Pyruvate formate-lyase-activating enzyme: Strictly anaerobic isolation yields active enzyme containing a [3Fe-4S](+) cluster

Pyruvate formate-lyase-activating enzyme (PFL-AE) from Escherichia coli (E. coli) catalyzes the stereospecific abstraction of a hydrogen atom from Gly 734 of pyruvate formate-lyase (PFL) in a reaction that is strictly dependen t on the cosubstrate S-adenosyl-L-methionine (AdoMet). Although Pn-AE is an iron-dependent enzyme, isolation of the enzyme with its metal center intac t has proven difficult due to the oxygen sensitivity and lability of the me tal center. We report here the first isolation of PFL-AE under nondenaturin g, strictly anaerobic conditions. Iron and sulfide analysis as well as W-vi sible, EPR, and resonance Raman data support the presence of a [3Fe-4S](+) cluster in the purified enzyme. The isolated native enzyme, but not ape-enz yme, exhibits a high specific activity (31 U/mg) in the absence of added ir on, indicating that the native cluster is necessary and sufficient for enzy matic activity, (C) Academic Press.