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1 Supplementary Information The direct role of selenocysteine in [NiFeSe] hydrogenase maturation and catalysis Marta C. Marques a, Cristina Tapia b, Oscar Gutiérrez-Sanz b, Ana Raquel Ramos a, Kimberly L. Keller cǂ, Judy D. Wall c, Antonio L. De Lacey b, Pedro M. Matias a,d*, Inês A. C. Pereira a * Authors Affiliations a Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, Oeiras, Portugal b Instituto de Catálisis y Petroleoquímica, CSIC. c/ Marie Curie 2, Madrid, Spain c Biochemistry Department, University of Missouri, Columbia, MO, USA and ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies), Berkeley, CA, USA d ibet, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, Portugal ǂ Current address: Biology Department, William Woods University, Fulton, MO, USA *corresponding authors: ipereira@itqb.unl.pt, matias@itqb.unl.pt

2 Supplementary Results Supplementary Table 1 Molar concentrations of iron (Fe) and nickel (Ni) determined by ICP-AES. Protein Fe content (M) Nickel content (M) Ratio (Fe:Ni) [NiFeSe] m hydrogenase 1.50 x x r[nifese] hydrogenase 2.61 x x Ni-Sec489Cys hydrogenase 3.94 x x

3 Supplementary Table 2 Data collection and processing statistics. rnifese-ox rnifese Sec489Cys-Ox Ni-Sec489Cys Ni-Sec489Cys-Ox Data collection Space group C2 C2 C2 C2 C2 Cell dimensions a, b, c (Å) , 63.46, , 62.77, , 62.66, , 62.86, , 63.18, ( ) 90, , 90 90, , 90 90, , 90 90, , 90 90, , 90 Resolution (Å)* ( ) ( ) ( ) ( ) ( ) R merge (0.315) (1.430) (0.723) (0.622) (1.177) I / I 13.7 (2.2) 22.3 (1.6) 11.6 (2.0) 16.1 (1.6) 10.0 (1.3) Completeness (%) 96.9 (70.8) (99.9) 99.4 (88.4) 96.9 (87.9) 97.7 (95.9) Redundancy 3.7 (2.0) 13.0 (11.5) 4.0 (3.9) 2.9 (2.5) 4.5 (4.2) Refinement Resolution (Å) ( ) ( ) ( ) ( ) ( ) No. reflections (5860) ** (27207) ** (3673) (9003) (4686) R work (0.228) (0.260) (0.215) (0.250) (0.232) R free (0.242) (0.274) (0.260) (0.273) (0.260) No. atoms Protein Ligand/ion Water *** B-factors Protein Ligand/ion Water *** R.m.s. deviations Bond lengths (Å) Bond angles ( ) * Values in parentheses are for highest-resolution shell; ** Friedel mates were treated as independent reflections; ***includes atoms from glycerol molecules

Supplementary Fig. 1. (a) Activity-stained native PAGE of crude extracts (50 μg): 1. D. vulgaris cells carrying membrane ([NiFeSe] m ) and soluble ([NiFeSe] s ) forms of the [NiFeSe] hydrogenase; 2.

4 Supplementary Fig. 1. (a) Activity-stained native PAGE of crude extracts (50 μg): 1. D. vulgaris cells carrying membrane ([NiFeSe] m ) and soluble ([NiFeSe] s ) forms of the [NiFeSe] hydrogenase; 2. IPMM01 cells expressing the r[nifese] hydrogenase; (b) Western blot of cell crude extracts (10 g of total protein) using antiserum raised against the native [NiFeSe] hydrogenase 29 : 3. IPMM01; 4. IPMM02; (c)12.5% SDS-Page stained with Coomassie blue: 5. LMW Marker; 6. r[nifese] hydrogenase; 7. Sec489Cys hydrogenase.

Supplementary Figure 2 I The NiFeSe active site and its surroundings in the crystal structure of the anaerobically purified and crystallized r[nifese] hydrogenase.

5 Supplementary Figure 2 I The NiFeSe active site and its surroundings in the crystal structure of the anaerobically purified and crystallized r[nifese] hydrogenase. (a) View of the partially refined structure with its corresponding 2 F o F c and F o - F c maps, showing evidence of one additional Ni site and two additional Sec positions. The 2 F o F c map (gray mesh) is drawn at the 1.5 map r.m.s. level and the F o F c map is represented at the 3.5 (green mesh) and -3.5 (red mesh) map r.m.s. levels. (b) View of the final refined structure superimposed with the anomalous Fourier map (orange mesh) drawn at the 4.5 map r.m.s. level and calculated with phases from the partially refined structure represented in (a). The [NiFe] binuclear center and the side-chains of its coordinating protein ligands are represented in ball-and-stick and the HysA protein chain is represented as a semitransparent cartoon. Atom colors are brown for iron, green for nickel, gold for sulphur, red for oxygen, light gray for carbon, blue for nitrogen and orange for selenium. Hydrogen atoms are omitted for clarity.

Supplementary Fig. 3. The active site and the proximal [4Fe-4S] cluster in the crystal structure of the aerobically purified and crystallized r[nifese] Hydrogenase (r[nifese]-ox).

6 Supplementary Fig. 3. The active site and the proximal [4Fe-4S] cluster in the crystal structure of the aerobically purified and crystallized r[nifese] Hydrogenase (r[nifese]-ox). The [NiFe] binuclear center, the proximal [4Fe-4S] cluster and their coordinating ligands are represented in ball-and-stick. Atom colors are brown for iron, green for nickel, gold for sulfur, red for oxygen, light gray for carbon, blue for nitrogen and orange for selenium. The HysA and HysB protein chains are represented as semitransparent cartoons. The 2 F o F c maps are drawn (gray mesh) at the 1.5 map r.m.s. level, the F o F c maps are represented at the

7 3.5 (green mesh) and -3.5 (red mesh) map r.m.s. levels and the anomalous Fourier maps are represented at the 4.5 (orange mesh) map r.m.s. level. (a) View of the final refined structure for the active site with three different side chain conformations for Sec489 and its corresponding 2 F o F c and F o F c maps. (b) View of the final refined structure for the active site with three different side chain conformations for Sec489 superimposed with its corresponding anomalous Fourier map. (c-e) Separate views of the three different side chain conformations for Sec489. (f) View of the final refined structure for the proximal [4Fe-4S] cluster and its corresponding 2 F o F c and F o F c maps. The [4Fe-4S-2O] oxygen-damaged form of the cluster is drawn with thinner sticks and smaller spheres. (g) View of the final refined structure for the canonical cubane form of the proximal [4Fe-4S] cluster and its corresponding anomalous Fourier map. (h) View of the final refined structure for the [4Fe-4S-2O] oxygen-damaged form of the proximal [4Fe-4S] cluster and its corresponding anomalous Fourier map. The yellow dashed line highlights the coordination of one of the Fe atoms by Glu77. In (f-h) the red spheres denote conserved water molecules in the crystal structures of [NiFe{Se}] hydrogenases located near the proximal [4Fe-4S] cluster.

8 Supplementary Fig. 4 Circular dichroism spectra of oxidized r[nifese] and Sec489Cys hydrogenases (0.1 μm in 20 mm Tris-HCl ph 7.6). Data are the average of five runs and are represented as black (r[nifese]) and salmon (Sec489Cys) curves after smoothing.

Supplementary Figure 5: H 2 production activities (μmol of H 2 per mg of Sec489Cys Hydrogenase) after treatment of Ni-depleted Sec489Cys hydrogenase with NiCl 2 in 20 mm Tris-HCl

9 Supplementary Figure 5: H 2 production activities (μmol of H 2 per mg of Sec489Cys Hydrogenase) after treatment of Ni-depleted Sec489Cys hydrogenase with NiCl 2 in 20 mm Tris-HCl buffer, during cell disruption and/or incorporation. Molar ratios relative to the hydrogenase are indicated on the x axis. The values are mean values ± s.d. from two independent experiments.

exchange production (H 2 ) with the single exchange production (HD).

10 Supplementary Figure 6 I ph dependence of enzymatic activities for the Ni-Sec489Cys variant versus the r[nifese] hydrogenase. (a) Dependence of the D 2 /H + isotope exchange activity (% maximal activity) of r[nifese] and Ni-Sec489Cys hydrogenases, and comparison of the rate double exchange production (H 2 ) with the single exchange production (HD). (b) ph dependence of the H 2 uptake activity (% maximal activity) of r[nifese] and Ni-Sec489Cys hydrogenases, at 37 C. The maximal enzyme activity for each hydrogenase is expressed as a percentage of the highest activity. Values represent mean values of at least three independent experiments ± s.d.

Supplementary Figure 7 I The active site and its surroundings in the crystal structure of the anaerobically purified and crystallized Ni-Sec489Cys hydrogenase.

11 Supplementary Figure 7 I The active site and its surroundings in the crystal structure of the anaerobically purified and crystallized Ni-Sec489Cys hydrogenase. View of the final refined structure superimposed with corresponding anomalous Fourier map. Red dashed lines represent presumed chemical bonds, not fully drawn for clarity. The [NiFe] binuclear center and its coordinating ligands are represented in ball-and-stick. Atom colors as in Figure 1. Hydrogen atoms were omitted for clarity. The HysA protein chain is represented as a semitransparent cartoon. The anomalous Fourier map is colored and contoured as in Figure 2.

Supplementary Fig. 8. The active site and the proximal [4Fe-4S] cluster in the crystal structure of the aerobically purified and crystallized Ni-reconstituted Sec489Cys hydrogenase (Ni-Sec489Cys-Ox).

12 Supplementary Fig. 8. The active site and the proximal [4Fe-4S] cluster in the crystal structure of the aerobically purified and crystallized Ni-reconstituted Sec489Cys hydrogenase (Ni-Sec489Cys-Ox). The [NiFe] binuclear center, the proximal [4Fe-4S] cluster and their coordinating ligands are represented in ball-and-stick. Atom colors are brown for iron, green for nickel, gold for sulfur, red for oxygen, light gray for carbon, blue for nitrogen and orange for selenium. The HysA and HysB protein chains are represented as semitransparent cartoons. The 2 F o - F c maps are drawn (gray mesh) at the 1.5 map r.m.s. level, the F o - F c maps are

13 represented at the 3.5 (green mesh) and -3.5 (red mesh) map r.m.s. levels and the anomalous Fourier maps are represented at the 4.0 (orange mesh) map r.m.s. level. (a) View of the final refined structure for the active site and its corresponding 2 F o - F c and F o - F c maps. (b) View of the final refined structure for the active site superimposed with its corresponding F o - F c and anomalous Fourier maps. The "x" marks the probable position of a second Ni site, less than 1 Å distant from Ni-1 and not included in the refinement. (c-e) The probable predominant structural forms of the active site, as interpreted from the final refined structure: (c) The Ni-1-containing component, with the Ni-1 site bound to Cys489 in its sulfinate form, Cys75 in one of its sulfenate forms and a bridging oxy ligand. (d) The Ni-2-containing component, with the Ni-2 site (represented by Ni-1) bound to Cys489, Cys75 in the other sulfenate form and a bridging oxy ligand. (e) The Ni-depleted major component, with Cys489 in its sulfinate form and two conformations of Cys75 forming a disulphide bond with Cys78. (f) View of the final refined structure for the proximal [4Fe-4S] cluster and its corresponding 2 F o - F c and F o - F c maps. The [4Fe-4S-2O] oxygen-damaged form of the cluster is drawn with thinner sticks and smaller spheres. (g) View of the final refined structure for the canonical cubane form of the proximal [4Fe-4S] cluster and its corresponding anomalous Fourier map. (h) View of the final refined structure for the [4Fe-4S- 2O] oxygen-damaged form of the proximal [4Fe-4S] cluster and its corresponding anomalous Fourier map. The yellow dashed line highlights the coordination of one of the Fe atoms by Glu77. In (f-h) the red spheres denote conserved water molecules in the crystal structures of [NiFe{Se}] hydrogenases located near the proximal [4Fe-4S] cluster.

Supplementary Fig. 9. The active site in the Sec489Cys variants of the D. vulgaris Hildenborough [NiFeSe] hydrogenase and standard [NiFe] hydrogenases.

14 Supplementary Fig. 9. The active site in the Sec489Cys variants of the D. vulgaris Hildenborough [NiFeSe] hydrogenase and standard [NiFe] hydrogenases. Stereoviews showing a superposition between the Ni- Sec489Cys crystal structure and the crystal structures of the standard [NiFe] hydrogenases from D. vulgaris Myiazaki F (PDB 1wuh), D. fructosovorans (PDB 1yqw) and D. gigas (PDB 1frv). Only one heterodimeric biological unit from 1yqw and 1frv is represented. Panels a and b display views rotated by 180º about an horizontal axis. The r.m.s. deviation between aligned C atoms in the large subunits w.r.t. the Ni-Sec489Cys structure is ca. 1.2 Å. Single-letter residue codes indicate different residues between Sec489Cys [NiFeSe] hidrogenases (black) and standard [NiFe] hydrogenases (red). The Ni and Fe atoms of the binuclear active site as well as the water molecules from all structures shown are represented as small spheres; the location of the four cysteine residues that coordinate the NiFe binuclear active site is indicated using the sequence numbering of the Ni-Sec489Cys structure; the proximal [4Fe-4S] cluster is labeled Prox; for all structures, non-carbon atom colors are blue for nitrogen, red for oxygen and gold for sulfur; carbon atoms are colored green for Ni-Sec489Cys, yellow for 1wuh, grey for 1yqw and salmon for 1frv.

SUPPLEMENTARY INFORMATION. doi: /nature07461

SUPPLEMENTARY INFORMATION. doi: /nature07461 Figure S1 Electrophysiology. a ph-activation of. Two-electrode voltage clamp recordings of Xenopus oocytes expressing in comparison to waterinjected oocytes. Currents were recorded at 40 mv. The ph of