Conversion of 3Fe-4S to 4Fe-4S clusters in native pyruvate formate-lyase activating enzyme: Mossbauer characterization and implications for mechanism

Pyruvate formate-ly ase activating enzyme utilizes an iron-sulfur cluster a nd S-adenosylmethionine to generate the catalytically essential glycyl radi cal on pyruvate formate-lyase. Variable-temperature (4.2-200 K) and variabl e-field (0.05-8 T) Mossbauer spectroscopy has been used to characterize the iron-sulfur clusters present in anaerobically isolated pyruvate formate-ly ase activating enzyme and in the dithionite-reduced form of the enzyme. Det ailed analysis of the Mossbauer data indicates that the anaerobically isola ted enzyme contains a mixture of Fe-S clusters with the cuboidal [3Fe-4S]() clusters as the primary cluster form, accounting for 66% of the total iro n. Other forms present include [2Fe-2S](2+) (12% of total Fe) and [4Fe-4S]( 2+) (8% of total iron). Careful examination of Mossbauer spectra recorded a t various applied fields reveal a fourth spectral component which is assign ed to a linear [3Fe-4S](+) (similar to 10% of total Fe). Reduction of the a s-isolated enzyme by dithionite, interestingly, converts all cluster types into the [4Fe-4S] form with a mixture of 2+ (66% of total iron) and 1+ (12% of total iron) oxidation states. These results are discussed in light of t he proposed role for the iron-sulfur cluster in radical generation.