ANAEROBIC CRYSTALLIZATION OF AN ISOPENICILLIN-N SYNTHASE-CENTER-DOT-FE(II)CENTER-DOT-SUBSTRATE COMPLEX DEMONSTRATED BY X-RAY STUDIES

Isopenicillin N synthase (IPNS) was cocrystallised with ferrous sulpha te and its substrate, delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valin e (Aad-Cys-Val). Vital to the successful procedure was the maintenance of a rigorously anaerobic environment. Hanging-drop vapour-diffusion crystallisation experiments, using lithium sulphate as the precipitant produced three crystal forms. Form I crystals, with a plate habit, di ffracted X-rays to at least 0.11-nm resolution at the European Synchro tron Radiation Facility and belong to the space group P2(1)2(1)2(1), w ith unit-cell dimensions a = 4.68, b = 7.15, c = 10.10 nm. Their asymm etric unit contains a single IPNS . Fe(II) . Aad-Cys-Val complex with a solvent content of 38.5 %. Form II crystals, with a hexagonal habit, diffract X-rays to at least 0.21 nm resolution at the European Synchr otron Radiation Facility and belong to the space group P3(1)21, with u nit-cell dimensions a = 10.10, b = 10.10, c = 11.567 nm. Their asymmet ric unit also contains a single IPNS . Fe(II) . Aad-Cys-Val complex wi th a solvent content of 69.5 %. Form III crystals, needles, do not sho w well-ordered diffraction. Although all three forms were initially pr oduced in crystallisation experiments under identical conditions, appr opriate micro and streak seeding allows selective crystallisation of f orm I or form II crystals. Extended X-ray-absorption fine-structure st udies on a crystalline slurry of the form I crystals demonstrate the p resence of an Fe-S(Aad-Cys-Val) bond length of 0.234 +/- 0.003 nm.