Reconstitution and characterization of the polynuclear iron-sulfur cluster in pyruvate formate-lyase-activating enzyme -: Molecular properties of the holoenzyme form

The glycyl radical (Gly-734) contained in the active form of pyruvate formate-lyase (PFL) of Escherichia coli is generated by the S-adenosylmethionine-dependent pyruvate formate-lyase-activating enzyme (PFL activase), A 5'-deoxyadenosyl radical intermediate produced by the activase has been suggested as the species that abstracts the pro-S hydrogen of the glycine 734 residue in PFL (Frey, M., Rothe, M., Wagner, A. F. V., and Knappe, J. (1994) J. Biol. Chem. 269, 12432-12437). To enable mechanistic investigations of this system we have worked out a convenient large scale preparation of functionally competent PFL activase from its apoform, The previously inferred metallic cofactor was identified as redox-interconvertible polynuclear iron-sulfur cluster, most probably of the [4Fe-4S] type, according to W-visible and EPR spectroscopic information, Cys --> Ser replacements by site-directed mutagenesis determined Cys-29, Cys-33, and Cys-36 to be essential to yield active holoenzyme, Gel filtration chromatography showed a monomeric structure (28 kDa) for both the apoenzyme and holoenzyme form, The iron-sulfur cluster complement proved to be a prerequisite for effective binding of adenosylmethionine, which induces a characteristic shift of the EPR signal shape of the reduced enzyme form ([4Fe-4S](+)) from axial to rhombic symmetry.