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

Abstract

Mycofactocin is a putative redox cofactor and is classified as a ribosomally synthesized and post-translationally modified peptide (RiPP). Some RiPP natural products, including mycofactocin, rely on a radical S-adenosylmethionine (RS, SAM) protein to modify the precursor peptide. Mycofactocin maturase, MftC, is a unique RS protein that catalyzes the oxidative decarboxylation and C-C bond formation on the precursor peptide MftA. However, the number, chemical nature, and catalytic roles for the MftC [Fe-S] clusters remain unknown. Here, we report that MftC binds a RS [4Fe-4S] cluster and two auxiliary [4Fe-4S] clusters that are required for MftA modification. Furthermore, electron paramagnetic resonance spectra of MftC suggest that SAM and MftA affect the environments of the RS and Aux I cluster, whereas the Aux II cluster is unaffected by the substrates. Lastly, reduction potential assignments of individual [4Fe-4S] clusters by protein film voltammetry show that their potentials are within 100 mV of each other.

Figure 1.

A) A representative crystal structure for the RS-SPASM protein family (PDBID: 4K36) showing components of the protein including the TIM barrel fold, RS domain (blue), the SPASM domain (red), [4Fe-4S] clusters (yellow/orange), and SAM (purple). B) A condensed reaction scheme for the formation of a 5′-deoxyadenosine radical, generated on the radical-SAM cluster (RS cluster) and the known biosynthetic modifications catalyzed by MftC and MftE.

Figure 2.

4.2-K Mössbauer spectra of the as-purified and chemically reconstituted wild-type Mu MftC. (Top) The spectrum of the as-isolated MftC (black) consists of a quadrupole doublet corresponding to the presence of [2Fe-2S] clusters (dark green highlight) and [4Fe-4S] clusters (light green highlight). The overall simulation is overlaid on the experimental spectrum as a black solid line. (Bottom) The spectrum of the chemically reconstituted with two equiv. of [Fe-S] clusters MftC (black) consists of a single quadrupole doublet corresponding to the presence of [4Fe-4S] clusters. The spectra were recorded in the presence of a small external magnetic field (0.078 T), applied parallel to the orientation of the γ beam.

Figure 3.

A) Activity assays consisting of 2 mM SAM, 10 mM DTT, 2 mM DTH, and 100 μM of the MftC variant or B) 2 mM SAM, 10 mM DTT, 2 mM DTH, 100 μM MftA, 100 μM MftB and 100 μM of the MftC variant suggest that Aux I and Aux II clusters are not required for SAM turnover and that all clusters are required for MftA modification.

Figure 4.

The extracted g values and g strain for the three [4Fe-4S] clusters from the difference spectra were used to simulate (red, cyan, magenta) the intact MftC protein (black, blue, and purple) in the absence and presence of SAM, MftA, and MftB. Spectra were obtained at 20 K.

Figure 5.

EPR spectra, measured at 20 K, for individual [4Fe-4S] cluster knockouts were subtracted from MftC, yielding difference spectra (A-C). The experimental difference spectra (black) represent the EPR spectra for the individual cluster knocked out and can be reasonably simulated (red). The EPR difference spectra in the presence of SAM (D-F) suggest that the removal of the RS cluster affects the environments or the remaining clusters when SAM is bound. Conversely, removal of the Aux I or Aux II cluster has little impact on the environments of the remaining clusters when SAM is bound. The experimental difference spectra (blue) for the RS cluster could not be simulated whereas the simulations (cyan) for the Aux I and Aux II cluster are plausible representations of [4Fe-4S]⁺ clusters. The EPR difference spectra in the presence of SAM, MftA, and MftB (G-I) have similar implications to when SAM is bound alone. Likewise, the removal of the RS cluster impacts the remining [4Fe-4S] clusters when SAM, MftA, and MftB is bound whereas the inverse is not observed. The experimental difference spectra are shown in violet and the simulated data is shown in magenta; the RS cluster could not be simulated.

Figure 6.

Voltammetry of MftC and variants measured at pH 7.5 and 4 ºC. (A) Cyclic voltammogram measured with a scan rate of 100 mV/s for wild-type MftC (solid line), fitting for three, one electron transfers (dotted line), and EPG baseline (dashed line). Square wave voltammograms measured with a frequency of 10 Hz and an amplitude of 20 mV for (B) wild-type MftC (solid line), (C) RS KO, and (D) Aux II KO with EPG baselines (dashed line).

Scheme 1.

MftC catalyzes the oxidative decarboxylation of MftA followed by the C-C bond formation between Val29 and Tyr30. The oxidative reaction is highlighted by the blue box and the redox neutral reaction is highlighted by the red box. It is currently unknown if the Aux I or Aux II clusters participate in the reaction via electron shuttling.