Designed Rubredoxin miniature in a fully artificial electron chain triggered by visible light

Abstract

Designing metal sites into de novo proteins has significantly improved, recently. However, identifying the minimal coordination spheres, able to encompass the necessary information for metal binding and activity, still represents a great challenge, today. Here, we test our understanding with a benchmark, nevertheless difficult, case. We assemble into a miniature 28-residue protein, the quintessential elements required to fold properly around a FeCys₄ redox center, and to function efficiently in electron-transfer. This study addresses a challenge in de novo protein design, as it reports the crystal structure of a designed tetra-thiolate metal-binding protein in sub-Å agreement with the intended design. This allows us to well correlate structure to spectroscopic and electrochemical properties. Given its high reduction potential compared to natural and designed FeCys₄-containing proteins, we exploit it as terminal electron acceptor of a fully artificial chain triggered by visible light.

Fig. 1. Crystal structure of V44A mutant of Cp Rd (PDB ID: 1C09).

The secondary structure is depicted as a gray ribbon, the iron ion as a brown sphere and the first (magenta) and second (pink) coordination sphere residues as sticks.

Fig. 2. Design of METPsc1.

a crystal structure of Cp Rd V44A mutant (PDB ID: 1C09). b miniaturized model, obtained by applying a C₂ longitudinal rotation to the Val38-Glu50 fragment of Cp Rd V44A. c superimposition of the 4-residue loops found from the fragment search. d single-chain METP prototype, obtained by combination of the C₂-symmetric dimer with the type I’ β-turn selected from the search. e designed model and sequence of METPsc1, in its complex with Zn²⁺. Cys and type I’ β-turn residues are highlighted in yellow.

Fig. 3. ZnMETPsc1 structural characterization.

a Metal ion, all sidechains, and N- and C-terminal capping groups are clearly visible in the electron density map (2F_o-F_c. map, 1.3 σ level). b The monomeric X-ray structure of ZnMETPsc1 (cyan, this work, PDB ID: 5SBG) closely matches the designed model (light brown). c Description of secondary structural elements found in ZnMETPsc1 structure (blue: β-strand; ocre: α-turn; gray: β-bulge; green: 3₁₀-helix; orange: type I’ β-turn). Dashed lines represent backbone to backbone H-bonds. d First coordination sphere shows the expected coordination bond distances between zinc and cysteine sulfur atoms. e Second coordination sphere involving amide of Ala7 and Ala22 exacerbates H-bond strength with respect to wt Cp Rd. f, The H-bond donors from sidechains of Asn19 and a symmetry-related Arg26 (in cyan) to METPsc1 partners are indicated.

Fig. 4. FeMETPsc1 spectroscopic characterization.

a UV-Vis titration of METPsc1 with Fe²⁺, spectra at increasing iron concentration are reported from violet to green. Absorbances at 311 nm are reported in the inset (black squares) and fitted by a 1:1 binding isotherm (red dashed line). Mohr’s salt (36 mM) aliquots were added to a 30 μM METPsc1 solution in a 20 mM HEPES buffer (pH 7) and 1 mM TCEP. b, c UV-Vis and CD spectra of the reduced (black line) and oxidized (red line) FeMETPsc1 (40 μM) species. d X-band CW-EPR spectrum of Fe³⁺METPsc1 (0.5 mM) in 20 mM phosphate buffer (pH 7) and 5 mM TCEP at 4.5 K. Source data are provided in a Zenodo repository under accession code 7748883.

Fig. 5. FeMETPsc1 redox characterization.

a UV-Vis monitoring of Fe²⁺METPsc1 (blue trace) aerobic oxidation to Fe³⁺METPsc1 (lime trace). Spectra were acquired every 3 min. 40 µM FeMETPsc1, 20 mM HEPES buffer 2 mM TCEP, pH 7. b Redox cycling of FeMETPsc1 (40 µM) in HEPES buffer (20 mM) and TCEP 2 mM, pH 7, monitored by absorption at 494 nm (corresponding to the ferric species). Cycles consist of successive (i) air purge of the Fe²⁺ complex to form the Fe³⁺ complex and (ii) argon purge and dithionite reduction to restore the Fe²⁺ complex. Source data are provided in a Zenodo repository under accession code 7748883.

Fig. 6. FeMETPsc1 electrochemical characterization.

a Cyclic voltammograms of FeMETPsc1 (80 μM) recorded at different scanning rate from 2.5 to 50 mV s⁻¹ (bright pink to violet), in 40 mM HEPES buffer (pH 7) and 0.3 M KCl. Each voltammogram is the last of three consecutive scans. b Anodic and cathodic peak currents derived from cyclic voltammetry experiments of FeMETPsc1 (80 µM) plotted as a function of the square root of the scan rate. Data points were fitted to the Randles-Ševčík equation, allowing to determine the diffusion coefficient of FeMETPsc1 both in the reduced (Dred = 0.92 10⁻⁶ cm² s⁻¹) and in the oxidized state (Dox = 1.4 10⁻⁶ cm² s⁻¹). Source data are provided in a Zenodo repository under accession code 7748883.

Fig. 7. Photoinduced electron transfer from ZnMC6*a (5 µM) to Fe³⁺METPsc1 (40 µM).

a Reaction scheme of the synthetic electron cascade. b experimental setup showing the LED strip wrapped around the UV cuvette under Ar atmosphere. c superimposed UV-Vis spectra of Fe³⁺METPsc1 (black trace) and Fe²⁺METPsc1 (red trace) in the presence of ZnMC6*a (5 µM) and triethylamine (2 mM). d redox cycling of FeMETPsc1 monitored at 496 nm and 311 nm (molar absorptivities are reported in Table 1). Pink boxes correspond to 20 min of green light irradiation, blue box corresponds to the dark control under Ar atmosphere. Source data are provided in a Zenodo repository under accession code 7748883.