Experimental Correlation of Substrate Position with Reaction Outcome in the Aliphatic

Transcription

1 Supporting Information for: Experimental Correlation of Substrate Position with Reaction Outcome in the Aliphatic Halogenase, SyrB2 Ryan J. Martinie, a Jovan Livada, a Wei-chen Chang, a Michael T. Green, a Carsten Krebs, a,b J. Martin Bollinger Jr., a,b and Alexey Silakov a* Departments of a Chemistry and b Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA. S1

2 SI Figures Figure S1. Representative HYSCORE spectra of NO-Aba (A) and NO-per-d 6 -Aba (B). Signals arising from 14 N, 1 H, and 2 H nuclei are indicated. Experimental conditions given in Figure 4 of the main text. S2

3 Figure S2. Experimental (A) and simulated (B) HYSCORE spectra for NO-4,5-d 5 -Nva. C) Antidiagonal skyline plots with experimental (blue) and simulated (red) spectra overlaid. Red lines in one-dimensional, absorptive EPR spectra (top) indicate magnetic field at which corresponding HYSCORE were collected/simulated. Experimental Conditions (listed from left to right): Magnetic Field: mt, mt, mt, mt; Microwave Frequency: GHz (602.2 mt), GHz (613.5 and mt), GHz (700.0 mt); mt spectrum collected with 200 points in x and y dimensions. Temperature: 4.2, 4.5, 4.2, and 4.0 K. S3

4 Figure S3. Comparison of HYSCORE spectra obtained for A) NO-4,5-d 5 -Nva and C) NO-5-d 3 - Nva. B) Simulated spectra. Red lines in one-dimensional, absorptive EPR spectra (top) indicate magnetic field at which corresponding HYSCORE were collected/simulated. Experimental parameters. NO-4,5-d 5 -Nva (see legend, Figure S4); NO-5-d 3 -Nva: Magnetic Field: mt, mt, mt; Microwave Frequency: GHz (602.2 mt), GHz (613.5 and mt); Temperature: 4.0 K. S4

5 Figure S4. Experimental (A) and simulated (B) HYSCORE spectra for NO-4-d 2 -Nva. C) Antidiagonal skyline plots with experimental (blue) and simulated (red) spectra overlaid. Red lines in one-dimensional, absorptive EPR spectra (top) indicate magnetic field at which corresponding HYSCORE were collected/simulated. Experimental Conditions (listed from left to right): Magnetic Field: mt, mt, mt, mt; Microwave Frequency: GHz (602.2 and mt), GHz (613.5 mt), GHz (950.0 mt); mt spectrum collected with 128 points in x and y dimensions. Temperature: 4.0 K. S5

6 Figure S5. Experimental (A) and simulated (B) HYSCORE spectra for NO-per-d 6 -Aba. C) Antidiagonal skyline plots with experimental (blue) and simulated (red) spectra overlaid. Red lines in one-dimensional, absorptive EPR spectra (top) indicate magnetic field at which corresponding HYSCORE were collected/simulated. Experimental Conditions (listed from left to right): Magnetic Field: mt, mt, mt, mt; Microwave Frequency: GHz (602.2, 611.9, and mt) GHz (950.0 mt); mt spectrum collected with 350 points in the x and y dimension with 24 ns step. Temperature: 4.65 K (602.2, 611.9, and mt), 4.2 K (950.0 mt). S6

7 Figure S6. Experimental (A) and simulated (B) HYSCORE spectra for NO-3-d 2 -Aba. C) Antidiagonal skyline plots with experimental (blue) and simulated (red) spectra overlaid. Red lines in one-dimensional, absorptive EPR spectra (top) indicate magnetic field at which corresponding HYSCORE were collected/simulated. Experimental Conditions (listed from left to right): Magnetic Field: mt, mt, mt, mt; Microwave Frequency: GHz (602.2 and mt), GHz (613.5 and mt); Temperature: 4.0 K. S7

8 Figure S7. Experimental (A) and simulated (B) HYSCORE spectra for NO-per-d 5 -Thr. C) Antidiagonal skyline plots with experimental (blue) and simulated (red) spectra overlaid. Red lines in one-dimensional, absorptive EPR spectra (top) indicate magnetic field at which corresponding HYSCORE were collected/simulated. Experimental Conditions (listed from left to right): Magnetic Field: mt, mt, mt, mt; Microwave Frequency: GHz (602.2, 613.6, and mt) GHz (950.0 mt); mt spectrum collected with 350 points in the x and y dimensions with 24 ns step; Temperature: 4.6 K (602.2, 613.6, and mt), 4.2 K (950.0 mt). S8

9 Figure S8. Experimental (A) and simulated (B) HYSCORE spectra for NO-2,3-d 2 -Thr. C) Antidiagonal skyline plots with experimental (blue) and simulated (red) spectra overlaid. Red lines in one-dimensional, absorptive EPR spectra (top) indicate magnetic field at which corresponding HYSCORE were collected/simulated. Experimental Conditions (listed from left to right): Magnetic Field: mt, mt, mt; Microwave Frequency: GHz; Temperature: 4.0 K. S9

10 Figure S9. Comparison of substrate docking models for Thr with NO displacing the following ligands (relative to the crystal structure): the water ligand (A), the chloride ligand (B), and C1 of 2OG (C). Adapted from ref. 37 in the main text by replacement of the ferryl O-atom with NO, and of acetate with 2-oxo-propionic acid S10

11 Figure S10. EPR pulse sequences employed: A) HYSCORE, B) Matched HYSCORE, C) Spinecho detected EPR (one-dimensional). S11

12 Figure S11. Cartoon representation of anti-diagonal skyline plot construction. A hypothetical HYSCORE spectrum, consisting of four peaks which are split by both hyperfine (A) and quadrupole (Q) interactions. The grey spectrum represents a skyline plot of the analyzed area (shaded grey), which is projected onto the frequency axis (red) to give the final result. For convenience, the projected result is shifted so as to be centered on the Larmor frequency. S12

13 Figure S12. Schematic representation of the model used to determine Fe- 2 H distances from a given hyperfine coupling tensor. S13

14 SI Tables Table S1. Spin Hamiltonian parameters used to simulate HYSCORE spectra. Parentheses indicate uncertainty in the last reported digit, determined by altering the parameter until the fit was deemed unacceptable by visual inspection. Variables are defined in the Materials and Methods. Hyperfine Coupling Quadrupole Coupling T (MHz) ϕ ( ) θ ( ) ψ ( ) K (MHz) ϕ ( ) θ ( ) ψ ( ) η d5-nva 0.40(5) - 58(5) 47(10) 0.035(5) - 51(10) 0(10) - d3-nva 0.40(5) - 58(5) 47(10) 0.035(5) - 51(10) 0(10) - d2-nva 0.28(5) - 75(15) 50(20) 0.025(5) - 65(20) 17(20) -0.5 d6-aba 0.29(4) - 80(10) 40(10) 0.050(5) - 55(10) 7(10) 0.2 d2-aba 0.14(2) - 80(10) 75(10) 0.043(5) - 45(10) 10(10) - d5-thr 0.20(3) - 80(10) 42(10) 0.040(5) - 53(10) 0(10) - d2-thr 0.14(3) - 75(10) 57(10) 0.043(5) - 30(20) 30(20) - Table S2. Geometric parameters obtained based on the hyperfine coupling tensors reported in Table S1. r eff represents the distance obtained from a simple point-dipole approximation, whereas r model represents that obtained from fitting of the hyperfine tensor using multiple dipolar coupling contriburions in an antiferromagnetic coupling model (see Materials and Methods). Parentheses indicate uncertainty in the last reported digit. Hyperfine Coupling r eff (Å) r model (Å) N-Fe-D ( ) d5-nva 3.1(2) 3.4(3) 64(7) d3-nva 3.1(2) 3.4(3) 64(7) d2-nva 3.5(3) 3.7(3) 81(15) d6-aba 3.5(2) 3.7(2) 85(10) d2-aba 4.4(3) 4.7(3) 85(10) d5-thr 3.9(3) 4.2(3) 85(10) d2-thr 4.4(4) 4.7(4) 81(10) S14