The nonheme iron oxidase isopenicillin N synthase catalyzes the formation of two new internal bonds in the tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form the β-lactam and thiazolidine rings of isopenicillin N. Concomitantly, O2 is reduced to 2 H2O. The recombinant enzyme from Cephalosporium acremonium (M(r) = 38,400), expressed as an apoenzyme in Escherichia coli, binds 1 g atom of Fe2+/mol of enzyme to reconstitute full activity. Mossbauer spectra of the 57Fe-enriched enzyme exhibit parameters (δ = 1.30 mm/s, ΔE(Q) = 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 δ (δ = 1.10 mm/s, ΔE(Q) = 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.
|Original language||English (US)|
|Number of pages||5|
|Journal||Journal of Biological Chemistry|
|State||Published - 1989|