Reconstitution and Characterization of the Polynuclear Iron-Sulfur Cluster in Pyruvate Formate-lyase-activating Enzyme

• DOI: 10.1074/jbc.273.9.4897
• Publication Date: 2002-07-26T14:47:21Z
• Authors: Raimund Külzer; Thomas Pils; Reinhard Kappl; Jürgen Hüttermann; Joachim Knappe
• Keywords: Iron-Sulfur Proteins; S-Adenosylmethionine; 0303 health sciences; Iron; Molecular Sequence Data; Electron Spin Resonance Spectroscopy; Sulfides; Ligands; S-Adenosylhomocysteine; Recombinant Proteins; Enzymes; 3. Good health; Enzyme Activation; 03 medical and health sciences; Acetyltransferases; Spectrophotometry; Escherichia coli; Mutagenesis, Site-Directed; Amino Acid Sequence; Cysteine; Apoproteins; Conserved Sequence

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 UV-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.