Contents. SI Figures: Figures S1-S13. SI Tables: Table S1-S3. SI References. Supporting Information

Transcription

1 Supporting Information Development of a rubredoxin-type center embedded in a de novo designed three-helix bundle Alison G. Tebo 1, Tyler B. J. Pinter 2, Ricardo García-Serres 3, Amy L. Speelman 2, Cédric Tard 4, Olivier Sénéque 3, Geneviève Blondin 3, Jean-Marc Latour 3, James Penner-Hahn 2, Nicolai Lehnert 2, Vincent L. Pecoraro 2 * 1 Program in Chemical Biology, University of Michigan, Ann Arbor, MI USA 2 Department of Chemistry and Biophysics, University of Michigan, Ann Arbor, MI USA 3 Univ. Grenoble Alpes, CNRS, CEA, BIG, LCBM (UMR 5249), F Grenoble, France. 4 LCM, CNRS, École Polytechnique, Université Paris-Saclay, Palaiseau Cedex, France. Contents SI Figures: Figures S1-S13 SI Tables: Table S1-S3 SI References S1

2 Figure S1 (a) Circular dichroism spectra of apo-α 3 DIV-L21C as well as in the presence of Fe(II) and Fe(III) indicates that no significant structural change accompanies Fe binding. Comparison of thermal denaturation as monitored by circular dichroism of apo-α 3 DIV-L21C (b) and α 3 DIV-L21C-Fe(II) (c) indicates a slight stabilization induced by Fe binding ( kcal/mol to kcal/mol). S2

3 Figure S2 Direct binding titrations measured by UV-visible absorption spectroscopy demonstrate phdependent Fe(II) binding. Experiments were carried out in a nitrogen atmosphere glovebox in 20 mm Tris buffer with 50 µm α 3 DIV-L21C and 100 µm TCEP. S3

4 Figure S3 (top) Simulations of the 600 G and 1 T applied-field Mossbauer spectra of α 3 DIV-L21C- Fe(III) from Figure 1 with different values of ZFS parameter D, with E/D = 0.15 and other parameters as in Tables 2 and S3. The spectra measured at 4. contain a superposition of six-line spectra from three Kramers doublets. Their relative intensities are highly sensitive to both the magnitude and the sign of D. Setting D = 1 cm -1 (green line) affords a spectrum with insufficient contribution of Kramers doublet 3, which is the highest in energy. Conversely, doublet 3 becomes too populated for D = -0.5 cm -1 (magenta lines), as it becomes lower in energy than doublets 1 and 2. The green and magenta simulations were not improved significantly by fitting the rest of the parameters. (bottom) X-band EPR spectra of 1.5 mm α 3 DIV-L21C with mm Fe(III) in 50 mm Tris buffer at ph 8.5 with ethylene glycol as glassing agent at 4., 12.5K, and 17K. S4

5 ε (M -1 cm -1 ) ε (M -1 cm -1 ) Wavelength / nm Figure S4. Comparison of the UV-Vis (at room temperature) versus the MCD spectrum (4K/1T) of α 3 - DIV-L21C. See Figure 3 for a fit of the data. S5

6 MCD Intensity / θ = mdeg nm 357 nm 385 nm 421 nm Energy / cm nm 520 nm 604 nm 7 T 6 T 5 T 4 T 3 T 2 T 1 T Figure S5. MCD spectrum of α 3 -DIV-L21C measured at and variable magnetic field MCD Intensity / θ = mdeg Energy / cm -1 Figure S6. MCD spectrum of α 3 -DIV-L21C measured at 1 T at variable temperatures. The temperature dependence of the MCD data indicates that all of the observed spectral features correspond to C-term MCD signals. S6

7 Band 1: 640 nm Band 2: 578 nm Band 3: 518 nm Band 4: 481 nm Band 5: 449 nm Band 6: 419 nm S7

8 Band 7: 386 nm Band 8: 357 nm Band 9: 329 nm Figure S7. VTVH saturation curves for MCD bands 1-9 (see Figure 3 and Table S1). Bands 1, 2, 7, and 9 show nested saturation behavior indicative of z-polarization, whereas bands 3-6 and 8 show overlaid saturation behavior indicative of xy polarization. Since bands 1 and 2 overlap slightly, saturation curves for band 1 were generated at a wavelength where band 2 does not have significant intensity. Fits for bands 2 and 6 are shown in Figure S7. S8

9 λ = 578 nm (Band 2) Fits λ = 419 nm (Band 6) Fits Figure S8. Fits of VTVH isotherms for (top) Band 2 (nested behavior; 1% xy polarized, 99% z-polarized) and (bottom) Band 6 (overlaid behavior; 72% xy polarized, 28% z-polarized). Other fit parameters: g = (2.0, 2.0, 2.0); E/D = 0.15; D = 0.5 cm -1 S9

10 100% x-polarized D = cm % x-polarized D = cm % y-polarized D = cm % y-polarized D = cm % z-polarized D = cm % z-polarized D = cm -1 Figure S9. Simulations of VTVH isotherms for electronic transitions with x (top), y (middle), and z (bottom) polarizations for positive values of D (left) and negative values of D (right). Other parameters: S = 5/2; g = (2.0, 2.0, 2.0); E/D = Note that if the sign of D is positive, the polarizations are inverted, with xy-polarized bands showing nested behavior and z-polarized bands showing overlaid behavior. S10

11 D = cm -1 D = cm -1 D = - cm -1 D = cm -1 D = cm -1 D = cm -1 Figure S10. Simulations of VTVH isotherms for electronic transitions with 100% z-polarization and negative D with variable magnitude as indicated. Other parameters: S = 5/2; g = (2.0, 2.0, 2.0); E/D = Note that larger values of D result in an increase in the nesting of the saturation curves. S11

12 1.5 Normalized Intensity energy, ev Figure S11. Fe K-edge XANES region of α 3 DIV-L21C-Fe(II) Figure S12. The redox reversibility of α3div-l21c-fe was tested by repeated exposure to air followed by reduction with β-mercaptoethanol. S12

13 current (A) Potential (V) vs. SCE Figure S13. (left) Representative cyclic voltammagram of α 3 DIV-L21C-Fe in solution with 100 mm Tris buffer, 100 mm Na 2 SO 4, ph 8.5, scan rate 20 mv s -1. (right) Dependence of apparent E on ph. S13

14 Table S1. Correlated fit of the UV-Visible spectrum (recorded at room temperature) and the MCD spectrum (recorded at ) of Fe(III)-α 3 DIV-L21C. A graphical representation of the fit is provided in Figure 3. UV-Vis MCD Band Energy (cm -1 ) ε (M -1 cm -1 ) FWHM (cm -1 ) Energy (cm -1 ) ε (M -1 cm -1 T -1 ) FWHM (cm -1 ) Table S2 Parameters from EXAFS fit of α 3 DIV-L21C Shell Fe-S Fit result R=2.32(5) σ 2 =0597 E 0 = F=130.3 S14

15 Table S3 Extended spectroscopic parameters of rubredoxin and designed proteins Protein UV-Vis λ nm ( ε M -1 cm -1 ) Rubredoxin 750 (350) 1 RM1 a 4 L ZR 5 Trx[Rd] 6 α 3 DIV-L21C- GSGA α 3 DIV-L21C- GSGC 570 (3200) 490 (6600) 370 (7710) 350 (7000, sh) 750 ( 500) a 600 ( 1500) 490 ( 2900) 370 ( 6800) 700 (220) 570 (2900) 491 (5250) 360 (7730) 500 ( 4500) a 360 ( 7000) 595 (1200) 491 (2700) 345 (5000) 595 (900) b 491 (1300) 345 (2500) Redox Potential (vs NHE) Mössbauer (ox) (δ and E Q in mm/s, D in cm -1, A in T) -100 to +50 δ=4 3 mv 2 E Q = -0.5 D=+1.9 E/D=3 η= A xx,yy,zz = (-16, -15.9, ) +55 mv N/A N/A +144 mv δ=4 E Q = -0.5 D=+1.9 E/D=3 η=0.13 A xx,yy,zz = (-15.8, -15.6, -19.9) -75 mv (ph 8.5) -80 mv (ph 8.5) δ=5(6) E Q = -0.5(3) D=+(2) E/D=0.14(3) η= A x,y,z = -16.6, -15.8, -17 δ=6(6) E Q = -0.5(3) D=+0.5(2) E/D=0.15(3) η= A xx,yy,zz = (-15.9, -16, - 17) Mössbauer (red) (δ and E Q in mm/s, D in cm -1, A in T) δ= E Q = D=+7.4 E/D=8 η=5 A xx,yy,zz = (-20.1, -8.3, -30.1) δ=9 E Q = D=+7.6 E/D=8 η=4 A xx,yy,zz = (-18.3, -8.3, -33) δ=0.73 E Q = D=+6.74 E/D=6 η=8 A xx,yy,zz = (- 16, -7, -25) δ=0 E Q = D=+2.59 E/D=4 η=10 A xx,yy,zz = (-5, - 4, -19) a From visual inspection of published spectra b molar extinction coefficients in absence of reductant S15

16 References for Supplemental Information: (1) Xiao, Z.; Lavery, M. J.; Ayhan, M.; Scrofani, S. D. B.; Wilce, M. C. J.; Guss, J. M.; Tregloan, P. A.; George, G. N.; Wedd, A. G. J. Am. Chem. Soc. 1998, 120 (17), (2) Meyer, J.; Moulis, J.-M. In Handbook of Metalloproteins; Messerschmidt, A., Huber, R., Eds.; Rubredoxin, vol, 2006; Vol. 1. (3) Wegner, P.; Bever, M.; Schünemann, V.; Trautwein, A. X.; Schmidt, C.; Bönisch, H.; Gnida, M.; Meyer-Klaucke, W. Hyperfine Interact. 2004, 156 (1-4), 293. (4) Nanda, V.; Rosenblatt, M. M.; Osyczka, A.; Kono, H.; Getahun, Z.; Dutton, P. L.; Saven, J. G.; DeGrado, W. F. J. Am. Chem. Soc. 2005, 127 (16), (5) Jacques, A.; Clemancey, M.; Blondin, G.; Fourmond, V.; Latour, J.-M.; Sénèque, O. Chem. Commun. 2013, 49 (28), (6) Benson, D.; Wisz, M.; Liu, W. Biochemistry 1998, 37, S16