Pyruvate formate-lyase-activating enzyme (PFL-AE) from Escherichia coli (E. coli) catalyzes the stereospecific abstraction of a hydrogen atom from Gly734 of pyruvate formate-lyase (PFL) in a reaction that is strictly dependent on the cosubstrate S-adenosyl-L-methionine (AdoMet). Although Pn-AE is an iron-dependent enzyme, isolation of the enzyme with its metal center intact has proven difficult due to the oxygen sensitivity and lability of the metal center. We report here the first isolation of PFL-AE under nondenaturing, strictly anaerobic conditions. Iron and sulfide analysis as well as W-visible, EPR, and resonance Raman data support the presence of a [3Fe-4S](+) cluster in the purified enzyme. The isolated native enzyme, but not ape-enzyme, exhibits a high specific activity (31 U/mg) in the absence of added iron, indicating that the native cluster is necessary and sufficient for enzymatic activity, (C) Academic Press.