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Assignment of the [4Fe-4S] clusters of Ech hydrogenasefrom Methanosarcina barkeri to individual subunits viathe characterization of site-directed mutantsLucia Forzi1,Ju¨rgen Koch1, Adam M. Guss2, Carl G. Radosevich2, William W. Metcalf2and Reiner Hedderich11 Max-Planck-Institute for terrestrial Microbiology, Marburg, Germany2 Department of Microbiology, University of Illinois, Urbana, IL, USAIn recent years a novel family of membrane-bound[NiFe] hydrogenases, now called energy-convertinghydrogenases, has been recognized [1]. These enzymesform a phylogenetically distinct group within the largefamily of [NiFe] hydrogenases [2]. Members of thishydrogenase family include hydrogenase 3 fromEscherichia coli, CO-induced hydrogenase from Rhodo-spirillum rubrum, Coo hydrogenase from Carboxydo-thermus hydrogenoformans [3,4] and Ech hydrogenasefrom Methanosarcina barkeri and Thermoanaerobactertengcongensis [5–7]. The hydrogenase large and smallsubunits of these enzymes show surprisingly littlesequence similarity to standard [NiFe] hydrogenases,except for the conserved residues coordinating theactive site and the proximal [4Fe-4S] cluster [8]. Inaddition to these subunits, which are conserved in all[NiFe] hydrogenases and which are essential for H2-activation, energy-converting hydrogenases contain atleast four other subunits: two hydrophilic proteins andtwo integral membrane proteins. These six subunitsform the basic structure of energy-converting hydro-genases and are conserved in all members of thisKeywords[NiFe] hydrogenases; NADH:quinoneoxidoreductase; complex I; Methanogenicarchaea; iron–sulfur proteinsCorrespondenceR. Hedderich, Max-Planck-Institute forterrestrial Microbiology, Karl-von-Frisch Str.,D-35043 Marburg, GermanyFax: +49 6421 178299Tel: +49 6421 178230E-mail: hedderic@staff.uni-marburg.de(Received 21 June 2005, revised 22 July2005, accepted 29 July 2005)doi:10.1111/j.1742-4658.2005.04889.xEch hydrogenase from Methanosarcina barkeri is a member of a distinctgroup of membrane-bound [NiFe] hydrogenases with sequence similarityto energy-conserving NADH:quinone oxidoreductase (complex I). Thesequence of the enzyme predicts the binding of three [4Fe-4S] clusters, oneby subunit EchC and two by subunit EchF. Previous studies had shownthat two of these clusters could be fully reduced under 105Pa of H2at pH7 giving rise to two distinct S½ electron paramagnetic resonance (EPR)signals, designated as the g ¼ 1.89 and the g ¼ 1.92 signal. Redox titra-tions at different pH values demonstrated that these two clusters had apH-dependent midpoint potential indicating a function in ion pumping. Toassign these signals to the subunits of the enzyme a set of M. barkerimutants was generated in which seven of eight conserved cysteine residuesin EchF were individually replaced by serine. EPR spectra recorded fromthe isolated mutant enzymes revealed a strong reduction or complete lossof the g ¼ 1.92 signal whereas the g ¼ 1.89 signal was still detectable asthe major EPR signal in five mutant enzymes. It is concluded that the clus-ter giving rise to the g ¼ 1.89 signal is the proximal cluster located in EchCand that the g ¼ 1.92 signal results from one of the clusters of subunitEchF. The pH-dependence of these two [4Fe-4S] clusters suggests that theysimultaneously mediate electron and proton transfer and thus could be anessential part of the proton-translocating machinery.AbbreviationsDDM, dodecyl-b-D-maltoside; Ech, energy-converting hydrogenase; EPR, electron paramagnetic resonance; FMD, formylmethanofurandehydrogenase.FEBS Journal 272 (2005) 4741–4753 ª 2005 FEBS 4741hydrogenase subfamily. Thus far the purification ofonly three members of this hydrogenase family hasbeen achieved: M. barkeri Ech hydrogenase [6], Coohydrogenase from C. hydrogenoformans [4] andT. tengcongensis Ech hydrogenase [7].From a biochemical perspective, the most thoroughlystudied member of energy-converting [NiFe] hydrogen-ases is Ech hydrogenase found in the methanogenicarchaeon M. barkeri. In vitro studies had shownthat a low-potential, soluble two-[4Fe-4S] ferredoxin(E0¢ ¼ )420 mV) isolated from M. barkeri functions asan electron donor and electron acceptor of Ech [6]. Thebiological role of Ech was studied using mutationalanalysis [9]. The following conclusions were made fromthe data obtained: under autotrophic growth condi-tions, the enzyme catalyses the reduction of the low-potential ferredoxin by H2. Reduced ferredoxin gener-ated by Ech hydrogenase donates electrons to varioussoluble oxidoreductases, e.g. formylmethanofurandehydrogenase (FMD), acetyl-CoA synthase ⁄ carbonmonoxide dehydrogenase complex, and pyruvate:ferre-doxin oxidoreductase. FMD, for example, catalyses thereduction of methanofuran and CO2to formyl-methanofuran. The overall reduction of CO2and meth-anofuran by H2is endergonic and is driven in vivoby reverse electron transport [10]. Reduction of ferre-doxin by H2, the partial reaction catalysed by Echhydrogenase, is the energy driven step [11].Purified Ech is composed of six subunits correspond-ing to the products of the echABCDEF operon [5].The EchA and EchB subunits are predicted to beintegral, membrane-spanning proteins, while the otherfour subunits are expected to extrude into the cyto-plasm. Amino acid sequence analysis of the cytoplas-mic subunits points to the presence of two [4Fe-4S]clusters in EchF and one [4Fe-4S] cluster in EchC. TheEchC subunit belongs to the family of [NiFe] hydro-genase small subunits. The EchE subunit contains thecharacteristic binding motif for the [NiFe] centre foundin the large subunits of all [NiFe] hydrogenases. Chem-ical analysis has revealed the presence of Ni, nonhemeFe and acid-labile S in a ratio of 1 : 12.5 : 12 [6], cor-roborating the presence of three [4Fe-4S] clusters.Characterization of the iron–sulfur clusters of theenzyme by electron paramagnetic resonance spectros-copy (EPR) showed that two of these clusters could befully reduced under 105Pa of H2at pH 7 giving rise totwo distinct S½ EPR signals, designated as the g ¼1.89 and the g ¼ 1.92 signal [12]. Redox titrations atvarious pH values demonstrated that the midpointpotentials of the [4Fe-4S] clusters responsible for theg ¼ 1.92 and g ¼ 1.89 signals are pH dependent indi-cating that they could be involved in ion pumping.A third minor EPR signal, designated the g ¼ 1.96 sig-nal, was tentatively assigned to the third iron–sulfurcluster of the enzyme. Redox titrations indicated thatthe g ¼ 1.96 signal has the lowest redox potential (wellbelow )420 mV at pH 7); therefore, this cluster couldonly be partly reduced.Ech hydrogenase is highly homologous to the cata-lytic core of complex I which is formed by the fourhydrophilic subunits NuoB, C, D and I and the mem-brane subunits NuoH and NuoL, M, N (following thenomenclature of the E. coli enzyme). The evolutionaryrelationship between complex I and energy-convertinghydrogenases has been addressed in recent reviews [13–16]. The catalytic core of complex I also contains threebinding motifs for [4Fe-4S] centres. The characteriza-tion of these clusters has been an important issue in thecomplex I field in the recent years. In NuoB (the homo-logue of EchC) three of the four Cys residues that areknown to ligate a [4Fe-4S] cluster in all [NiFe] hydro-genases are conserved. In NuoB, these Cys residues,together with a fourth unidentified residue, provide theligands for the EPR-detectable iron–sulfur cluster N2[17,18]. Cluster N2 exhibits a pH-dependent midpointpotential and therefore is thought to be involved in H+pumping [19]. Subunit NuoI shares two conservedfour-Cys motifs for the binding of [4Fe-4S] clusterswith EchF, which is the homologue of NuoI. In com-plex I, however, these clusters are not detectable byEPR spectroscopy and could only be detected byUV ⁄ Vis redox difference spectroscopy [20]. The mid-point potentials of these clusters are pH independent.Hence, the properties of the iron–sulfur clusters presentin the catalytic core of complex I seem to differ fromthose of the homologous clusters in Ech. In the latterenzyme two [4Fe-4S] clusters were detected by EPR,both exhibiting a pH dependent midpoint potential[12]. To ensure that the two EPR signals detected inEch are derived from two different clusters and toassign these clusters to distinct subunits we have per-formed a systematic mutagenesis study in M. barkeri inwhich seven of eight conserved cysteine residues inEchF were individually changed to serine. These studieswere possible due to the recent development of genetictechniques for use in Methanosarcina species [21–23].Here we apply this system for the first time to constructsite-directed mutants in Methanosarcina barkeri.ResultsGeneration of echF mutantsWe have constructed a set of M. barkeri mutants inwhich seven of eight conserved cysteine residues inAssignment of iron-sulfur clusters in Ech hydrogenase L. Forzi et al.4742 FEBS Journal 272 (2005) 4741–4753 ª 2005 FEBSEchF, expected to be involved in iron–sulfur clustercoordination, were individually replaced by serine(Fig. 1, Table 1). The construction of an M. barkerimutant with a deletion of the echABCDEF operon haspreviously been described. The mutant was still able togrow on methanol as the sole energy substrate butfailed to grow on H2⁄ CO2,H2⁄ methanol or acetate [9].Methanol-grown cells were therefore used for the gen-eration of echF point mutants. The echF mutationswere constructed in vitro and recombined into thechromosome as described (Fig. 2). The echF gene wassequenced from each clone to ensure that it carried theappropriate nucleotide sequence for the individualmutations.Ech hydrogenase activity in cell extracts of the EchFmutants and the strain carrying a wild-type copy ofechF was determined using the ferredoxin assay, whichis specific for Ech (Table 2). In all EchF mutants Echhydrogenase activity was strongly reduced. The highestactivities were observed in cell extracts of EchF6,EchF7 and EchF8 mutants. The activity of heterodi-sulfide reductase, an essential enzyme of the energymetabolic pathway, was determined for internal calib-ration. The low Ech activity in the mutants was notdue to a down-regulation of the enzyme as shown bywestern blot analysis of total cell extracts using anantiserum to detect the catalytic subunit EchE (Fig. 3).The serum shows cross reactivity with subunit HdrDof heterodisulfide reductase, which was used as aninternal standard [9].Cells used in this study were cultivated on methanolin single cell morphology. It was observed that Echactivity in wild-type cells grown under these conditionsFig. 1. Representation of the two four-Cys motifs of subunit EchF.The mode of binding of the two putative [4Fe-4S] clusters is indica-ted. The M. barkeri strains carrying cysteine to serine mutationsare indicated on the top.Table 1. M. barkeri strains used in this study.Strain Genotype SourceFusaro(DSM804)Wild type DSMZ, Braunschweig,GermanyEchF1 echFC42S-pac This studyEchF2 echFC45S-pac This studyEchF3 echFC48S-pac This studyEchF5 echFC73S-pac This studyEchF6 echFC76S-pac This studyEchF7 echFC79S-pac This studyEchF8 echFC83S-pac This studyEchF9 echF(wt)-pac This studyFig. 2. Generation of M. barkeri chromosomal echF mutations.Plasmids carrying echF point mutations were digested with ApaIand BamHI and then transformed into M. barkeri. Each echF muta-tion was stably integrated into the chromosome via two homo-logous recombination events, which was selected by puromycinresistance.Table 2. Ech Hydrogenase and heterodisulfide reductase activity incell extracts of EchF mutants and wild type M. barkeri. Ech hydro-genase activity was measured by following the H2- and ferredoxin-dependent reduction of metronidazole as described in methods.Heterodisulfide reductase (Hdr) activity was measured as describedpreviously [38].StrainHydrogenase activity Hdr activity(UÆmg)1)(%) (UÆmg)1)(%)EchF9 (WT) 0.340 100 1.7 100EchF1 0.010 2.9 1.0 59EchF2 0.009 2.6 1.7 100EchF3 0.011 3.2 1.1 65EchF5 0.012 3.5 1.7 100EchF6 0.038 11.2 1.1 65EchF7 0.018 5.3 1.4 82EchF8 0.036 10.6 1.7 100L. Forzi et al. Assignment of iron-sulfur clusters in Ech hydrogenaseFEBS Journal 272 (2005) 4741–4753 ª 2005 FEBS 4743was approximately six- to 10-fold lower than thoseof Ech activity in cells grown on either methanol oracetate in low salt medium where cells grow as cell-aggregates [6,9].Isolation of Ech hydrogenase from the echFmutant strainsEch was purified from wild type and the echF mutantstrains using a modified version of the proceduredescribed previously [6]. After protein solubilization,purification of Ech was carried out by chromatographyon DEAE Sepharose, Q Sepharose and hydroxy-apatite. The mutant enzymes studied showed the samechromatographic properties as wild-type Ech through-out all purification steps. Approximately 1.5 mg pro-tein was obtained from 30 g of cells. The preparationsthus obtained were analysed by SDS ⁄ PAGE (Fig. 4).In preparations obtained from mutant strains EchF2,EchF6 and EchF8 all six subunits of Ech were detect-able in Coomassie stained gels (Fig. 4A). The prepara-tions contained contaminating protein bands withapparent molecular masses of 63 kDa, 75 kDa and90 kDa (only in EchF8). In the preparations obtainedfrom mutant strains EchF1, EchF3, EchF5 and EchF7the small subunits EchC, EchF and EchD were notclearly detectable in the Commassie stained gel(Fig. 4A), but became detectable after silver staining(Fig. 4B). Subunit EchD was only visible as a fuzzyband migrating directly below EchF. In general thepurity of the enzyme from these mutants was lowerthan the enzyme isolated from the EchF2, EchF6 andEchF8 mutants.Hydrogenase activity of the purified enzymes wasdetermined by the H2-uptake assay using the M. bark-eri ferredoxin, which is the physiological substrate ofFig. 3. Western blot detection of EchE subunit in cell extracts ofthe different EchF mutants. Immunodetection was performed usingrabbit anti-Ech sera, as described. The antiserum also detects HdrDof heterodisulfide reductase, which was used as internal standard.The upper band corresponds to HdrD (43 kDa) and the lower bandto EchE (39 kDa). In each lane 6 lg protein from cell extracts ofthe EchF mutant strains were loaded. Purified wild-type Ech (WTEch; 40 ng) was loaded for comparison.ABFig. 4. SDS ⁄ PAGE analysis of Ech hydro-genase preparations from M. barkeri EchFmutants. Proteins were denaturated by incu-bation in Laemmli buffer containing 5 mMdithiothreitol and 2% SDS for 60 min atroom temperature and were subsequentlyseparated in 14% slab gels (8 · 7 cm). Gelswere stained with (A) Coomassie brilliantblue R250 or (B) silver. In each lane 5 lgofprotein were loaded. The highly purifiedenzyme from acetate-grown cells was usedas wild-type Ech (WT). The molecular mas-ses of low-molecular-mass marker proteinsare given on the left, the Ech hydrogenasesubunits are indicated on the right.Assignment of iron-sulfur clusters in Ech hydrogenase L. Forzi et al.4744 FEBS Journal 272 (2005) 4741–4753 ª 2005 FEBSthe enzyme, as electron acceptor. In addition,H2-uptake activity was determined with benzylviologenas an artificial electron acceptor (Table 3). As deter-mined by both assays the EchF8 mutant had the high-est activities, with approximately 10% of the activityof the wild-type enzyme. The enzymes from the EchF7,EchF5, and EchF2 mutants showed between 4% and6% of the wild-type activity. Almost no activity wasdetectable in the enzymes from the EchF1, EchF3 andEchF6 mutants. The specific activities of the purifiedenzymes generally correlate with the activities observedin cell extracts. An exception is the EchF6 mutant inwhich relatively high Ech activity was determined incell extract but almost no activity could be detectedwith the purified enzyme. Wild-type Ech catalysed thereduction of benzylviologen under the experimentalconditions at fourfold higher rates than the reductionof ferredoxin. This activity ratio was nearly constantin the different mutant enzymes.EPR analysis of Ech hydrogenase isolated fromEchF mutant strainsThe iron–sulfur centres of Ech isolated from the differ-ent EchF mutant strains and the strain carrying awild-type copy of EchF were characterized by EPRspectroscopy (Fig. 5). Samples were reduced under anatmosphere of 100% H2and EPR spectra wererecorded at 10 K and 2 mW microwave power. Thewild-type enzyme exhibited a spectrum identical to thatdescribed previously [12]. Based on the EPR line shapeand differences in temperature dependence this spec-trum had been shown to be an overlap of two majorEPR signals originating from S ¼ ½ reduced [4Fe-4S]Table 3. Ech hydrogenase activity of purified Ech hydrogenasefrom EchF mutants and wild-type M. barkeri. Hydrogenase uptakeactivity was measured by following the H2- and ferredoxin-depend-ent reduction of metronidazole (Fd assay) or the H2-dependentreduction of benzyl viologen (BV assay) as described.StrainHydrogenase activityFd assay BV assay(UÆmg)1)(%) (UÆmg)1)(%)WT 30 100 128 100EchF1 0.4 1.3 0.7 0.5EchF2 1.2 4 3.4 3EchF3 0.2 0.7 0.6 0.5EchF5 1.9 6 9.9 8EchF6 0.2 0.7 0.4 0.5EchF7 1.8 5 4.0 3EchF8 3.0 10 11.7 9Fig. 5. EPR spectra of Ech hydrogenase isolated from wild-typeand EchF mutants. Enzymes (4.6 mgÆmL)1) were dissolved inMops buffer pH 7.0 and reduced by incubation for 10 min at 30 °Cunder 100% H2(1.4 · 105Pa). EPR conditions: microwave fre-quency, 9460 MHz; microwave power, 2 mW; modulation ampli-tude, 0.6 mT; temperature, 10 K. The g ¼ 1.89 position is indicatedby a dotted line. The intensity of the spectrum of the wild-typeenzyme (EchF9) was reduced twofold.L. Forzi et al. Assignment of iron-sulfur clusters in Ech hydrogenaseFEBS Journal 272 (2005) 4741–4753 ª 2005 FEBS 4745clusters, one signal with gxy¼ 1.921 and gz¼ 2.050(designated the 1.92 signal) and the second with gxy¼1.887 and gz¼ 2.078 (designated the 1.89 signal). EPRspectra recorded from the enzyme of the EchF8,EchF7 and EchF2 mutants show a strong signalwith gxy¼ 1.890 and gz¼ 2.078, corresponding to theg ¼ 1.89 signal found in the wild-type enzyme. Theg ¼ 1.92 signal was still detectable in these mutantenzymes but its spin concentration was stronglyreduced relative to the g ¼ 1.89 signal. The EPR spec-trum obtained for the enzyme from the EchF8 mutantshowed the most intense g ¼ 1.89 signal. Both iron–sulfur signals were simulated (Fig. 6). The parametersfor the g ¼ 1.89 signal are slightly different than thosereported previously [12] because the cluster is notinvolved in spin–spin interaction with other clusters.The 1.92 signal was simulated with the same parame-ters as before as it makes only a small contribution tothe overall spectrum. The overall spin concentration inthe iron–sulfur cluster region, corrected for the g ¼2.03 ⁄ 2.00 radical-like signals (see below) was 10 lm,the enzyme concentration was 19 lm. The g ¼ 1.89and g ¼ 1.92 signals are present in a ratio of 9 : 1 asestimated from the simulated EPR spectra. As in thewild-type enzyme the g ¼ 1.92 signal and the g ¼ 1.89signal showed a different temperature dependence. Theg ¼ 1.92 signal was optimally sharpened at 17 Kwhereas the g ¼ 1.89 signal was already considerablybroadened at 17 K as shown for the enzyme from theEchF8 mutant in Fig. 7.To determine if the mutations had turned the spinof the ground state of the cluster(s) of subunit EchF toS ¼ 3 ⁄ 2, EPR spectra were recorded in the low fieldregion (50–2000 G) at low temperature (4.5 K) andhigh power (20 mW). Pronounced signals wereFig. 6. Simulation of the EPR spectrum of the EchF8 mutantenzyme. The experimental spectrum (EchF8), the simulated g ¼1.89 and g ¼ 1.92 spectra and the difference spectrum obtainedafter subtraction of the two simulated spectra from the experimen-tal spectrum, are shown. Simulation of the g ¼ 1.89 signal of thespectrum from the EchF8 mutant enzyme with parameters gzyx¼2.07750, 1.90223, 1.89000 and widths (zyx) 3.4, 2.6 and 5.9 mT.Simulation of the g ¼ 1.92 signal with parameters gzyx¼ 2.04721,1.93799, 1.91821 and widths 2.66, 2.7 and 2.77.Fig. 7. EPR spectra of the EchF8 mutant enzyme at different tem-peratures in comparison to the wild-type spectrum. The g ¼ 1.92position is indicated by a dotted line. For EPR conditions see Fig. 5.Assignment of iron-sulfur clusters in Ech hydrogenase L. Forzi et al.4746 FEBS Journal 272 (2005) 4741–4753 ª 2005 FEBSobserved only at g ¼ 4.3 which could be due to adven-titious Fe(III).EPR spectra recorded from the enzyme of theEchF5 mutant showed only a weak g ¼ 1.89 signal,but the amplitude of the g ¼ 1.92 signal was compar-able to that found in the enzyme from the EchF2,EchF7 and EchF8 mutants. The EchF6 mutant alsoshowed a weak g ¼ 1.89 signal but only a very weakg ¼ 1.92 signal. In EPR spectra recorded from theEchF1 and EchF3 mutant enzymes no iron–sulfurcluster signals could be detected.EPR spectra of the EchF mutants all showed signalswith g-values at 2.033 and 2.003. The two signalsshowed different saturation properties (Fig. 8). The2.03 signal could not be saturated at 4 K and 20 mW(10 dB) whereas the 2.00 signal was already saturatedat 10 K and 2 mW (20 dB). The signals could beobserved at temperatures up to 130 K without signalbroadening. The line width of both signals wasapproximately 1.2 mT, which is typical for radical spe-cies. The identical line width indicates that both signalscould belong to the same paramagnetic species. Theg ¼ 2.03 signal has been previously detected in wild-type Ech where it is only present at very low intensity.However, this signal is much stronger in the EchFmutants. The spin concentration of this signal wasdetermined in the EchF1 and EchF3 mutants, whichshowed no iron–sulfur cluster signal. Here the spinconcentration was approximately 0.7 lm, assuming anS½ species. The enzyme concentration was 19 lm.Bycomparing the signal amplitudes it could be estimatedthat the spin concentration of the g ¼ 2.03 ⁄ 2.00 signalsin the EchF8 mutant is 0.4–0.5 lm corresponding to4–5% of the spin concentration of the iron–sulfur clus-ter signals. In general the spin concentration of theg ¼ 2.03 ⁄ 2.00 signals was approximately 1.6 timeshigher in the enzyme from those mutants whichshowed no or very low intensity signals for the iron–sulfur clusters. The g ¼ 2.03 ⁄ 2.00 signals shown inFig. 8 were observed for the enzyme reduced by 100%H2. Addition of 20 mm sodium dithionite did notchange the intensity of these signals. When 1 mm duro-quinone (E0¢ ¼ +86 mV) was added to the enzymeunder N2, the g ¼ 1.89 signal was no longer detect-able, indicating an oxidation of this iron–sulfur center.The intensities of the g ¼ 2.03 ⁄ 2.00 signals were, how-ever, not altered by duroquinone oxidation.DiscussionThe characterization of the iron–sulfur clusters of Echhydrogenase by EPR spectroscopy, performed previ-ously, revealed the presence of two axial like EPR sig-nals fully reducible under 100% H2. The two signalswere designated as the g ¼ 1.89 and the g ¼ 1.92 sig-nal. Importantly, both species have a pH-dependentmidpoint potential. The E0¢ value of the g ¼ 1.92signal decreased by 53 mV per pH unit; that of theg ¼ 1.89 signal decreased by 62 mV per pH unit [12].These values are reasonably close to the theoreticalvalue of )59 mV per pH unit for a redox titrationinvolving a stoichiometric amount of electrons andprotons. The g ¼ 1.89 and the g ¼ 1.92 signal showedslightly different temperature optima, the g ¼ 1.89signal being optimally sharpened at 12 K and the g ¼1.92 signal being optimally sharpened at 17 K. At tem-peratures below 15 K a twofold splitting of the Nia–Lsignal was observed due to the interaction of theNi-based unpaired electron with the S ¼ ½ system ofthe reduced proximal [4Fe-4S] cluster. The temperaturedependence of the splitting of the Nia–L signal paral-leled the temperature dependence of the g ¼ 1.89 sig-nal. It was therefore tentatively concluded that theg ¼ 1.89 signal is due to the reduced proximal clusterFig. 8. Normalized EPR spectra of the EchF8 mutant enzyme atdifferent powers. For EPR conditions see Fig. 5.L. Forzi et al. Assignment of iron-sulfur clusters in Ech hydrogenaseFEBS Journal 272 (2005) 4741–4753 ª 2005 FEBS 4747in EchC [12]. The experiments described here substan-tiate this former assignment. Seven of eight cysteineresidues predicted to ligate the iron–sulfur clusters inEchF were systematically changed to serine. For twoof the mutant enzymes, EchF2 and EchF8, the g ¼1.89 signal was the major signal and only residual spinintensities of the g ¼ 1.92 signal were observed. Thespin concentration of the g ¼ 1.89 signal of the EchF8mutant was highest and accounted for approximately50% of the enzyme concentration. The determinationof the spin concentration is based on the total proteinconcentration of the sample. Because the preparationstill contained three contaminating protein bands(Fig. 4), the spin concentration is probably underesti-mated. EPR spectra recorded from the enzymes isola-ted from the EchF5, EchF6 and EchF7 mutantsalso contained the g ¼ 1.89 signal, however, at lowerspin intensities. The formation of the g ¼ 1.89 and theg ¼ 1.92 clusters was not dependent on whether themutation was in the first (EchF2 and EchF8) orthe second (EchF5 and EchF7) iron–sulfur clusterbinding motif of EchF (Fig. 1). EPR spectra of theEchF2, EchF5, EchF7 and EchF8 mutant enzymesshowed a weak signal in the g ¼ 1.92 region which canbe attributed to the gxyof the g ¼ 1.92 signal. Studieswith the wild-type enzyme had shown that the thirdcluster of the enzyme, assigned to the g ¼ 1.96 signal,has a low redox potential and thus could only bereduced to a low extent under 100% H2. It is thereforedifficult to judge if the intensity of this signal haschanged in the mutant enzymes. One possible explan-ation for the formation of low amounts of the g ¼1.92 cluster in some of the EchF mutants could beligand exchange. Subunit EchF contains an additionalfree Cys residue in position 87 which could function asa ligand in some of the mutants. Likewise, the intro-duced Ser residues could also function as a ligand ofthe cluster as suggested for a Cys to Ser mutant ofE. coli nitrate reductase [24]. For the R. capsulatusNuoI mutants (see below) it was also proposed that insome of the mutants the introduced Ser residue couldbe a direct ligand to a [4Fe-4S] cluster [25].The cluster ligating Cys residues conserved in EchFhave also been mutagenized in the homologous subunitof complex I, NuoI, from R. capsulatus [25] and E. coli[17]. In R. capsulatus five Cys residues were individu-ally changed to Ser. Four of these mutants hadretained significant amounts of complex I activity inthe membrane fraction (up to 72% of the wild-typeactivity). Purification of the mutant enzymes was notattempted as even the wild-type enzyme was found tobe unstable upon purification. The eight cluster-ligat-ing Cys residues of the closely related E. coli complexI were individually mutated to Ala. With the exceptionof the C102A mutant, which had retained 17% of thewild-type activity, all other mutants had lost complex Iactivity. The comparison indicates that Cys to Sermutations are more likely to produce active enzyme incomparison with Cys to Ala mutations.In most of the EchF mutants the intensity of theg ¼ 1.89 signal was also reduced and in two of themutants no signal due to an iron–sulfur cluster couldbe detected indicating that the mutation in EchF alsohad a strong effect on iron–sulfur cluster assembly inEchC. This is analogous to mutations of the clusterligating cysteine residues of the NuoI subunit of E. colicomplex I, which in most cases also resulted in a lossof the iron–sulfur cluster N2 located on subunit NuoB,a homologue of EchC [17]. It has also been observedwith other systems, e.g. two [4Fe-4S] ferredoxins, thata substitution of one of the cluster ligands in a subunitoften significantly affects incorporation of the neigh-bouring clusters [26].The characterization of the different EchF mutantstrains revealed that subunit EchF, homologous tocomplex I subunit NuoI (or TYKY), contains anEPR-detectable iron–sulfur cluster which exhibits apH-dependent midpoint potential. In contrast, no EPRsignal could be attributed to one of the [4Fe-4S] clus-ters located on NuoI (or TYKY) of complex I. Instudies performed with complex I from E. coli andNeurospora crassa, a redox-group was identified bymeans of UV⁄ Vis spectroscopy and was assigned tothe two [4Fe-4S] clusters located on NuoI (or TYKY)[20]. A redox titration of this group, which was fol-lowed by UV ⁄ Vis spectroscopy, revealed a pH-inde-pendent midpoint potential of these clusters with anE0¢ value of )270 mV. It is thought that these clustersare magnetically coupled in the reduced state andtherefore are difficult to detect by EPR spectroscopy.From these results it was concluded that NuoI (orTYKY) has redox properties very similar to those of8Fe-ferredoxins, e.g. the one from Clostridium pasteu-rianum. Therefore, NuoI was proposed to be involvedin simple electron transfer. Other studies focusing onthe characterization of the iron–sulfur clusters of NuoIwhere performed with the homologous protein fromParacoccus denitrificans, termed NQ09 [27]. NQ09 washeterologously produced in E. coli. The isolated sub-unit was found to bind two [4Fe-4S] clusters whichwhen reduced gave rise to a set of two relatively broadaxial-type EPR signals at g ¼ 2.08, 2.05 and 1.93 and1.90. The two sets of EPR signals could either bederived from two distinct species of [4Fe-4S] clustersor alternatively one signal could be derived from thetwo S ¼ 1 ⁄ 2 [4Fe-4S] clusters in NQ09 which exhibitAssignment of iron-sulfur clusters in Ech hydrogenase L. Forzi et al.4748 FEBS Journal 272 (2005) 4741–4753 ª 2005 FEBSsimilar EPR spectra and the second signal could arisefrom spin–spin interaction between the former twoparamagnetic species. The midpoint potentials of theseclusters were, however, < )600 mV indicating thattheir redox properties changed considerably in the het-erologously produced subunit. In the entire complex Ifrom P. denitrificans these signals were not observed.In Ech the [4Fe-4S] cluster located on subunit EchCand one of the [4Fe-4S] clusters on subunit EchF exhi-bit a pH-dependent midpoint potential. This indicatesthat oxidation ⁄ reduction of these clusters depends oncharge compensation of an acidic residue close to thecluster. These subunits therefore could also play a cru-cial role in coupling electron transfer to proton trans-location. Acidic residues that could be involved in thisprocess have been identified in multiple sequence align-ments of EchF or EchC with their homologues fromother energy-converting hydrogenases and the corres-ponding subunits of complex I from various sources.In EchF the second 4· Cys-binding motif was foundto contain a Glu, Asp or His residue in all members ofthe protein family [8]. In addition, a highly conservedGlu residue is found in the proximity of the second4· Cys-binding motif (C-X(2)-C-X(2)-C-X(3)-C-P-X(8–10)-E). The cluster on EchC corresponds to clusterN2 located on the homologous subunit of complex I(NuoB or PSST). Recently, electrochemically inducedFT-IR-difference spectroscopy of site-directed mutantsof E. coli complex I revealed that the reduction ofiron–sulfur cluster N2 is accompanied by the protona-tion of Y114 and Y139 of subunit NuoB [28,29]. TheFT-IR data also indicated that the oxidation of clusterN2 is coupled with the protonation of one or morecarboxylic amino acids. The residues corresponding toY114 and Y139 are also conserved in Ech and otherenergy-converting hydrogenases. The FT-IR data alsoindicate that the oxidation of cluster N2 is coupledwith the protonation of one or more carboxylic aminoacids. Both, EchC and NuoB contain highly conservedGlu and Asp residues, which are not conserved in thehomologous subunit of standard [Ni-Fe] hydrogenasessuggesting a common mechanism as well.The hydrogenase activity of the EchF mutants, withferredoxin as well as with benzylviologen as electronacceptor, was strongly reduced. The relative hydro-genase activity, with regards to which electron acceptorwas used, indicates that the EchF mutants and wild-type Ech use the same set of iron–sulfur clusters forthe electron transfer from the [Ni-Fe] centre. In thiscontext the analysis of the EchF8 mutant is of partic-ular interest. In this mutant the spin concentration ofthe proximal cluster was highest and accounted for atleast 50% of the enzyme concentration. The hydro-genase activity of this mutant enzyme determined withboth the physiological and the artificial electron accep-tor, however, was only about 10% of that of the wild-type enzyme. This indicates that the g ¼ 1.92 clusterand probably also the g ¼ 1.96 cluster are requirednot only for the reduction of the ferredoxin but alsofor the reduction of the artificial electron acceptor ben-zylviologen. Reduction of the g ¼ 1.89 cluster by H2was still possible in the mutant enzymes, which pro-vides further evidence that this cluster directly interactswith the [Ni-Fe] centre and that the [Ni-Fe] centre isintact in the mutant enzymes. Hence, the electrontransfer reaction mediated by the enzyme can be sum-marized as follows: H2is activated at the [Ni-Fe] cen-tre, and electrons are transferred via the proximalcluster located on subunit EchC (g ¼ 1.89 signal) to thecluster(s) located on subunit EchF, where ferredoxinis reduced. Our studies show that at least the clustergiving rise to the g ¼ 1.92 signal is required for ferre-doxin reduction. At the current stage, the exact role ofthe low-potential g ¼ 1.96 signal is not known. Evenin the wild-type enzyme, the intensity of this signal isvery low because of its very low redox potential.Unlike in complex I, quinones are not involved in theelectron transfer reaction pathway mediated by energy-converting hydrogenases. The comparison betweencomplex I and Ech hydrogenase rather indicates thatthe [Ni-Fe] centre and the quinone have complement-ary functions. The characterization of several complexI mutants which carry mutations at conserved posi-tions in the NuoD (or 49 kDa) subunit has shown thatthis subunit of complex I carries a significant part ofthe quinone binding pocket and that this bindingpocket could have evolved directly from the [Ni-Fe]centre binding site of the hydrogenases [16]. ThepH-dependence of the two [4Fe-4S] clusters in Echsuggests that these clusters simultaneously mediateelectron and proton transfer and thus could be anessential part of the proton translocating machinerywhich delivers protons to proton transfer pathwaysformed within the membrane part. The protonsrequired for H2formation at the active site arethought to be delivered by a distinct proton channellocated within the hydrogenase large subunit [30,31].A question that remains is if the paramagnetic spe-cies that gives rise to the 2.03 ⁄ 2.00 signals is anintrinsic part of the enzyme (e.g. a yet unknownredox group) or if this species is artificially generatedto a greater extent in the EchF mutants. The observa-tion that the signals do not respond to oxidation orreduction favours the second possibility. Also the spinconcentration is rather low. In freshly prepared wild-type Ech the 2.03 signal is hardly detectable (Fig. 5)L. Forzi et al. Assignment of iron-sulfur clusters in Ech hydrogenaseFEBS Journal 272 (2005) 4741–4753 ª 2005 FEBS 4749whereas the signal becomes more intense upon agingof the enzyme (R. Hedderich, unpublished results).The line shape and the temperature dependence of thesignals could indicate a free radical, but such highg-values have only been described for sulfur-basedradicals [32]. Such radicals are, however, very unsta-ble and are normally observed only under presteady-state conditions. Because the g ¼ 2.03 component ofthe signal could not be saturated at 4.2 K and fullpower, it was suggested that the signal could be dueto a radical in close proximity to a very rapidly relax-ing paramagnet, e.g. high spin Fe3+[12]. EPR spectrawith similar g-values (g ¼ 2.032 and g ¼ 2.004) andtemperature behaviour have been observed for iron–nitrosyl–histidyl complexes which have for examplebeen observed upon disassembly of the [3Fe-4S] clus-ter of mitochondrial aconitase upon anaerobic NOaddition [33]. If such a species is formed upon reac-tion of a partially assembled iron–sulfur cluster ofEch with NO, which could be reductively generatedfrom contaminating amounts nitrate or nitrite, needsto be shown.Experimental proceduresPlasmid and strain constructionStandard techniques were used throughout for isolation andmanipulation of plasmid DNA in E. coli, using DH10B(Invitrogen, Carlsbad, CA, USA) as the host strain. Allinferred plasmid sequences are available upon request.echDEF was amplified by PCR from M. barkeri chromo-somal DNA using primers 5¢-GGCGCGCCGGGCCCACGGAGTAGTGGCAGCACTT-3¢ and 5¢-GGCGCGCCCTCGAGGGAGAACATTCAGTATTGTTTTTCAAG-3¢(restriction sites are underlined), digested with ApaI andXhoI, and ligated into pBluescriptSK (Stratagene, La Jolla,CA, USA) cut with ApaI and XhoI, resulting in pAMG57.Point mutations were generated by the QuickChangeTMmethod (Stratagene) using pAMG57 as the PCR template.The insert of interest was sequenced to verify that the selec-ted clones only contained the desired mutations. The down-stream region of M. barkeri ech was amplified by PCRusing primers 5¢-GGCGCGCCCTGCAGGGTCTAAATTTGGCAGTTAAGGAA-3¢ and 5¢-GGCGCGCCGGATCCCCTGCACCTTTCCTGATTTT-3¢, digested with Bam HIand PstI (restriction sites underlined), and ligated into pJK3[23] cut with BamHI and PstI, resulting in pAMG77. Eachof the seven point mutations generated from pAMG57 andthe insert from the original pAMG57 were then subclonedinto pAMG77 using the ApaI and XhoI restriction sites,resulting in pCGR1 to pCGR3 and pCGR5 to pCGR9,respectively. These plasmids were then digested with ApaIand BamHI and transformed into M. barkeri using standardtechniques (Fig. 2) [21–23,34], selecting puromycin resist-ance. After single-colony purification, clones were screenedfor the correct genotype by PCR amplification andsequenced using primers 5¢-ACTTATGTTACCGGGCGTCA-3¢ and 5¢-CCTCGAGGGAGAACATTCAG-3¢, result-ing in the strains listed in Table 1. M. barkeri strains weregrown in single cell morphology [35] at 37 °C in high-saltmedia under strictly anaerobic conditions, as described pre-viously [36]. Methanol (125 mm) was added to high-saltmedia as carbon and energy source. Puromycin was addedto 2 mgÆmL)1as appropriate.Preparation of cell extracts and isolationof Ech hydrogenaseEch hydrogenase was purified from methanol-grownM. barkeri echF mutant strains under strictly anaerobicconditions using a modification of the procedure describedpreviously [6]. Late exponential-phase M. barkeri singlecells were lysed by resuspension in 50 mm Mops ⁄ NaOHpH 7.0 containing 2 mm dithiothreitol (buffer A), to whicha few crystals of DNase I were added (spontaneous lysisoccurs due to osmotic shock). Complete lysis was ensuredby sonication four times at 200 W for 3 min. Intact cellsand cell debris were removed by centrifugation at 10 000 gfor 30 min at 4 °C. The cell extracts thus generated wereused for activity measurements and western blot analysis.For the isolation of the membrane fraction, cell extractswere loaded on a DEAE Sephacel column (2.6 · 15 cm).The column was washed with 100 mL buffer A. Themajority of Ech hydrogenase activity was recovered in theturbid void volume of the column whereas most solubleproteins were bound to the column material. This proce-dure resulted in higher Ech yields as compared to an ul-tracentrifugation step, as membranes of methanol-grownM. barkeri were difficult to sediment by ultracentrifuga-tion, probably due to their high glycogen content.Membranes thus obtained were solubilized by dodecyl-b-d-maltoside (15 mm, 4.5 mg detergentÆmg protein)1) for 12 hat 4 °C. After centrifugation at 150 000 g for 30 min thesolubilized membrane proteins present in the supernatantwere loaded on a DEAE–Sepharose column (2.6 · 10 cm)equilibrated with buffer A containing 2 mm dodecyl-b-d-maltoside (buffer A + DDM). The column waswashed with 50 mL buffer A + DDM and proteins wereeluted with NaCl in buffer A + DDM using a step gradi-ent: 0.24 m (150 mL) and 0.4 m (150 mL). Ech hydro-genase was recovered in the fractions eluting with 0.24 mNaCl. Further purification of Ech was carried out bychromatography on Q Sepharose and hydroxyapatite asdescribed [6]. The enzyme eluted from the Q Sepharosecolumn with 0.19 m NaCl and from the hydroxyapatitecolumn with 500 mm potassium phosphate. These fractionswere concentrated and the buffer was exchanged to buf-fer A + DDM by ultrafiltration using Amicon Ultra-4Assignment of iron-sulfur clusters in Ech hydrogenase L. Forzi et al.4750 FEBS Journal 272 (2005) 4741–4753 ª 2005 FEBS[...]... reduction to CH4 Eur J Biochem 186, 309–316 11 Stojanowic A & Hedderich R (2004) CO2 reduction to the level of formylmethanofuran in Methanosarcina barkeri is non-energy driven when CO is the electron donor FEMS Microbiol Lett 235, 163–167 4751 Assignment of iron-sulfur clusters in Ech hydrogenase 12 Kurkin S, Meuer J, Koch J, Hedderich R & Albracht SP (2002) The membrane-bound [NiFe] -hydrogenase (Ech) from. .. [6] The 0.8-mL assays contained 50 mm Mops ⁄ NaOH pH 7.0, 2 mm dithiothreitol, 2 mm DDM, 20 lm ferredoxin (isolated from M barkeri as described [6], 150 lm metronidazole (e320 ¼ 9.3 mm)1 cm)1) and protein (purified Ech hydrogenase, membrane fraction or cell extract) One unit of hydrogenase activity corresponds to the oxidation of 1 lmol H2 measured by the ferredoxin-dependent reduction of 1 ⁄ 3 lmol of. .. membrane-bound [NiFe] -hydrogenase (Ech) from Methanosarcina barkeri: unusual properties of the iron-sulphur clusters Eur J Biochem 269, 6101– 6111 13 Friedrich T & Weiss H (1997) Modular evolution of the respiratory NADH: ubiquinone oxidoreductase and the origin of its modules J Theor Biol 187, 529–540 14 Friedrich T & Scheide D (2000) The respiratory complex I of bacteria, archaea and eukarya and its... Quinkal I (1996) The coordination sphere of iron-sulfur clusters: lessons from site-directed mutagenesis experiments J Biol Inorg Chem 1, 2–14 27 Yano T, Magnitsky S, Sled VD, Ohnishi T & Yagi T (1999) Characterization of the putative 2x[4Fe-4S]-binding NQO9 subunit of the proton-translocating NADHquinone oxidoreductase (NDH-1) of Paracoccus denitrificans Expression, reconstitution, and EPR characterization. .. Gunsalus RP (1993) Disaggregation of Methanosarcina spp & Growth as Single Cells at Elevated Osmolarity Appl Environ Microbiol 59, 3832–3839 36 Metcalf WW, Zhang JK, Shi X & Wolfe RS (1996) Molecular, genetic, and biochemical characterization FEBS Journal 272 (2005) 4741–4753 ª 2005 FEBS Assignment of iron-sulfur clusters in Ech hydrogenase of the serC gene of Methanosarcina barkeri Fusaro J Bacteriol 178,... plasmidbased complementation in the methanogenic archaeon Methanosarcina acetivorans C2A demonstrated by genetic analysis of proline biosynthesis J Bacteriol 184, 1449–1454 22 Boccazzi P, Zhang JK & Metcalf WW (2000) Generation of dominant selectable markers for resistance to pseudomonic acid by cloning and mutagenesis of the ileS gene from the archaeon Methanosarcina barkeri fusaro J Bacteriol 182,... (2004) Energy-converting [NiFe] hydrogenases from archaea and extremophiles: ancestors of complex I J Bioenerg Biomembr 36, 65–75 2 Vignais PM, Billoud B & Meyer J (2001) Classification and phylogeny of hydrogenases FEMS Microbiol Rev 25, 455–501 3 Fox JD, He Y, Shelver D, Roberts GP & Ludden PW (1996) Characterization of the region encoding the COinduced hydrogenase of Rhodospirillum rubrum J Bacteriol... properties of a CO-oxidizing: H2-evolving enzyme complex from Carboxydothermus hydrogenoformans Eur J Biochem 269, 5712–5721 5 Kunkel A, Vorholt JA, Thauer RK & Hedderich R ¨ (1998) An Escherichia coli hydrogenase- 3-type hydrogenase in methanogenic archaea Eur J Biochem 252, 467–476 6 Meuer J, Bartoschek S, Koch J, Kunkel A & Hedderich ¨ R (1999) Purification and catalytic properties of Ech hydrogenase from Methanosarcina. .. Friedrich T (2000) FT-IR spectroscopic characterization of NADH: ubiquinone oxidoreductase (complex I) from Escherichia coli: oxidation of FeS cluster N2 is coupled with the protonation of an aspartate or glutamate side chain Biochemistry 39, 10884–10891 29 Flemming D, Hellwig P & Friedrich T (2003b) Involvement of tyrosines 114 and 139 of subunit NuoB in the proton pathway around cluster N2 in Escherichia... Fernandez VM & Rousset M (2004) A glutamate is the essential proton transfer gate during the catalytic cycle of the [NiFe] hydrogenase J Biol Chem 279, 10508–10513 32 Lawrence CC, Bennati M, Obias HV, Bar G, Griffin RG & Stubbe J (1999) High-field EPR detection of a disulfide radical anion in the reduction of cytidine 5¢-diphosphate by the E441Q R1 mutant of Escherichia coli ribonucleotide reductase Proc . Assignment of the [4Fe-4S] clusters of Ech hydrogenase from Methanosarcina barkeri to individual subunits via the characterization of site-directed mutants Lucia. directly below EchF. In general the purity of the enzyme from these mutants was lowerthan the enzyme isolated from the EchF2, EchF6 andEchF8 mutants. Hydrogenase
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