Figure 1.
Cfr consumes two AdoMet equivalents per reaction cycle to support both methyl transfer to Cfr Cys338 (AdoMet1; step 1) and subsequently generation of the 5′dA⋅ radical (AdoMet2; step2).
Figure 2.
AdoMet Binding to the Cfr [4Fe-4S] Cluster.
Interaction of AdoMet with the Cfr [4Fe-4S] cluster as monitored by absorbance spectroscopy. A. Absorbance spectra of reconstituted wild-type Cfr (60 µM; red trace) and on serial addition of AdoMet (black traces) to a final concentration of 260 µM (blue trace). Inset: difference spectrum obtained by subtracting last (blue; AdoMet) from first (red; no AdoMet) spectrum. B. Absorbance of reconstituted wild-type Cfr at 390 nm (corrected for baseline effects at 600 nm) plotted as a function of AdoMet concentration. Solid line is fit to equation 3 (R2 = 0.87) giving a dissociation constant of 8.4 µM. C. Absorbance spectra of reconstituted Cys338 Ala Cfr (60 µM; red trace) and on serial AdoMet addition (black traces) to 260 µM (blue trace). Inset: difference spectrum obtained by subtracting last (blue; AdoMet) from first (red; no AdoMet) spectrum. D. Absorbance of reconstituted Cys338 Ala Cfr at 390 nm (corrected for baseline effects at 600 nm) plotted as a function of AdoMet concentration. Solid line is fit to equation 3 (R2 = 0.83) giving dissociation constant of 11.7 µM.
Figure 3.
EPR Spectra of the Cfr [4Fe-4S] cluster.
Cw-EPR spectra of wild-type Cfr (left hand column) and Cfr Cys338 Ala (right hand column). Experimental spectra are shown as black lines with corresponding simulations beneath (blue lines). A. Wild-type Cfr (105 µM) B. Wild-type Cfr (105 µM) plus AdoMet (300 µM). C. Wild-type Cfr plus AdoHcy (300 µM). D. Cfr Cys338 Ala (105 µM). E. Cfr Cys338 Ala plus AdoMet (300 µM). F. Cfr Cys338 Ala plus AdoHcy (300 µM). Note that all samples were reduced with sodium dithionite (1 mM).
Table 1.
g-Values for EPR Spectra of Cfr and Ligand Complexes.
Figure 4.
DOA production by Wild-type and Cys338 Ala Cfr.
Production of 5′-deoxyadenosine (DOA) from uncoupled cleavage of AdoMet by wild-type (filled circles) or Cys338 Ala (open circles) Cfr assayed by reverse-phase HPLC. Solid line shows fit of wild-type data to equation 2 (R2 = 0.99) yielding a kcat value of 42×10−4 s−1. Data points are the means of two independent measurements of separately prepared samples, shown with their standard error.
Figure 5.
Mechanisms for Radical Generation and Uncoupled AdoMet Turnover by Cfr.
Oxidative cleavage of AdoMet generates 5′dA⋅, 2, which abstracts a hydrogen atom to yield the detected 5′-deoxyadenosine product, 3. Uncoupled formation of 5′-deoxyadenosine could occur by quenching of 5′dA⋅ through abstraction of a hydrogen atom from either i) mCys338, 1, yielding the thermodynamically favourable radical intermediate 4 (pathway A, shown in red) or ii) a non-specific functional group (pathway B, shown in blue). Further details are given in the text.