Deconvoluting the Reduction Potentials for the Three [4Fe-4S] Clusters in an AdoMet Radical SCIFF Maturase

Abstract

Enzymes in the S -adenosyl- l -methionine (AdoMet) radical enzyme superfamily are metalloenzymes that catalyze a wide variety of complex radical-mediated transformations with the aid of a [4Fe-4S] cluster, which is required for activation of AdoMet to generate the 5′-deoxyadenosyl radical to initiate the catalytic cycle. In addition to this cluster, some enzymes share an additional domain, the SPASM domain, that houses auxiliary FeS clusters whose functional significance is not clearly understood. The AdoMet radical enzyme Tte1186, which catalyzes a thioether cross-link in a cysteine rich peptide (SCIFF), has two auxiliary [4Fe-4S] clusters within a SPASM domain that are required for enzymatic activity but not for the generation of the 5′-deoxyadenosyl radical intermediate. Here we demonstrate the ability to measure independently the midpoint potentials of each of the three [4Fe-4S] clusters by employing Tte1186 variants for which only the first, second, or AdoMet binding cluster is bound. This allows, for the first time, assignment of reduction potentials for all clusters in an AdoMet radical enzyme with a SPASM domain. Our results show that the clusters have midpoint potentials that are within 100 mV of each other, suggesting that their electrochemical properties are not greatly influenced by the presence of the nearby clusters.

Spectroscopic and Electrochemical Characterization of the Mycofactocin Biosynthetic Protein, MftC, Provides Insight into Its Redox Flipping Mechanism

“…The ability to redox flip is unique to the RS-SPASM family and suggests that MftC has means to modulate the electrochemical environment within its active site for each catalytic step. The resting state reduction potential for the Aux I cluster is similar to what was measured for the same cluster in SCIFF maturase 52 . This similarity suggests that the midpoint potential of the Aux I cluster in MftC may be modulated through substrate interaction or active site rearrangement to accommodate both oxidative and redox neutral chemistries.…”

Mechanism of Reduction of an Aminyl Radical Intermediate in the Radical SAM GTP 3′,8-Cyclase MoaA

“…Elucidation of the catalytic function of auxiliary clusters in radical SAM enzymes in the SPASM-twitch family remains difficult. While reduction potentials of several other members of the SPASM-twitch family have been reported, ,,, ambiguity remains about their functions in catalysis due to the lack of understanding in the reduction potentials of reaction intermediates and the redox state of the auxiliary clusters during catalytic turnover. In this study, we used a combination of PFV, EPR, and DFT calculation to provide experimental evidence that the reduced 1+ state auxiliary cluster of MoaA reduces the aminyl radical intermediate to form the final product.…”

“…So far, the best-characterized systems are SPASM subfamily enzymes responsible for modifying ribosomally synthesized peptide natural products (RiPPs). Among such enzymes, SCIFF maturase and MftC were reported to have two [4Fe-4S] AUX clusters with ∼50 and ∼100 mV more negative reduction potentials than that of [4Fe-4S] RS . , However, these potentials have not yet been correlated to the function of [4Fe-4S] AUX as an electron acceptor. More recently, a study on another SPASM subfamily member SuiB provided experimental evidence for the redox function of auxiliary cluster .…”

Photoinduced Electron Transfer in a Radical SAM Enzyme Generates an S-Adenosylmethionine Derived Methyl Radical

“…ENDOR conditions: T = 2 K; modulation amplitude, 4 G; scan speed, 3 MHz/s; scan direction, forward; microwave frequency, 34.8 GHz. EasySpin EPR simulations: function, pepper; g = [2.021, 2.0047,1.987]; Gaussian line widths 30 MHz; for spectrum (B), A( 13 C) = [20,30,20] MHz; H strain = [60,30,60] MHz. Temperature dependence of the • CH 3 signal.…”