+ of the enzyme.ACV.NO complex acquires Fe3+ character and that the cysteinyl thiol moiety of ACV coordinates to the iron. Similar substrate thiol coordination to the iron of the enzyme.ACV complex is the most probable explanation for the large decrease in isomer shift observed. These results provide the first evidence for the direct involvement of iron in this unique O2-dependent reaction and suggest novel roles for iron and oxygen in biological catalysis.
authors.affiliation: Lilly Research Laboratories, Indianapolis, Indiana 46285.
authors.forename: V J
authors.initials: VJ
authors.lastname: Chen
authors.forename: A M
authors.initials: AM
authors.lastname: Orville
authors.forename: M R
authors.initials: MR
authors.lastname: Harpel
authors.forename: C A
authors.initials: CA
authors.lastname: Frolik
authors.forename: K K
authors.initials: KK
authors.lastname: Surerus
authors.forename: E
authors.initials: E
authors.lastname: Münck
authors.forename: J D
authors.initials: JD
authors.lastname: Lipscomb
mesh_terms: D000164:Acremonium / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D004798:Enzymes / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007501:Iron / Q000032:analysis; D007700:Kinetics; D010088:Oxidoreductases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D013057:Spectrum Analysis
chemical_list: D004798:Enzymes; D011994:Recombinant Proteins; D007501:Iron; D010088:Oxidoreductases; C038370:isopenicillin N synthetase