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A designed heme-[4Fe-4S] metalloenzyme catalyzes sulfite reduction like the native enzyme

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Science  14 Sep 2018:
Vol. 361, Issue 6407, pp. 1098-1101
DOI: 10.1126/science.aat8474
  • Fig. 1 Design of a [4Fe-4S] binding site in CcP to mimic the heme-[4Fe-4S] center in native SiR.

    From left to right: A search structure generated from the binding cavity of the [4Fe-4S] in the siroheme-[4Fe-4S] cofactor from the hemoprotein subunit of native Escherichia coli SiR [Protein Data Bank (PDB) ID 2GEP] (9) was used to search the PDB for suitable hemoprotein scaffolds. Yeast CcP was identified as a suitable scaffold, and a binding site for a heme-[4Fe-4S] cofactor was designed by a combination of computational and rational design methods. The resulting computational model of the designed heme-[4Fe-4S] center in SiRCcP.1 is shown on the right. Three Cys mutations (T180C, W191C, L232C) coordinate Fe atoms, and the H175C mutation acts as a bridging Cys ligand between the heme and [4Fe-4S] cofactors.

  • Fig. 2 Spectroscopic properties of SiRCcP.1 with [4Fe-4S], heme, and heme-[4Fe-4S] cofactors confirm binding of [4Fe-4S] and heme-[4Fe-4S] cofactors.

    (A) X-band EPR spectrum of FeS-SiRCcP.1 reduced with an excess of sodium dithionite (black) and simulated spectrum (magenta) indicating an S = ½ species consistent with a [4Fe-4S]+: gx = 1.891, gy = 1.919, gz = 2.035; linewidths (G) Ax = 42, Ay = 27, Az = 25. The spectrum shown was measured at a frequency of 9.173 GHz and modulation amplitude of 10 Gauss; a microwave power of 10 mW, and a temperature of 15 K. No paramagnetic species at g = 2 are observed before reduction, suggesting a [4Fe-4S]2+ (S = 0) state as prepared (oxidized). A [3Fe-4S]+ species could be generated by reoxidation of the reduced species with an excess of potassium ferricyanide (fig. S10). (B) Magnitude of the phase-uncorrected k3-weighted Fourier transform (FT) and EXAFS (inset) of the Fe K-edge spectra for FeS2+-SiRCcP.1 (black) and the best fit (magenta). The EXAFS spectrum is consistent with a symmetrical cubane structure (three scattering Fe atoms per absorber Fe and four scattering S atoms in the first coordination shell). Fitting parameters are provided in table S1, and the EXAFS of FeS+-SiRCcP.1 are plotted in fig. S11. R, scattering shell distance. (C) UV-visible spectra of Fe-S–reconstituted SiRCcP.1 (FeS-SiRCcP.1) and heme-Fe-S–reconstituted SiRCcP.1 (heme-FeS-SiRCcP.1) in the presence and absence of potassium cyanide. As prepared, FeS-SiRCcP.1 has weak, broad absorption at 400 nm, which decreases upon reduction (fig. S9). Ferric heme-FeS-SiRCcP.1 has a blue-shifted Soret peak (378 nm) relative to that of native CcP (408 nm) and exhibits a Soret maximum and Q-bands (inset) consistent with penta-coordinate, thiolate-ligated high-spin ferriheme. The ferrous heme-FeS-SiRCcP.1 spectrum obtained by incubation with sodium dithionite (fig. S12) is distinct from the species obtained by photoreduction (fig. S13) and is identical to the spectrum of heme2+-FeS+-SiRCcP.1 incubated with sulfite under nonturnover conditions (fig. S14), indicating sulfite-bound hexacoordinate ferroheme. The photoreduced spectrum (fig. S13) represents the ferrous pentacoordinate species. (D) X-band EPR spectra of heme-FeS-SiRCcP.1 in the presence of cyanide. CN-heme3+-FeS2+-SiRCcP.1 is predominantly low-spin ferric heme (gx = 1.87, gy = 2.26, gz = 2.45) with no visible [4Fe-4S] features. In the all-ferrous state, heme features disappear (presumably S = 0 ferrous heme) while the S = ½ [4Fe-4S] feature reappears. Similar behavior has been reported for CN-bound SiR. EPR spectra were measured at 18 K with a microwave power of 10 mW at a frequency of 9.24 GHz.

  • Fig. 3 Sulfite reduction activity of SiRCcP mutants is modulated by substrate binding and [4Fe-4S] secondary sphere mutations.

    (A) Computational model of the SiRCcP substrate-binding site with mutations W51K, H52R, and P145K (cyan) made to mimic native SiR residues K217, R153, and K215, respectively (fig. S15). The native CcP residue R48 (orange) is positioned similarly to R83 in native SiR. (B) Computational model of the mutations D235V (SiRCcP.1, cyan), D235N (SiRCcP.2, green), and D235C (SiRCcP.3, magenta) in SiRCcP. (C) Sulfite reduction activity of four SiRCcP mutants in comparison to a native sulfite reductase from M. tuberculosis (Mtb NiRA). “K+R” denotes the presence of the three mutations W51K, H52R, and P145K. Reported activities represent the average of triplicate measurements with standard error (see table S2).

Supplementary Materials

  • A designed heme-[4Fe-4S] metalloenzyme catalyzes sulfite reduction like the native enzyme

    Evan N. Mirts, Igor D. Petrik, Parisa Hosseinzadeh, Mark J. Nilges, Yi Lu

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods 
    • Supplementary Text
    • Figs. S1 to S19
    • Tables S1 and S2
    • Listings S1 to S4
    • References 

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