Interactions and electron transfer involved in pyruvate formate-lyase activation
Crain, Adam Vernon
MetadataShow full item record
Pyruvate formate-lyase activating enzyme (PFL-AE) is one of the best-characterized members of the radical S-adenosyl-L-methionine (SAM) superfamily. The radical SAM enzymes utilize an iron-sulfur cluster and SAM to catalyze a diverse set of reactions such as vitamin synthesis, enzyme activation, DNA repair, and sulfur insertion to name a few. PFL-AE contains one [4Fe-4S] cluster coordinated by cysteine residues from a canonical CX3CX2C radical SAM motif. Iron-sulfur cluster-initiated reductive cleavage of S-adenosylmethionine results in a highly reactive 5'-deoxyadenosyl radical that abstracts a pro-S hydrogen from glycine 734 on PFL creating a stable glycyl radical. PFL utilizes this glycyl radical to catalyze the reaction pyruvate + CoA  formate + acetyl-CoA, thereby providing the main source of acetyl-CoA for the citric acid cycle under anaerobic conditions. We have undertaken experiments using circular dichroism and isothermal titration calorimetry to characterize interactions between flavodoxin (Fld) and its cofactor (FMN). These experiments show that cofactor binding significantly increases flavodoxin stability and structure, which is required for protein-protein interactions. Anaerobic surface plasmon resonance experiments were used to provide insight into protein-protein interactions between the enzymes involved in PFL activation and in all cases, the proteins interact with low micromolar affinity. SAM binding experiments with PFL-AE were performed in the presence and absence of PFL, which demonstrate that PFL binding to PFL-AE does not alter SAM binding affinity for PFL-AE. PFL activation studies using PFL-AE in the presence of PFL substrates/analogues show that they are not required for PFL activation, however they do play a large role in activation and their inclusion resulted in 3.7 fold higher glycyl radical concentrations. In vivo concentrations were calculated for proteins and small molecules involved in PFL activation and activity in E. coli to provide a context for our measured equilibrium constants and to determine the amount of bound protein in vivo. Activity assays and UV-vis electron transfer assays show that pyruvate:flavodoxin oxidoreductase (PFOR) is capable of activating the PFL system. The aggregate data suggests that electron transfer from Fld to PFL-AE only occurs when SAM and PFL are bound to PFL-AE.