Metalloenzymes in the S-adenosyl-L-methionine (AdoMet) radical enzyme (ARE) superfamily catalyze a vast array of complex radical-mediated transformations via a reactive [4Fe-4S] cluster, which is involved in the homolytic cleavage of AdoMet to generate the 5′- deoxyadenosyl radical to initiate the catalytic cycle. In addition to this cluster, AdoMet-SPASM enzymes contain an elongated C-terminal SPASM domain that typically binds two auxiliary [4Fe-4S] clusters whose functional significance is not clearly understood.<b> </b>While the auxiliary clusters are known to be essential for substrate modification by the enzymes, their mechanistic role has yet to be clearly defined. Because all AREs are activated through this initial cleavage step, we have hypothesized that the diverse chemistries that these enzymes catalyze are determined by the thermodynamic characteristics of the corresponding auxiliary FeS clusters found within the SPASM domain. A comparative study utilizing protein film voltammetry (PFV) has been executed to investigate the reduction potentials of the auxiliary clusters of two SPASM domain AdoMet enzymes, MftC and Tte1186. Each enzyme contains two auxiliary [4Fe-4S] clusters that are required for enzymatic activity but are not essential for the generation of the 5′-deoxyadenosyl radical intermediate. Identifying the diversity in reduction potentials of these clusters will provide insight into the possible roles that these clusters play in tuning the function of these particular AREs. Here, we demonstrate the ability to measure the midpoint potentials of each individual [4Fe-4S] cluster by generating either double (Tte1186) or single (MftC) cluster knockout variants. The studies of MftC and Tte1186 will be discussed thoroughly using electrochemical methods to assign reduction potentials to each [4Fe-4S] cluster in order to gain better understanding of what role the clusters play in enzyme function. Further emphasis will be placed on the effect AdoMet and substrate have on the assigned reduction potentials in Tte1186, such that role of the individual [4Fe-4S] clusters are understood in regards to catalytic mechanism, and drawing comparisons to other members of the superfamily.

Electrochemical characterization of AdoMet radical enzymes

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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