Supplementary Information

Transcription

1 Supplementary Information Activation and Reduction of Carbon Dioxide by Nitrogenase Iron Proteins Johannes G. Rebelein, Martin T. Stiebritz, Chi Chung Lee, Yilin Hu* Department of Molecular Biology and Biochemistry, University of California, Irvine, California *Correspondence to:

2 Supplementary Results Supplementary Table 1. Changes of reduction potential of the NifH and VnfH [4Fe4S] 1+/2+ observed upon nucleotide binding in the presence of CO2. Protein No Nucleotide +MgATP NifH VnfH Potentials E 0, mv/she. 2

3 Supplementary Figure 1 In vitro reduction of CO 2 to CO by Fe proteins. (a) Time-dependent formation of CO by dithionite-reduced VnfH (black) or NifH (gray). (b) GC-MS analysis of CO generated by VnfH (upper) or NifH (lower) upon reduction of 12 CO2 (left) or 13 CO2 (right). The isotope distributions of CO ( 12 CO, m/z=28; 13 CO, m/z=29) generated in these reactions are shown in the insets. Activities (a) were determined based on 3 independent experiments (n = 6) and presented as mean s. d. GC-MS experiments (b) were conducted in duplicate, and representative results are shown. 3

4 Supplementary Figure 2 Increase of specific activities of Fe proteins during purification. (a, b) Purification tables showing the activities of VnfH (a) and NifH (b) in CO2-reduction in the presence of Eu II -DTPA at different purification stages. (c, d) Increase of the specific activities of VnfH (c) and NifH (d) in CO2-reduction in the presence of Eu II -DTPA (black bars) and in COoxidation in the presence of IDS (gray bars). Activities are expressed as per mg of total proteins at different purification steps as per convention of purification tables. The maximum activities of purified proteins after the last step of purification (gel filtration chromatography) were: VnfH, nmol CO/mg/h (CO2-reduction) and nmol CO2/mg/h (CO-oxidation); NifH, nmol CO/mg/h (CO2-reduction) and nmol CO2/mg/h (CO-oxidation). These activities are set as 100%, and the activities at earlier steps of the purification procedure (crude extract, anion exchange chromatography) are expressed accordingly (c, d). Each Fe protein was purified 6 times, 3 times from the wildtype strains and 3 times from the variant strains containing deletions of genes encoding the catalytic partners of the Fe proteins. Activities were determined based on 6 independent experiments (n = 12) and presented as mean s. d. 4

5 Supplementary Figure 3 Linear correlation between the activity and amount of the purified Fe protein and reproducibility of activity from different protein preparations. (a-c) Activities of VnfH (solid circles) and NifH (open circles) in CO2-reduction in the presence of Eu II -DTPA (a) and dithionite (b), and in CO-oxidation in the presence of IDS (c). Activities of proteins were determined based on 2 independent experiments (n = 4) and presented as mean s. d. (d-f) Average activities of VnfH (light gray bars) and NifH (dark white bars) in CO2-reduction in the presence of Eu II -DTPA (d) and dithionite (e), and in CO-oxidation in the presence of IDS (f). Each Fe protein was purified 6 times, 3 times from the wildtype strains (solid bars) and 3 times from variant strains containing deletions of genes encoding the catalytic partners of the Fe proteins (striped bars). Activities were determined based on 3 independent experiments (n = 6) and presented as mean s. d. 5

6 Supplementary Figure 4 Interactions of CO 2 with the [Fe 4 S 4 ] clusters of the Fe proteins. Perpendicular-mode EPR spectra of nucleotide-free (left) and ATP-bound (right) VnfH and NifH in Eu II -DTPA-reduced states in the presence of CO2. Spectra were recorded at 10 K. The g values are indicated. All EPR experiments were conducted in duplicate, and representative results are shown. Supplementary Figure 5 Coordination of CO 2 to the [Fe 4 S 4 ] cluster of NifH as obtained from DFT optimizations (TPSS/def2-SVP). Binding of CO2 to a Fe atom of the [Fe4S4] 0 cluster in singlet spin state (S = 0). Element color code: O, red; C, gray; S, yellow; and Fe, orange. 6

7 Supplementary Figure 6 In vitro oxidation of CO to CO 2 by Fe proteins. (a) Time-dependent formation of CO2 by IDS-oxidized VnfH (black) or NifH (gray) without repeated additions of IDS and (b) increase of CO2-formation by VnfH (black) or NifH (gray) upon repeated additions of IDS. Activities in b were determined 360 min after each addition IDS. (c) Yield of CO2 from COoxidation after 5 repeated additions of IDS by VnfH or NifH in nucleotide-free states. Activities were determined based on 3 independent experiments (n = 6) and presented as mean s. d. Supplementary Figure 7 Western blot analysis of Fe protein expression. Lane 1, purified VnfH; lane 2, crude extract of VnfH-expressing A. vinelandii strain grown in the absence of ammonia; lane 3, crude extract of VnfH-expressing A. vinelandii strain grown in the presence of ammonia; lane 4, purified NifH; lane 5, crude extract of NifH-expressing A. vinelandii strain grown in the absence of ammonia; lane 6, crude extract of NifH-expressing A. vinelandii strain grown in the presence of ammonia. The western blot analysis was conducted in triplicate, and the representative result is shown. 7

8 Supplementary Figure 8 In vivo reduction of CO 2 to CO by Fe proteins. GC-MS analysis of CO generated by A. vinelandii strains expressing VnfH (upper) or NifH (lower) in the absence of ammonia (black traces) upon reduction of 12 CO2 (left) or 13 CO2 (right). No CO was detected by GC-MS analysis when these strains were grown in the presence of ammonia (gray traces). The isotope distributions of CO ( 12 CO, m/z=28; 13 CO, m/z=29) generated in these reactions are shown in the insets. GC-MS experiments were conducted in duplicate, and representative results are shown. Supplementary Figure 9 Formate analysis by GC-MS. Production of formate in the reactions of CO2-reduction by VnfH (red trace) and NifH (blue trace) in the presence of Eu II -DTPA was measured by treating the protein-free solutions by pentafluorobenzyl bromide and determining whether pentafluorobenzyl-formic ester could be traced at m/z = 226 ion in the SIM mode of GC- MS. A solution of 0.1 mm formate was treated by the same procedure and used as the standard of GC-MS analysis (black trace). The detection limit of formate was mm, which was equivalent to less than 0.09 turnover of CO2. GC-MS experiments were conducted in duplicate, and representative results are shown. 8