Supplementary Text - PDF document

1 Supplementary Text Comparison of the FeS
Supplementary Text Comparison of the FeS clusters and subunit arrangements of SH with those of complex I and group I [NiFe]hydrogenases Although the N3 [4Fe4S] cluster in Nqo1 is conserved in HoxF (termed F1), the N1a [2Fe2S] cluster in Nqo2 is not found (Fig. 1, A and B), consistent with the fact that three of four cysteines serve as ligands of the cluster are not conserved in HoxF (fig. S1). In contrast, the conservation of all four cysteines in HoxF of photosyntheticbacteria implies that the N1a [2Fe2S] cluster exists in SH from some organisms . HoxU is small compared with Nqo3, sharing ~30% of the terminal portion of Nqo3, and lacking the N7 cluster in the Cterminal portion of Nqo3 present in complex I of limited species includinthermophilus. The other three FeS clusters (one [2Fe2S] and two [4Fe4S] clusters) are conserved. The U3 [4Fe4S] cluster in HoxU is at the same position as thN5 [4Fe4S] cluster in Nqo3 and is similarly coordinated by three cysteines and one histidine, whereas all other FeS clusters have four cysteine ligands. The U3 cluster is located next to the Y1 [4Fe4S] clusterin HoxY, which ismost proximal to the NiFe active site(Fig. 1B)he most distant [4Fe4S] cluster from the NiFe active site among the three F

2 eS clusters in the small subunit of the
eS clusters in the small subunit of the group I [NiFe]hydrogenases, hitherto the best studied [NiFe]hydrogenases , is also coordinated by three cysteines and one histidine, although the functional significance of the histidine coordination remains to be elucidated. HoxY harbors one [4FeS] cluster (Y1) as doesits complex I homolog Nqo6. While the Y1 cluster is accommodated by two CXXC motifs,as is typical for [4Fe4S] clusters,the N2 cluster in Nqo6 includes one CXXC motif and two consecutive cysteine residues. It has been proposed that the observed redoxdependent conformational change of the nonconventional N2 cluster is crucial for the protonpumping function of complex I . HoxY is also evolutionarily related to the small subunit of the group I [NiFe]hydrogenases, but it is 5080% smaller. Furthermore, there are three FeS clusters in the small subunit of the group I [NiFe]hydrogenases and the Freducing [NiFe]hydrogenase of group III [NiFe]hydrogenases , whereas HoxY has one FeS cluster that spatially corresponds to the cluster most proximal to the NiFe active site of the group I [NiFe]hydrogenases. The NiFe active site in HoxH, but its complex I homolog Nqo4 contains no metals, despite good structural similarity with the correspon

3 ding region around the Fe active siteof
ding region around the Fe active siteof hydrogenases. Compared with the large subunit of the group I [NiFe]hydrogenases that accommodate the NiFe active site, HoxH is more compact due to several deletions. The subunit orientation between HoxF and HoxU is similar to that between Nqo1·2 and Nqo3. Similarly, the quaternary structure of the HoxHY subcomplex is close to that of the Nqo6·4 subcomplex. In contrast, the orientation between the HoxFU and HoxHY subcomplexes is very different from that between the Nqo13 and Nqu6·subcomplexes (Fig. 1 and fig. S2). Consequently, although the electron transfer chain of complex I includes the N6a and N6b clusters in Nqo9 between the N5 and N2 clusters, the U3 (corresponding to N5 in complex I) and Y1 (N2 in complex I) clusters of SH are closely positioned, allowing direct electron transfer (Fig. 1). Materials and Methods Bacterial strain A thermophilic hydrogenoxidizing bacterium,Hydrogenophilusthermoluteolusstrain TH1, was isolated from soil around a hot spring intheIzu eninsula, Shizuoka, Japan . A draft genome sequence analysis was performed using Genome Analyzer GAIIx (Illumina Inc.) and FLX Titanium (Roche Ltd), providing34,180,094 and 304,337 readings, respectively. The nucleotide seq

4 uences of the structural genes of the NA
uences of the structural genes of the NADreducing [NiFe]hydrogenase, hoxFhoxUhoxYand hoxHhave been submitted to the DNA Data Bank of Japan with the GInumbers 675402511675402513675402515,and 675402517, respectively Production and preparation of crystals The procedures for bacterial cultivation, purification,and crystallizationof the enzymeand crystallographic experimentfor crystals of the airoxidized state have been described previouslyFor the crystals in the Hreduced state, the procedures were modified as follows. thermoluteolus1 was cultivated in a 200 L jar fermenter (MPFU200, B. E. Marubishi Co. Ltd.) with a medium containing sodium acetate (4 g), sodium phosphate (9 g), potassium phosphate (1.5 g), glycerol (4 mL), ammonium chloride (2 g), magnesium sulfate (0.2 g), ironchloride (5 mg), calcium chloride (10 mg), nickel chloride (0.2 mg), and SL6 trace element solution (5 mL) per liter at 322 K for 21 h. In the purification, the dialysis before the first ion exchange chromatography has been replaced by dilution with 25 mM TrisHCl (pH 7.4)In addition, the pooled fractions of gelfiltration chromatography have further been purified by a TSKgel SuperQ5PWanion exchange columnTOSOHwith 25 mM TrisHCl (pH 7.4) and 0.050.2 M NaCl. The

5 Hreduced crystals were prepared from th
Hreduced crystals were prepared from the airoxidized crystals as follows. The crystals were transferred into a sealed vial containing 0.1 M TrisHCl (pH 8.5), 0.2 M MgCl, 1 mM BV, and 35%(w/v) PEG3350. After removing Oin the vial by repeating degassing with a vacuum line and purging with 1 bar of Ar three times, 1 bar of Hwas purged for 5 min and the vial was left for 24 h at room temperature. ray diffraction data collection and structure determination The Xray diffraction data were collected at beamlines BL32XU, BL38B1, BL41XUand BL44XU of SPring8 (Hyogo, Japan). The crystals were maintained at 100 K with Ngas stream coolers during data collection. The diffraction spots were integrated by using XDS , and scaled and merged withSCALA from the CCP4 program suite . Phases and initial models were ained by the singlewavelengthanomalous dispersion(SAD)method with intrinsic iron atoms using the PHENIX program suite The iterative model building and structure refinement procedures were performed by using COOT and Refmac5 , respectively. ighresolution data hases with highenergy Xray= 0.9000 ) were obtained by the molecular replacement method using MolRep from the CCP4 suite with the model obtained from theSAD data as a search model. Data

6 collection and refinement statistics are
collection and refinement statistics are summarized in Supplementary Table 1. The simulated annealing refinements and the omit map calculations have beeperformed using the PHENIX program. Figures were prepared with PyMol (DeLano Scientific LLC). Spectroscopic measurement For the electron paramagnetic resonance (EPR) measurements, purified soluble [NiFe]ydrogenase from thermoluteolusSH) wasconcentrated to 0.25 mMunder air buffer [25 mM TrisHCl pH 7.4150 mM NaCl], andtransferred into 5 mm EPR tube. The reduced enzyme was prepared by degassingwith a vacuum line, purgingwith 1 bar of H, and subsequent incubationat 50 °C for 1 hMeasurements were performedat 77 K with an EPR spectrometer (JESFA100, JEOL), where thefrequency, microwave power, modulation width, and time constant were 9.042 GHz, 5 mW, 1 mT, and 0.1 s, respectively. Table S1.Data collection and refinement statistics.DatasetAiroxidizedreducedLow energyCrystal parameters Space groupCell dimensions (Å)131431897112459132751924713082131190222483 β(º)10910508109Data collection Wavelength (Å)0.90000.9000 7300 Resolution range (Å) a 95.048 (725896.24857020.002020 Total reflections a 317516 (46192331,962(48,212180324 (26119) Unique reflections a 90 , 430 ( 13 , 17

7 6 ) 86,103 ( 12,516 ) 47 , 442 (
6 ) 86,103 ( 12,516 ) 47 , 442 (6934 ) ergea, b0.091 (0.4860.98 (0.4700.088 (0.455) as a, c 0.121(0.700.139(0.64)0.119(0.614 R pim a, d 0.064 (0.3570.098 (0.469)0.080 (0.410) CC 1/2 a, e 0.992 (0.765 ) 0.994 (0.932) 0.994 (0.792) Average a, (2. Completeness a 0.997(1.0000.993(0.9900.99(0.999 Redundancy a .8 (3.RefinementSAD phasing Resolution range (Å) a 95.048 (655896.247770No. of Fe sites29 R cryst / R free R - factor a, g , h 0.1990.2900.20.3330.1980.3390.240.409 Figure of m erit 0.307 Reflections used for refinement85,90181,782Atoms in an asymmetric unitProtein2222022,229 Ligand 152 90 Water768126 B values 2 ) Protein5370Ligand3941Water3849Deviatios from ideal geometryBond distances (Å)0.0080.010 Bond angle s ( º ) 1.1 5 1. 3 7 Ramachandran plotFavored(%)9694Allowed(%) Outlier (%) 0.0 0.0 Values in parentheses are for highest resolution shells. mergehklhklhkl�|)/hklhkl meashklklN/(N-1)]1/2hklhkl�|)/hklhkl pimhkll1/(N-1)]1/2hklhkl)� |/hklhkl1/2is a correlation coefficient between intensities from random half datasets. Signalnoise ratio of intensities. |)/ Five percent of reflections were randomly chosen for calculati

8 ng the free factor 4 5
ng the free factor 4 5 6 7 8 FigAmino acid sequence alignments of the NADreducing [NiFe]hydrogenases.The darker backgroundof each amino acid represent higher degreeof conservation. losed red circles mark the residues that coordinate the FeS clusters, which arestrictly conserved without exception. esidues within the distance of 3.5 from the counterpart subcomplexare marked by closed rectanglesthe colorsof whichrepresentthesubunit of the closeresiduein the counterpart subcomplexHoxFHoxUHoxY,and HoxHare indicated by brown, pink, greenand light blue, respectively. The residues corresponding to the four cysteines that serve as ligands for the N1a [2Fe2S] cluster in Complex I are denoted by closed blue circles. From the top, the sequences are HoxFHoxUHoxY,and HoxHfrom Synechocystissp. PCC 6803(GI: 16330689163306871633068616330683for HoxFHoxUHoxYHoxH), SynechococcuselongatusPCC7942(GI: 81299090813013668130136581301364), Thiocapsaroseopersicina(GI: 37787352377873533778735437787355), Allochromatiumvinosum(GI: 28894158288941583288941584288941585), Hydrogenophilusthermoluteolus1 (GI: 675402511675402513675402515675402517), RalstoniaeutrophaH16(GI: 38637753386377543252709332527094), Rhodococcus opacus(GI: 161757

9 0161757116175721617573), Thiocapsaroseop
0161757116175721617573), Thiocapsaroseopersicina(GI: 300432247300432248300432249300432250and Methylococcuscapsulatusstr. Bath(GI: 53803150538031495380314853802627). The upper foursequences enclosed by green rectangles are classified as photosynthetic bacterial heteropentameric types, and the lower fivesequences enclosed by cyan rectangles are heterotetrameric types (RalstoniaeutrophaH16additionally contains a HoxIhomodimer unit ). Thiocapsaroseopersicinais exceptional in that it contains both types FigS2. Comparison of the subcomplex orientation between and omplex IOverall structure of ) Structure of complex I (PDB ID: 4HEA), whereonly subunits homologous to are shown for clarity. For the two structures, homologous subunits are represented by the same colorswith the exception of Nqo2 in complex I. Thetwo rotational operations, shown red and blue arrows in (A) for the HoxYH subcomplex with the fixed HoxFU subcomplex, make the same subcomplex orientation as complex I. 10 FigS3.Structure comparison of the NiFe active site with previously reported model compounds mimicking the active site of the [NiFe]hydrogenase) and ) The crystal structures of the inactive model compounds reporteby Ohki (CSD ID: 673707(A) and 9 (B)he NiFe activ

10 e site of SH in the airoxidized state) T
e site of SH in the airoxidized state) The NiFe active site of SH in the Hreduced state. The bond distances are shown black numerals, and the distance between Ni and Fe atoms are shown pinknumerals. The units are ngstrhe NiFe distances of the inactive model compounds are longer (3.1) thanoseof the hydrogenases (~2.8 and ~2.5 for inactive and activestates, respectively). 11 FigS4.EPR spectra of Hreducedat 77 K) The protein was concentrated to 0.16 mM in buffer containing 25 mM TrisHCl (pH 7.4) and 150 mM NaCl, and transferred a 5 mm EPR tube. The Hatmosphere was prepared by degassing witha vacuum line, purging with 1 bar of Hand incubation at C for 1 h. () After measuring (A), the sample solution was degassed with the vacuum line and purged with 1 bar of N) After measuring (B), the sample was irradiated for 30 min with white light (500 W Xe lamp) at 77 K. () After measuring (C), the sample was thawed at room temperature. The intensities of Nilike weak signals (= 2.21and= 2.14; the signal was overlapped with other signalsincreased when the atmosphere was changed from H(A) to N(B). Furthermore, upon light irradiation of the Nilike state under an Natmosphere at 77 K, two like states (= 2.28 and 2.31, = 2.11, = 2.05) c

11 haracteristic group I [NiFe]drogenaseswe
haracteristic group I [NiFe]drogenaseswere also observed (. The Nilikestates returned to the Nilike state after raising the temperature and refreezing Signals at = 1.94and 2.03 are attributed to reduced [2Fe2S]. The asterisk signal at = 2.00 is due to an unknown species. 12 specifically activated by NADPHJ. Bacteriol.). doi:10.1128/JB.187.9.3122Medline MarótiFarkasI. K.NagyMarótiKondorosiRákhelyK. L.Kovácssecond soluble Hoxtype NiFe enzyme completes the hydrogenase set in Thiocapsa roseopersicinaBBSAppl. Environ. Microbiol.). doi:10.1128/AEM.00351Medline J. M.BerrisfordL. A.SazanovStructural basis for the mechanism of respiratory complex IBiol. Chem.). doi:10.1074/jbc.M109.032144Medline P. J.HoltR. G.EfremovNakamaruOgisoL. A.SazanovReversible FMN dissociation from Escherichia coli respiratory complex IBiochim. Biophys. Acta). doi:10.1016/j.bbabio.2016.08.008Medline van der LindenB. W.FaberBleijlevensBurgdorfBernhardFriedrichS. P. AlbrachtSelective release and function of one of the two FMN groups in the cytoplasmic NADreducing [NiFe]hydrogenase from Ralstonia eutrophaEur. J. Biochem.). doi:10.1111/j.1432Medline AubertJoussetCanoGuedeneyRichaudCournacRole of HoxE subunit in Synechocystis PCC6803 hydrogenaseFEBS J.

12 278). doi:10.1111/j.1742Medline VittWa
278). doi:10.1111/j.1742Medline VittWarkentinMollA. J.PierikShimaErmlerThe F420reducing [NiFe]hydrogenase complex from Methanothermobacter marburgensis, the first ray structure of a group 3 family memberJ. Mol. Biol.). doi:10.1016/j.jmb.2014.05.024Medline TaketaNakagaHabukawaOsukaKihiraKomoriShibataIshiiIgarashiNishiharaYoonOgoShomuraHiguchiCrystallization and preliminary ray analysis of the NADreducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus THActa Crystallogr. F Struct. Biol. Commun.). doi:10.1107/S2053230X14026521Medline KabschActa Crystallogr. D Biol. Crystallogr.). doi:10.1107/S0907444909047337Medline Collaborative Computational Project, Number 4The CCP4 suite: Programs for protein crystallographyActa Crystallogr. D Biol. Crystallogr.). doi:10.1107/S0907444994003112Medline P. D.AdamsP. V.AfonineBunkócziV. B.ChenI. W.DavisEcholsJ. J.HeaddHungG. J.KapralR. W.GrosseKunstleveA. J.McCoyN. W.MoriartyOeffnerR. J.ReadD. C.RichardsonJ. S.RichardsonT. C.TerwilligerP. H.ZwartPHENIX: A comprehensive Pythonbased system for macromolecular structure solutionActa Crystallogr. D Biol. Crystallogr.221). doi:10.1107/S0907444909052925Medline EmsleyCowtanCoot: Modelbuilding tools for molecular graphicsActa Crystallog

13 r. D Biol. Crystallogr.). doi:10.1107/S0
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14 ges at its nonstandard NiFe site during
ges at its nonstandard NiFe site during activation of the NADreducing hydrogenase from Ralstonia eutrophadetected by ray absorption, EPR, and FTIR spectroscopyJ. Am. Chem. Soc.). doi:10.1021/ja0461926 Medline SchneiderCammack. G.SchlegelContent and localization of FMN, FeS clusters and nickel in the NADlinked hydrogenase of Nocardia opacaEur. J. Biochem.). doi:10.1111/j.14321033.1984.tb08252.xMedline R. P.HappeRoseboomEgertC. G.FriedrichMassanzFriedrichS. P. J.AlbrachtUnusual FTIR and EPR properties of the H2activating site of the cytoplasmic reducing hydrogenase from Ralstonia eutrophaFEBS Lett.). doi:10.1016/S00145793(99)01799Medline Van der LindenBurgdorfBernhardBleijlevensFriedrichS. P. J.AlbrachtThe soluble [NiFe]hydrogenase from Ralstonia eutrophacontains four cyanides in its active site, one of which is responsible for the insensitivity towards oxygenJ. Biol. Inorg. Chem.). doi:10.1007/s00775Medline GermerZebgerSagguLendzianSchulzAppelOverexpression, isolation, and spectroscopic characterization of the bidirectional [NiFe] hydrogenase from Synechocystis sp. PCC 6803J. Biol. Chem.). doi:10.1074/jbc.M109.028795Medline HorchLauterbachSagguHildebrandtLendzianBittlLenzZebgerProbing the active site of an Otolerant N

15 ADreducing [NiFe]hydrogenase from Ralsto
ADreducing [NiFe]hydrogenase from Ralstonia eutropha H16 by in situ EPR and FTIR spectroscopyAngew. Chem. Int. Ed. Engl.). doi:10.1002/anie.201002197Medline S. P. J.AlbrachtNickel hydrogenases: In search of the active siteBiochim. Biophys. Acta). doi:10.1016/00052728(94)90036Medline LubitzOgataRüdigerReijerseHydrogenasesChem. Rev.). doi:10.1021/cr4005814Medline ShomuraHiguchiStructural aspects of [NiFe]hydrogenasesRev. Inorg. Chem.). doi:10.1515/revic References and NotesP. M.VignaisBilloudOccurrence, classification, and biological function of hydrogenases: An overviewChem. Rev.). doi:10.1021/cr050196rMedline J. C.FontecillaCampsVolbedaCavazzaNicoletStructure/function relationships of [NiFe]and [FeFe]hydrogenasesChem. Rev.). SchneiderH. G.SchlegelPurification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16Biochim. Biophys. Acta). doi:10.1016/00052744(76)90058Medline HorchLauterbachLenzHildebrandtZebgerNAD(H)coupled hydrogen cycling BöhmSauterBöckNucleotide sequence and expression of an operon in Escherichia coli coding for formate hydrogenlyase componentsMol. Microbiol.). doi:10.1111/j.13652958.1990.tb00590.xMedline 6E. doi:10.1016/S0005 2728(02)00214 RákhelyA. T.KovácsMarótiB.

16 D.FodorCsanádiLatinovicsK. L.KovácsCy
D.FodorCsanádiLatinovicsK. L.KovácsCyanobacterialtype, heteropentameric, NADreducing NiFe hydrogenase in the purple sulfur photosynthetic bacterium Thiocapsa roseopersicinaAppl. Environ. Microbiol.). doi:10.1128/AEM.70.2.722Medline L. A.SazanovHinchliffeStructure of the hydrophilic domain of respiratory complex I from Thermus thermophilusScience1436). BaradaranJ. M.BerrisfordG. MinhasL. A.SazanovCrystal structure of the entire respiratory complex INature). doi:10.1038/nature11871Medline BowienStructural analysis of the fds operon encoding the NADlinked formate MalkiSaimmaimeDe LucaRoussetDermounJ. P.BelaichCharacterization of an operon encoding an NADPreducing hydrogenase in Desulfovibrio fructosovoransJ. Bacteriol.). doi:10.1128/jb.177.10.2628 2636.1995 Medline www.sciencemag.org/content/6354928/suppl/DC1SupplementaryMaterialStructural basis of the redox switches in the NADreducing soluble [NiFe]hydrogenaseShomuraTaketaNakashimaTaiNakagawaIkedaIshiiIgarashiNishiharaYoonOgoHirotaHiguchi*Corresponding author. Email: hig@sci.uhyogo.ac.jp; yasuhito.shomura.s@vc.ibaraki.ac.jpPublished 1 SeptemberScience357928(20DOI:10.1126/science.aan4497This PDF file includes:Materials and MethodsSupplementary TextFigs. S1 to Tabl