The membrane-bound [NiFe]-hydrogenase (Ech) from Methanosarcina barkeri : unusual properties of the iron-sulphur clusters Sergei Kurkin 1 ,Jo¨ rn Meuer 2 ,Ju¨ rgen Koch 2 , Reiner Hedderich 2 and Simon P. J. Albracht 1 1 Swammerdam Institute for Life Sciences, Biochemistry, University of Amsterdam, the Netherlands; 2 Max-Planck-Institut fu ¨ r Terrestrische Mikrobiologie, Marburg, Germany The purified membrane-bound [NiFe]-hydrogenase from Methanosarcina barkeri was studied with electron para- magnetic resonance (EPR) focusing on the properties of the iron–sulphur clusters. The EPR spectra showed signals from three different [4Fe)4S] clusters. Two of the clusters could be reduced under 101 kPa of H 2 , whereas the third cluster was only partially reduced. Magnetic interaction of one of the clusters with an unpaired electron localized on the Ni–Fe site indicated that this was the proximal cluster as found in all [NiFe]-hydrogenases. Hence, this cluster was assigned to be located in the EchC subunit. The other two clusters could therefore be assigned to be bound to the EchF subunit, which has two conserved four-Cys motifs for the binding of a [4Fe)4S] cluster. Redox titrations at different pH values demonstrated that the proximal cluster and one of the clusters in the EchF subunit had a pH-dependent midpoint potential. The possible relevance of these properties for the function of this proton-pumping [NiFe]-hydrogenase is discussed. Keywords: Ech; hydrogenase; iron-sulphur; pH dependence; redox properties. Hydrogenases catalyse the simplest chemical reaction in nature: H 2 « 2H + +2e – . They are found in wide variety of microorganisms. Hydrogenases enable some organisms to use H 2 as a source of reducing equivalents under both aerobic and anaerobic conditions. In other organisms the enzyme is used to reduce protons to H 2 , thereby releasing the reducing equivalents obtained from the anaerobic degradation of organic substrates [1,2]. On basis of the transition-metal content, hydrogenases can be divided into two major classes [3]: the [Fe]-hydrogenases [4] and the [NiFe]-hydrogenases [5–7]. The large subunit of [NiFe]- hydrogenases harbours the binuclear Ni–Fe active site, which is coordinated by two conserved CxxC motifs, one locatedintheN-terminalregionandthesecondlocatedin the C-terminal region of the polypeptide [5]. The small subunit of all [NiFe]-hydrogenases displays a conserved amino acid sequence pattern, CxxCx n GxCxxxGx m GCPP (n ¼ 61 to 106, m ¼ 24 to 61 [5]), binding one [4Fe)4S] cluster. This cluster is within 14 A ˚ oftheactivesite[8]andis called the proximal cluster. In most, but not all enzymes, the small subunit contains six to eight additional cysteine residues, which harbour two more clusters: in the Desulf- ovibrio gigas enzyme these are a second [4Fe)4S] cluster (distal cluster) and a [3Fe)4S] cluster (medial cluster). The combination of the Ni–Fe active site and the proximal [4Fe)4S] cluster seems to be important for the catalytic action of [NiFe]-hydrogenases [7]. The study of hydrogenases in methanogens led to the discovery of a third class of hydrogenases, not containing any metals [9]. This class of enzyme is active only in the presence of its second substrate, N 5 ,N 10 -methenyltetra- hydromethanopterin. There is evidence for an unknown nonmetal prosthetic group in this enzyme [10,11]. Metha- nogens also contain [NiFe]-hydrogenases and the expression of the several enzymes depends on the available energy sources [12,13]. Some time ago a membrane-bound [NiFe]- hydrogenase was isolated from methanogenic archaea [14], which consists of six subunits much like hydrogenase-3 of Escherichia coli. Hydrogenase-3 in E. coli is part of the formate-hydrogen lyase complex and is composed of seven different subunits [15]. This hydrogenase shows surprisingly little sequence homology with other [NiFe]-hydrogenases, except for the conserved residues coordinating the active site and the proximal Fe–S cluster. The enzyme showed a high sequence similarity with the CO-induced hydrogenase of Rhodospirillum rubrum [16,17]. The latter bacterium can grow anaerobically on CO and its [NiFe]-hydrogenase is thus expected to be insensitive towards CO. The same is expected for the ÔE. coli-like hydrogenaseÕ (Ech) from Methanosarcina barkeri [14,18]. From growth characteristics of R. rubrum and from cell-suspension experiments with M. barkeri, it can be inferred that the [NiFe]-hydrogenases in these organisms probably act as a proton pumps [16,19]. Ech is the only enzyme of this subclass which has been purified and partly characterized. Purified Ech consists of six subunits, encoded by genes organized in the echABCDEF operon. The EchA and EchB subunits are predicted to be integral, membrane-spanning proteins, while the other four subunits are expected to extrude into the cytoplasm (Fig. 1). Amino acid sequence analysis of the cytoplasmic subunits points to the presence of two classical [4Fe)4S] clusters in EchF and one [4Fe)4S] Correspondence to S. P. J. Albracht, Swammerdam Institute for Life Sciences, Biochemistry, University of Amsterdam, Plantage Muidergracht 12, NL-1018 TV Amsterdam, the Netherlands. Fax: + 31 20 5255124, Tel.: + 31 20 5255130, E-mail:asiem@science.uva.nl Abbreviations:Ech,membrane-boundhydrogenaseofMethanosarcina barkeri; EPR, electron paramagnetic resonance; Hdr, heterodi- sulphide reductase. (Received 8 August 2002, revised 4 October 2002, accepted 21 October 2002) Eur. J. Biochem. 269, 6101–6111 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03328.x cluster in EchC. The EchE subunit belongs to the family of the large subunits in [NiFe]-hydrogenases and shows the characteristic binding motif for the Ni–Fe site found in the large subunits of all [NiFe]-hydrogenases. Chemical analysis revealed the presence of Ni, nonheme Fe and acid-labile S in a ratio of 1 : 12.5 : 12 [18], corroborating the presence of three Fe–S clusters. A low-potential, soluble ferredoxin (E 0 ¢ ¼ ) 420 mV) was found to be the natural donor/ acceptor of electrons for Ech [18]. Kinetic analyses revealed that purified Ech is inactivated by O 2 and, like most [NiFe]- hydrogenases, is inhibited by CO [18]. The biological role of Ech was recently studied using mutational analysis [20]. There are several functions proposed for Ech, depending on the growth conditions and cell energy requirements. In acetoclastic methanogen- esis, Ech catalyses H 2 formation from reduced ferredoxin, generated by the oxidation of the carbonyl group of acetate to CO 2 . Under autotrophic growth conditions, the enzyme catalyses the energetically unfavourable reduction of ferre- doxin by H 2 , most probably driven by energy-induced reversed electron transport, and the reduced ferredoxin thus generated functions as the low potential electron donor for the synthesis of pyruvate in an anabolic pathway. The reduced ferredoxin also provides the reducing equivalents for the first step of the methanogenesis, namely the reduction of CO 2 to formylmethanofuran. The six subunits of Ech show a striking amino acid sequence similarity with six subunits of proton-pumping NADH : ubiquinone oxidoreductase (complex I) [14–16]. Complex I catalyses electron transfer from NADH to ubiquinone and couples it to the translocation of four to five protons across a membrane. Studies of submitochondrial particles have demonstrated that of all the Fe–S clusters of complex I, only two, called the clusters 2 or N-2, which are presumably located in TYKY subunit (homologous to EchF) [21], are directly involved in energy transduction. It is known that the redox potential of these Fe–S clusters is pH dependent ()60 mVÆpH unit )1 ) [22], which is rare for Fe–S clusters. The TYKY and EchF subunits belong to a family of polypeptides, which are found exclusively in complex I and proton-pumping hydrogenases [23]. The amino acid sequences of the proteins in this family are so unique and conserved, that the two [4Fe)4S] clusters held by this protein were proposed to function as the direct electrical driving unit for a proton pump [23]. To delineate a possible roleoftheFe–SclustersintheEchofM. barkeri in this action, the electron paramagnetic resonance (EPR) and redox properties of these Fe–S clusters were investigated. MATERIALS AND METHODS Purification of M. barkeri Ech and sample preparation Ech was purified as described elsewhere [18]. The enzyme was routinely dissolved in 50 m M Mops pH 7.0, 2 m M dithiothreitol and 2 m M dodecylmaltoside under an atmo- sphere of 4% (v/v) H 2 . For redox titrations the concentra- tion of dithiothreitol in the enzyme solution was reduced to 2 l M . The following buffers were used for redox titrations: 100 m M Tris/HCl pH 8.0, potassium phosphate pH 7.0, Tris/Mes pH 6.5, or Mes pH 6.0. The standard enzyme solution was concentrated and then diluted with new buffer; this was repeated several times. Samples for all spectro- scopic measurements were handled anaerobically i.e. all operations were performed in anaerobic box at 4% (v/v) H 2 . Membranes were obtained from cells grown on acetate at 37 °C and were prepared as described [18]. They were suspended in 50 m M Mops/NaOH pH 7.0, containing 2m M dithiothreitol. Ferredoxin was purified as described by Fischer and Thauer [24]. Redox titrations Redox titrations of Ech were performed using a Pt vs. Calomel electrode system (Radiometer, Copenhagen) in a device analogous to that of Dutton [25]. The redox potential was measured using a digital voltmeter RW9408 (Philips). All redox potentials mentioned here are expressed vs. the normal hydrogen electrode. Correction for the temperature dependence of the reference electrode was performed as in Ives and Janz [26]. As Ech is rapidly inactivated by O 2 , several precautions were taken to avoid the introduction of O 2 into the titration cell. First, the cell was flushed with 100% (v/v) H 2 (freed from traces of O 2 by passing through a column with a Pd catalyst; Degussa, type E236P). There- after, a solution of Ech (incubated under 100% H 2 )was transferred anaerobically into the titration cell. Two types of titrations were performed, one in the presence of redox dyes and one in the absence of these dyes. In both cases the cell was continuously flushed with a water-saturated mixture of H 2 and He, used to adjust the redox potential in the system. The home-built H 2 /He mixer produced mixtures from 0.1% to 100% (v/v) H 2 [27]. In this system the potential values read from the Pt electrode were within 10 mV of the theoretical redox potentials calculated from the gas mixture using the formula: E h ¼À RT F log e pH À RT 2F log e log P H 2 where R is the gas constant, F is the Faraday constant and T is the temperature in Kelvin. Fig. 1. Schematic representation of the possible organization of the subunits of Ech in membranes from M. barkeri. The Ni–Fe active site in the EchE subunit together with the proximal cluster located in EchC subunit form the centre for hydrogen production. Two transmembrane proteins EchA and EchB are supposed to be involved in the transfer of protons across the membrane. The two [4Fe)4S] clusters in subunit EchF, which is related to the TYKY subunit in bovine complex I, have been suggested to be involved in proton translocation coupled to electron transfer [23]. 6102 S. Kurkin et al. (Eur. J. Biochem. 269) Ó FEBS 2002 In redox titrations in the presence of mediating dyes the following dyes were present in a final concentration of 50 l M : 2,3,5,6-tetramethyl-p-phenylendiamine dihydrochlo- ride (E 0 ¢ ¼ +275 mV), 2,6-dichlorophenol-indophenol (E 0 ¢ ¼ +230 mV), 1,2-naphtoquinone-4-sulfonic acid (E 0 ¢ ¼ +215 mV), phenazine methosulfate (E 0 ¢ ¼ +80 mV), 1,4-naphtoquinone (E 0 ¢ ¼ +36 mV), methylene blue (E 0 ¢ ¼ +11 mV), duroquinone (E 0 ¢(1,2) ¼ ) 5/ +35 mV), indigodisulfonate (indigo carmine; E 0 ¢ ¼ )125 mV), 2-hydroxy-1,4-naphtoquinone (E 0 ¢(1,2) ¼ )139/)152 (mV), lapachol (E 0 ¢ ¼ )179 mV), antraqui- none-2-sulfonate (E 0 ¢ ¼ )225 mV), safranin T (E 0 ¢ ¼ )289 mV), benzyl viologen (E 0 ¢ ¼ )358 mV) and methyl viologen (E 0 ¢ ¼ )449 mV). All redox potentials are given at pH 7. As some of the redox dyes have a pH-dependent redox potential, these values are not valid for the titrations performed at pH 6 or pH 8. However the mixture of these dyes still covers the whole redox-potential range at pH 6 or pH 8. Also in this case the redox potentials were set by aH 2 /He gas mixture. As this limits the potential range, potentials higher than that of 0.1% (v/v) H 2 were achieved by addition of aliquots of potassium ferricyanide (250 m M ) as oxidizing agent or, to bring the potential down again, by aliquots of a solution of sodium dithionite (100 m M ) as reducing agent. After stabilization of the redox potential, samples were withdrawn with a gas-tight syringe through a suba-seal rubber stopper and injected into EPR tubes. The tubes, sealed with latex tubing, were preflushed with the same gas (mixture) of the titration cell. After filling, the tubes were rapidly frozen by immersion in cold isopentane (133 K). EPR measurements EPR spectra at X-band (9 GHz) were obtained with a Bruker ECS 106 EPR spectrometer with a field-modula- tion frequency of 100 kHz. Cooling of the sample was attained with an Oxford Instruments ESR 900 cryostat with a ITC4 temperature controller. The sample-tempera- ture indication from this instrument was correct from 4.2 K to 100 K within ± 2% as ascertained from the Curie dependence of a copper standard (10 m M CuSO 4 Æ5- H 2 O, 2 M NaClO 4 ,10m M HCl). The magnetic field was calibrated with an AEG Magnetic Field Meter. The X-band frequency was measured with a HP 5350B microwave frequency counter. The microwave power incident to the cavity was measured with a HP 432B power meter and was 260 mW at 0 dB. Simulations were performed as described [28]. Quantification of EPR signals was carried out by direct double integration of the experimental spectra [29,30] or by comparison with a good-fitting simulation. Analysis of titration data The midpoint potentials of the Fe–S clusters were estimated using the amplitudes in the EPR spectra at two different g-values: for one signal (here termed the Ôg ¼ 1.92Õ signal) the peak at g ¼ 1.947 (see Fig. 3, trace A) was used; for a second signal (Ôg ¼ 1.89Õ signal) the amplitude of the trough at g ¼ 1.88 was taken. The amplitudes were plotted against the applied potential and each data set was then fitted to the Nernst equation: E h ¼ E 0 0 þð59=nÞ: log [ox]=[red] where E 0 ¢ is the midpoint potential in mV at the pH used, E h is the applied potential, n is the number of electrons involved in the redox reaction. IGOR PRO software (WaveMetrics, Inc.) was used for the curve-fitting analysis. Quantification of the EPR signal to obtain the total concentration of Fe–S clusters was performed with the samples obtained under 100% H 2 at pH 8 in the absence of redox mediators. The Ni content of the enzyme, determined by Atomic Absorption Spectroscopy, was used as the basis for the enzyme concentration. Metal content determination Nickel was determined with an Hitachi 180-80 polarized Zeeman Atomic Absorption spectrophotometer using either internal standards or a standard series. The enzyme concentrations, calculated on basis of a protein determin- ation with the Bradford method assuming molecular mass of 180 kDa, correlated well with the values based on the Ni contents. RESULTS EPR properties of Fe–S clusters in Ech EPR spectra of purified Ech. A sample of the purified enzyme equilibrated with 4% (v/v) H 2 ,eitherinMes buffer at pH 6.0, or in Mops buffer at pH 7.0, showed signals only in the g ¼ 2.3 to g ¼ 1.8 region, apart from a small g ¼ 4.3 signal due to high-spin 3d 5 metal ions in a rhombic ligand field (usually adventitious Fe 3+ ). From the temperature dependence of the signals in the g ¼ 2 region for fully reduced enzyme under 100% H 2 (Fig. 2, left panel), it is concluded that the spectrum is due to at least two, possibly three, different signals of reduced [4Fe)4S] clusters. All signals broadened considerably above 17 K. Below 30 K one signal was optimally sharpened at 17 K. It has a trough around g ¼ 1.921 andistermedhereastheÔg ¼ 1.92 signalÕ.Itsg z value is at 2.050. The second major signal only sharpened optimally at 12 K and has a trough at g ¼ 1.887 (termed the Ôg ¼ 1.89 signalÕ). Its g z value is at 2.078. At 17 K and lower, there was also a clear shoulder (peak) detectable around g ¼ 1.959. As in redox titrations (see below) this signal behaved independently of the other two signals, it is termed the Ôg ¼ 1.96 signalÕ. At this point it is unclear where the g z and g x lines of this signal are. No additional signals were observed down to 4.2 K. At 70 K a minute signal could be observed (at a larger magnification) with a major line around g ¼ 2.3, which is reminiscent of Co 2+ in methyltransferase [31]. This signal was also observed in membranes of M. barkeri (see below). At pH 7.0 the Fe–S signals had about twice the intensity of that found at pH 6.0; the overall line shape of the spectrum was the same at both pH values. Direct double integration of the Fe–S signals at 12 K at pH 7 amounted to a total spin concentration of % 51 l M ; the enzyme concentration was 25 l M . As the amino-acid sequence of Ech points to the presence of three [4Fe)4S] clusters, the sample was apparently only partially reduced under the conditions used (100% (v/v) H 2 at pH 7.0). Ó FEBS 2002 The membrane-bound [NiFe]-hydrogenase (Ech) from M. barkeri (Eur. J. Biochem. 269) 6103 To a first approximation, the spectrum of Ech under 4% (v/v) H 2 at pH 7.0 could be simulated rather well on the basis of the two main components mentioned above (Fig. 2, right panel). Using the simulated spectra, it could be calculated that the relative spin concentrations of the g ¼ 1.89 signal was % 1.6 times that of the g ¼ 1.92 signal. In addition, a rather isotropic signal at g ¼ 2.03 was apparent, especially at higher temperatures, where the other signals broadened. The line shape and the temperature dependence of the signal indicate a free radical. Its g-value, however, indicates that the radical cannot be a truly ÔfreeÕ electron (with a g-value close to the free-electron value). We also noticed that this signal could not be saturated at 4.2 K and full microwave power. This suggests that it might be due to a radical close to a very rapidly relaxing paramagnet, e.g. high-spin Fe 2+ . Another method to obtain a rough impression of the line shape of two overlapping signals with different relaxation rates is the one described by Hagen and Albracht [32]. By setting the observing amplifier around 90° out of phase, while partly saturating the signals with a suitable microwave power at a suitable temperature, first one and then, at a slightly different phase, the other signal could be virtually eliminated from the spectrum. This is demonstrated in Fig. 3. The g ¼ 1.92 signal (trace B) shows an apparent g z at 2.05. The perpendicular region (g xy between 1.90 and 1.97) shows more structure than assumed in the simulation of Fig. 2. Also the peak at g ¼ 1.96 is clearly detectable, as well as a g z -like peak at 2.01. We tentatively conclude that the spectrum represents an overlap of two different signals, i.e. the g ¼ 1.92 signal and the g ¼ 1.96 signal. The g ¼ 1.89 signal (trace C) apparently has its g z value around 2.07 (top), while g xy line has a trough at g ¼ 1.89. This agrees with the interpretation shown in Fig. 2. The radical- like signal at g ¼ 2.03 is present in trace C, but not in trace B. This indicates that the species causing it has a relaxation rate at 10 K which is of the same order of magnitude as that of the g ¼ 1.92 species. We also note that the g-values and the temperature dependence of the g ¼ 1.92 signal are similar to those of the clusters N-2 in bovine-heart complex I [21,33]. As the g ¼ 1.89 signal appears to interact with the observed Ni a –L* signal (see below), it is concluded that this signal is presumably due to the proximal cluster in the EchC subunit and so the remaining Fe–S signals present in the spectrum at 17 K are ascribed to the [4Fe)4S] clusters in the EchF subunit. Two of the subunits of Ech, EchE and EchC, bear a large resemblance to the large and the small subunits, respect- ively, of [NiFe]-hydrogenases, suggesting the presence of a Fig. 2. Temperature dependence of the Fe–S signals of purified Ech reducedwith101kPaH 2 (left panel) and a simulation of the 12 K EPR spectrum (right panel). Spectra were recorded at nonsaturating microwave powers and replotted normalized for microwave frequency, microwave power, temperature and receiver gain; hence they can be quantitatively compared. EPR conditions: microwave frequency, 9416.2 MHz; microwave powers incident to the cavity, 50, 40, 30, 30, 30, 20, 20 dB for spectra from top to bottom (0 dB ¼ 260 mW); modulation amplitude, 1.27 mT; the temperature is indi- cated for each spectrum. In the right panel the following spectra are presented: (A) Experimental spectrum of Ech dissolved in Mops buffer pH 7.0 under 4% H 2 and further reduced with a few grains of solid dithionite. EPR conditions: microwave frequency, 9415.8 MHz; microwave power, 30 dB; modulation amplitude, 0.64 mT; tempera- ture, 12 K. (B) Simulation of the g ¼ 1.89 signal with parameters g xyz ¼ 1.88391, 1.90223, 2.06977 and widths (xyz) ¼ 5.2, 3.7, 6.0 mT. (C) Difference spectrum A minus B. This difference spectrum was used to fit the remaining signal (g ¼ 1.92 signal). (D) Simulation of the g ¼ 1.92 signal (trace C) with parameters g xyz ¼ 1.91821, 1.93799, 2.04721 and width (xyz) ¼ 2.77, 2.70, 2.66 mT. (E) Difference spec- trum C minus D. Fig. 3. Three EPR signals that can be detected in the spectrum of purifiedEchreducedwith101kPaH 2 at pH 8.0 by varying the detecting phase of the amplifier. (A) Normal EPR spectrum. (B) Approximate line shape of the g ¼ 1.92 plus g ¼ 1.96 signal obtained by using an amplifier phase to minimize the g ¼ 1.89 signal. (C) Approximate line shape of the g ¼ 1.89 signal obtained by using an amplifier phase to minimize the g ¼ 1.92 signal. EPR conditions: microwave frequency, 9426.6 MHz; microwave power, 10 dB; modulation amplitude, 1.27 mT; temperature, 10 K. 6104 S. Kurkin et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Ni–Fe active site. Hence, under 4% (v/v) H 2 an EPR signal due to the Ni a –C* state (usually with g xyz ¼ 2.20, 2.15, 2.01) is expected, as apparent in many other [NiFe]-hydrogenases under that H 2 -partial pressure. No such signal was observed, however (data not shown). Even minute signals due to Ni a –C* can usually be detected in a background of large overlapping signals, by making use of its light sensitivity [34]. Hence a sample was illuminated for 25 min at 45 K. Curiously, a difference spectrum of light minus dark showed only the induction of a signal typical for the Ni a –L* state (g xyz ¼ 2.0486, 2.101, 2.270), but no disappearance of its expected parent Ni a –C* signal could be detected (Fig. 4). The signal, which could be readily simulated, amounted to a concentration of only 1.1 l M ;it could be clearly seen in the spectrum, however, due to its sharp lines. When studied below 15 K, a clear two-fold splitting of the g y and g x lines, but not of the g z line, was apparent (A x ¼ 3.9 mT, A y ¼ 5.2 mT). The splitting was blurred at 15 K and was not apparent at 20 K or higher temperatures (Fig. 4). This temperature dependence paral- lels the temperature dependence of the g ¼ 1.89 signal. In [NiFe]-hydrogenases the Ni a –L* EPR signal shows a small splitting due to interaction of the Ni-based unpaired electron with the S ¼ 1/2 system of the reduced proximal [4Fe)4S] cluster signal [35–37]. It is therefore tentatively concluded that the g ¼ 1.89 signal is due to the reduced proximal cluster in Ech. Usually, in [NiFe]-hydrogenases containing a [3Fe)4S] cluster, the relaxation of the proximal cluster is very much enhanced by coupling to the nearby S ¼ 2 system of the reduced [3Fe)4S] cluster. As a result the effective relaxation of the proximal cluster Ôcools downÕ only at temperatures below 7 K, and it is only then when the Ni a –C* and Ni a –L* signals in these enzymes show the twofold splitting. Ech does not contain a 3Fe cluster and therefore the proximal cluster has relaxation properties normally associated with [4Fe)4S] clusters. Oxidation of the sample by stirring with air for a few minutes resulted in the complete disappearance of the signals due to Co 2+ and the reduced Fe–S clusters. Only traces of lines reminiscent of the EPR signals of the Ni r * state (observed here around g x ¼ 2.31 and g y ¼ 2.178) and the Ni u * state (observed here at g x ¼ 2.28 and g y ¼ 2.24) appeared (data not shown). There was only a trace of a signal reminiscent of that of an oxidized [3Fe)4S] cluster and it is concluded that Ech does not contain a [3Fe)4S] cluster. EPR spectra of membranes of M. barkeri. As Ech (a membrane-bound protein) constitutes up to 3% of the total cell protein, we have also inspected membranes of M. bark- eri with EPR. Initial EPR measurements failed due to the presence of large signals from Mn 2+ , which is added to the growth medium. Omission of Mn 2+ from the medium did not result in any noticeable changes in the growth or specific activity of Ech in the membranes, and now only trace signals due to Mn 2+ remained. Wide-scan spectra (600 mT) at 12 K of reduced membranes thus obtained revealed only three main lines (Fig. 5). The line around g ¼ 2.3 is due to the g xy lines of the Co 2+ , presumably from a membrane- bound methyltransferase. The other two lines (around g ¼ 2.05 and g ¼ 1.9) are due to reduced [4Fe)4S] clusters. As signals from [4Fe)4S] clusters usually disappear at 45 K due to relaxation broadening, whereas the Co 2+ signal does not (Fig. 5, trace B), a difference spectrum (Fig. 5, trace C) reveals the spectrum of these clusters only. This is shown in more detail in the right panel of Fig. 5. A difference of the spectra at 12 K and 45 K shows similarities to the spectrum of the purified Ech in Fig. 5, right panel. As with the purified enzyme, no additional signals could be observed in the membranes between 12 K and 4.2 K. Membranes of M. barkeri contain additional metallo- proteins, like heterodisulphide reductase (Hdr), with Fe–S clusters showing EPR signals in the same region [38] and a b-type cytochrome [39], methanophenazin-reducing (F 420 - nonreducing) [NiFe]-hydrogenases (VhoGAC and Vht- GAC) [40], as well as a methyltransferease. The Vho, but not the Vht hydrogenase, is present in high amounts in acetate-grown cells. Upon cell lysis, however, Vho hydro- genase loses its contact to the b-type cytochrome, which anchors this enzyme in the membrane. The amount of the other hydrogenases (which only reduce dyes but not ferredoxin) was estimated by activity measurements. Based on these determinations it can be concluded that the amount Fig. 4. Temperature dependence of the splitting of the Ni a –L* signal. An EPR tube with Ech (in Mops buffer pH 7.0 under 4% H 2 )wasfrozen in liquid nitrogen and then kept in the dark at 200 K for 10 min. A spectrum was recorded at the indicated temperatures and then the sample was illuminated for 25 min in the EPR cavity at 45 K [34]. After switching off the light, a second set of spectra was recorded under identical conditions. The difference spectra light minus dark are plotted in the figure. EPR conditions: microwave frequency, 9415.5 MHz; microwave power, 20dB (70K, 45K),30dB (20K, 15K, 12K, 10 K) or 10 dB for the bottom spectrum; modulation amplitude, 1.27 mT. Spectra A–F were normalized for temperature, microwave power and gain. Vertical dashed lines indicate the positions of the g y and g x lines. Ó FEBS 2002 The membrane-bound [NiFe]-hydrogenase (Ech) from M. barkeri (Eur. J. Biochem. 269) 6105 of Vho and Vht hydrogenases in washed membranes is very low. When membranes were oxidized with air, the signals of Co 2+ and of the reduced Fe–S clusters disappeared and now two main signals dominated the spectrum (data not shown). They were recognized as an Ni u * signal, typical for [NiFe]-hydrogenases in the oxidized, unready state, and a peculiar signal earlier encountered by us in purified, oxidized preparations of Hdr in the presence of H-S-CoM (g xyz ¼ 2.013, 1.991, 1.938) from M. barkeri and Metha- nothermobacter marburgensis [38]. Both signals could be readily simulated, showing that the remainder of the spectrum consisted of small signals presumably due to an oxidized, low-spin cytochrome (with g z ¼ 2.32) and some contaminating Mn 2+ . No trace of a signal due to the Ni r * state (with g z ¼ 2.31 and g y ¼ 2.16) could be spotted. The simulations enabled quantification of the signals. The spin concentration represented by the Ni u * signal amounted to 11.6 l M ; the Hdr signal was calculated to represent a spin concentration of 16.3 l M . This indicates that the EPR signals of the reduced Fe–S clusters from Hdr, which have similar g-values [38], heavily interfere with those of Ech in the used membranes and hence these membranes are not suited for the study of the Fe–S clusters of Ech. As the membranes contain only very low amounts of other hydrogenases, the Ni u * signal is considered to be due to Ech only; its concentration was at least 2.1 g pure enzyme proteinÆL )1 . Estimating the total protein concentration to be % 80 gÆL )1 , this is about 2.7% of the total membrane protein. Redox titration in the absence of redox dyes at pH 6.5, 7 and 8 Because in some cases redox dyes have been shown to change the redox properties of [NiFe]-hydrogenases we performed redox titrations in the absence of redox dyes. These titrations were performed at three different pH values (6.5, 7 and 8) using pure Ech preparations at 25 °C. As an example of the spectral changes we have compiled spectra obtained at pH 8 and pH 7 (Fig. 6). The enzyme was first incubated in the titration cell under 100% H 2 .AtallpH values the lines at g ¼ 2.05, 1.92, 2.065 and 1.89 were decreasing in amplitude with increasing potential. The ratio between the g ¼ 1.92 signal and the g ¼ 1.89 signal was different at different pH values. The g ¼ 1.89 signal was more pronounced at pH 6.5 and 7. At pH 8 a small apparent shift or the line at g z ¼ 2.05 to smaller values was noticed when the potential was increasing. The amplitude changes of the g ¼ 1.96 line were accompanied by the changes of a ÔkinkÕ in the 1.92 line suggesting a contribution of the g ¼ 1.96 signal at that position, as also suggested by trace B in Fig. 3. The peak at g ¼ 2.01 was present in titrations at all three pH values and disappeared with increasing potential. At pH 8 it was no longer detectable at H 2 concentrations < 10%. This signal was less pronounced at pH 7 and 6.5. The spin concentrations, estimated for enzyme under 100% (v/v) H 2 , were 45, 65 and 56 l M at pH 6.5, 7 and 8, respectively. As the enzyme concentration used for all titrations was 25 l M , the amount of spins per enzyme molecule represented by the Fe–S signals was 1.8, 2.6 and 2.24 for pH 6.5, 7 and 8, respectively. At all three pH values a plot of the amplitude of the g ¼ 1.92 and g ¼ 1.89 signals fitted best to n ¼ 2Nernst curves (Fig. 7). The amplitudes at pH 6.5 were smaller and the data were rather scattered; hence the estimated E 0 ¢ values are less reliable, while the n-values could not be Fig. 5. Wide-scan EPR spectra of membranes of M. barkeri (left panel) and details of the g = 2region(rightpanel).Membranes from acetate- grown cells were prepared as described in Materials and methods and equilibrated with 100% H 2 before freezing in liquid nitrogen. Spectra were recorded between 5 and 605 mT (left panel) and a scan range of only 80 mT was used in the g ¼ 2 region (right panel). Left panel: (A) spectrum at 12 K; (B) spectrum at 45 K; spectra were normalized for microwave frequency, microwave power, temperature and receiver gain; (C) difference A minus B. Right panel: (A) spectrum at 12 K; (B) spectrum at 45 K; spectra were normalized for microwave frequency, microwave power, temperature and receiver gain; (C) difference A minus B. (D) Spectrum of the purified enzyme at pH 7.0 under 4% (v/v) H 2 . EPR conditions: microwave frequency, 9416.5 MHz; microwave power, 40 dB for A and D, 30 dB for B; modulation am- plitude, 1.27 mT (left panel) and 0.64 mT (right panel); temperatures are indicated in the figure. Fig. 6. EPR spectra of samples from a titration of Ech hydrogenase at pH 8 (left panel) and at pH 7 (right panel) with H 2 /He mixtures (in the absence of redox dyes). EPR conditions: microwave frequency, 9460 MHz; microwave power, 30 dB; modulation amplitude, 1.27 mT; temperature, 12 K. All spectra are normalized for the gain,the tube-calibration factor, and the microwave frequency and hence they can be directly compared. The redox potentials are indi- cated in the figure. 6106 S. Kurkin et al. (Eur. J. Biochem. 269) Ó FEBS 2002 determined. The midpoint potentials of the two signals obtained at all three pH values are summarized in Table 1. For both signals there was a pH dependence of )38 to –50 mVÆpH unit )1 . Redox titrations in the presence of redox dyes at pH 6, 6.5, 7 and 8 EPR spectra under 101 kPa H 2 at different pH values in the presence of redox dyes. The titrations at all four pH values were started with 100% H 2 -reduced enzyme, which was transferred anaerobically to the titration vessel under a continuous flow of O 2 -free H 2 . Hence, the starting redox potential was that of the hydrogen potential at each pH value. Spectra taken under these conditions are summarized in Fig. 8, left panel. Comparison of the four spectra shows that the degree of reduction diminished with decreasing pH. The spin concentrations obtained by direct double integra- tion showed that at pH 6 the intensity was only % 30% of that at pH 8. There were also clear changes in the overall line shapes of the spectra. At pH 6 two separate g z lines at 2.078 and 2.050 were observed (Fig. 8, left panel). At pH 6.5, the 2.05 line markedly increases together with the trough around g ¼ 1.92. This reinforces the earlier inter- pretation that these two lines form the g z and the g xy region of the g ¼ 1.92 signal, whereas the g z ¼ 2.078 and the trough at g ¼ 1.887 form the g z and g xy lines of the g ¼ 1.89 signal. At pH 7.0 both of the two g z lines as well as the two g xy lines increased noticeably. Also the g ¼ 1.96 signal could now be discerned as a shoulder. At pH 8.0 this shoulder at g ¼ 1.959 is much better defined and forms a separate peak. As the region between the two g z lines at 2.078 and 2.050 seems to Ôfill upÕ, one might conclude that this is caused by a g z line (around g ¼ 2.06) of the g ¼ 1.96 signal. Spectra encountered during the redox titrations (see below) made this interpretation less likely. At this point we tentatively conclude that the g ¼ 1.96 species has its g z line either at 2.06 or at 2.01. Comparison of the EPR spectra at different pH values at )340 mV. It is interesting to compare the EPR spectra at different pH values and the same potential ()340 mV). The comparison showed that the overall reduction level was roughly the same (Fig. 8, right panel) although there were clear spectral differences. At none of the pH values was any trace of the g ¼ 1.96 signal observed. The relative ratio of the other two signals was clearly dependent on the pH. At pH 6.0 the g ¼ 1.89 signal dominated the spectrum, while at pH 8.0 the g ¼ 1.92 signal was the most pronounced. The g ¼ 1.96 species apparently has a redox potential considerably lower than those of the g ¼ 1.92 and g ¼ 1.89 species (see below). Redox titrations results. The overall behaviour of the 2.05/ 1.92 lines of the g ¼ 1.92 signal and the 2.065/1.89 lines of the g ¼ 1.89 signal in the titrations in the presence of redox dyes was comparable to the titration in the absence of these dyes (Fig. 9). The g ¼ 2.01 signal found in the absence of dyes was not detectable in the EPR spectra in the presence of dyes as it was obscured by the strong radical signals round g ¼ 2.00, originating from the redox dyes. The spin concentrations estimated by double integration of the experimental EPR spectra of enzyme under 101 kPa H 2 were 0.51, 1.8, 2 and 1.9 spins per molecule at pH 6, 6.5, 7 and 8, respectively. These values are slightly overestimated due to the contribution of the radical signals. The amplitudes of the g ¼ 1.92 and g ¼ 1.89 signals changed with pH; they were smaller at lower pH values (see Table 1). This reflects the overall decrease in the level of reduction of the enzyme at lower pH values. The line at g ¼ 1.96 disappeared on shifting from pH 8 to pH 6, in line with the Fig. 7. Redox behaviour of the g = 1.92 and g = 1.89 signals in a titration in the absence of mediating dyes at pH 6.5, 7 and 8. The amplitudes (arbitrary units) of the g ¼ 1.92 signal (left panel) and the g ¼ 1.89 signal (right panel) are plotted against redox potential. Solid curves indicate theoretical Nernst lines with n ¼ 2. The estimated E 0 ¢ and n-values and the maximal amplitudes of the signals are listed in Table 1. Table 1. Summary of the redox properties of the Fe–S clusters in Ech as obtained from the redox titrations with H 2 /He mixtures in the presence and in the absence of the redox dyes. g ¼ 1.92 signal g ¼ 1.89 signal pH Dyes n-value Amplitude under 1 bar H 2 a E 0 ¢ (mV) n-value Amplitude under 1 bar H 2 a E 0 ¢ (mV) 6 + 2 0.29 ) 328 2 0.40 ) 323 6.5 + 2 0.57 ) 340 2 0.49 ) 343 7 + 2 1.13 ) 348 2 0.78 ) 352 8 + 1 1.00 ) 368 1 1.12 ) 413 6.5 – 2 0.60 ) 304 2 0.67 ) 337 7 – 2 1.04 ) 350 2 1.10 ) 360 8 – 2 0.87 ) 388 2 0.91 ) 410 a Arbitrary units. Ó FEBS 2002 The membrane-bound [NiFe]-hydrogenase (Ech) from M. barkeri (Eur. J. Biochem. 269) 6107 conclusions from the EPR spectra at )340 mV at different pH values (Fig. 8, right panel). Fig. 9 shows that the g ¼ 1.96 signal appeared only at the lowest potentials. Its E 0 ¢ value is estimated to be well below )420 mV. In all titrations, but especially in those at pH 6.0 and 6.5, weak signals due to Ni were observed at H 2 -partial pressures of £ 10%. The signals had the characteristic g-values of the Ni a –C* state (g xyz ¼ 2.21, 2.13, 2.01) and the light-induced Ni a –L* state (g xyz ¼ 2.05, 2.11, 2.3), as observed in other [NiFe]-hydrogenases [34]. The total spin concentration amounted to maximally 10% of the enzyme concentration. The data obtained in the presence of dyes (Fig. 10) were not as clear-cut as those obtained in the absence of dyes. At all pH values, except pH 8, the g ¼ 1.92 and 1.89 signals both titrated as n ¼ 2systems.AtpH8thebestfitwas obtained with n ¼ 1 and this result is different from the titration in the absence of dyes, where the best fit was obtained with n ¼ 2 Nernst curves. DISCUSSION Iron-sulphur clusters The best way to study membrane-bound enzymes, especially for those expected to pump protons, is to use intact membranes. As demonstrated, % 3% of the protein content of membrane preparations of M. barkeri consisted of Ech, but the concentration of Hdr was also quite high. This prevented a specific study of the Fe–S clusters in Ech. We therefore turned to the purified enzyme. From the EPR line shape and the temperature depend- ence of spectra from H 2 -reduced Ech, it can be concluded that signals due to three different S ¼ 1/2 species from reduced [4Fe)4S] clusters are present. We have labelled them as the g ¼ 1.92 signal, the g ¼ 1.89 signal and the g ¼ 1.96 signal. Only insignificant signals due to a [3Fe)4S] + cluster could be detected in air-oxidized enzyme. This result is in line with the presence of two four-Cys motifs for the binding of [4Fe)4S] clusters in the amino acid sequences of the EchF subunit and one such motif in the EchC subunit. It also is in good agreement with the content of Fe and acid-labile sulphur of the purified enzyme. The redox titrations indicated that the g ¼ 1.96 signal has the lowest redox potential (well below )420 mV at pH 7); therefore this cluster could only partly be reduced. This is in line with the maximal amount of spins detected in the spectra of the reduced Fe–S clusters (% 2–2.6 spins per enzyme molecule at pH 8). The temperature dependence of the splitting of the Ni a – L* signal paralleled the temperature dependence of the g ¼ 1.89 signal. We hence conclude that the unpaired spin located at the Ni site has magnetic interaction with the Fe–S cluster responsible for the g ¼ 1.89 signal. This indicates that this [4Fe)4S] cluster is the proximal cluster located in the EchC subunit. It then follows that the two [4Fe)4S] clusters causing the g ¼ 1.92 and g ¼ 1.96 signals are located in the EchF subunit. A major disadvantage of the use of redox mediators in redox titrations is that they sometimes dramatically change the redox properties of [NiFe]-hydrogenases [27,41]. The interaction of H 2 with hydrogenases offers the possibility to study redox changes in enzyme in the absence of redox mediators simply by varying the H 2 -partial pressure in a known mixture of H 2 and He. This method minimizes the possible artefacts introduced by redox dyes. This laboratory has used the method before for the hydrogenases from M. marburgensis and Allochromatium vinosum.Itwas Fig. 8. EPR spectra of Ech under 101 kPa H 2 in the presence of me- diating dyes at different pH values (left panel) and EPR spectra of Ech from titrations poised at )340 mV ± 5 mV (right panel). The measured potential at each pH value is given in the figure and this legend. The theoretical potential of 101 kPa H 2 isgiveninthislegendinparen- theses. Left panel: (A) pH 8, )463 mV ()472 mV); (B) pH 7, )405 mV ()413 mV); (C) pH 6.5, )383 mV ()383 mV); (D) pH 6, )360 mV ()360 mV). Right panel: (A) pH 8; (B) pH 7; (C) pH 6.5 and (D) pH 6. The EPR conditions were the same as in Fig. 6. Fig. 9. EPR spectra (Fe–S region) of Ech during redox titrations at potentials below )282 mV at pH 8 in the presence of redox dyes. EPR conditions were as in Fig. 6. All spectra are normalized for gain, tube factor and microwave frequency. 6108 S. Kurkin et al. (Eur. J. Biochem. 269) Ó FEBS 2002 observed that the presence of dyes had a major effect on the reversible redox transition between the Ni a –C* and the Ni a – SR states. The reaction was an n ¼ 1 transition involving one proton when performed with a H 2 /He mixture in the presence of redox dyes [27,41]. When the dyes were omitted, however, the reaction was found to be n ¼ 2 and involved two protons. In addition, in the absence of redox dyes, there was no redox equilibrium between the Ni a –S and Ni a –C* states. A limitation of the titrations in the absence of dyes is the limited potential range, which can be covered by the 2H + /H 2 couple. The maximal obtainable potential is approximately 120 mV above that of the hydrogen poten- tial at a given pH. In the redox titratations with Ech nearly all curves fitted best to n ¼ 2 Nernst lines. As all titrations were performed with H 2 /He mixtures, H 2 is directly involved in all reduction and oxidation reactions; hence n ¼ 2 lines are expected. There is a notable difference in the results of the redox titrations performed at pH 8: in the presence of dyes the curve fitted best to a n ¼ 1 Nernst line; when the dyes were omitted the reaction was found to be n ¼ 2. According to previous studies this could be due to the artefacts caused by the redox dyes. The titrations at different pH values using two different methods show that there is definite pH dependence of the midpoint potentials of the Fe–S clusters responsible for the g ¼ 1.92 and the g ¼ 1.89 signals (Fig. 11). This effect was best observed in the titrations in the absence of redox dyes at pH 8 and pH 7. For the g ¼ 1.92 signal the E 0 ¢ value decreased by 53 mV per pH unit; this value was 62 mV per pH unit for the g ¼ 1.89 signal. For the titrations in the presence of redox dyes these values were 20 mV and 45 mV per pH unit for the g ¼ 1.92 and g ¼ 1.89 signals, respectively. This pH dependence for the proximal cluster (g ¼ 1.89 signal) is in agreement with the pH dependence of the E 0 ¢ value of the proximal cluster in standard [NiFe]-hydro- genases [42]. The values obtained for both signals were reasonably close to the theoretical value of )59 mV per pH unit for a redox reaction involving a stoichiometric amount of elec- trons and protons. E 0 ¢ values with such a large pH dependency are rare for [4Fe)4S] clusters with a classical Cys coordination [43–45]. No firm conclusion is possible for the cluster causing the g ¼ 1.96 signal. The data indicate that the redox potential of this cluster is considerably lower than those of the other two clusters. The existence of two [4Fe)4S] clusters with different midpoint potentials in one polypeptide is not unprecedented. It was found in the ferredoxin of A. vinosum [46]. The g-values (g z ¼ 2.05 and g xy ¼ 1.92) and pH dependence of )53 mV per pH unit of the g ¼ 1.92 signal, ascribed to one of the [4Fe)4S] clusters in the EchF subunit, is reminiscent of the g-values (g z ¼ 2.054 and g xy ¼ 1.922) and the pH dependence of )60 mV per pH unit of the signal ascribed the cluster(s) N-2 of bovine complex I [22]. There is a debate in the literature as to the precise location of this cluster N-2 [21,23,47–50]. Ech contains only three [4Fe)4S] clusters and one of them (causing the g ¼ 1.89 signal) is close to the Ni–Fe site and thus located in the EchC subunit. Hence, in Ech the other two [4Fe)4S] clusters are in the EchF subunit which shows a very high amino acid sequence similarity to the TYKY subunit of the bovine complex I [23]. This strengthens our earlier suggestion [23] that the Fe–S clusters in these subunits might be involved in an electron- transfer driven proton-pumping unit. Further studies are required to verify this. The data presented are a good starting point towards an understanding of the behaviour of Fe–S clusters in proton-pumping [NiFe]-hydrogenases. Point mutations of amino acid residues close to the several Fe–S clusters can give more insight into the mechanism of action. At the same time the results obtained with Ech can be helpful to a better understanding of similar studies in the field of complex I. ACKNOWLEDGEMENTS S. P. J. Albracht is indebted to the Netherlands Organization for Scientific Research (NWO) for funding provided via the Section for Chemical Sciences. R. Hedderich acknowledges the Max-Planck- Gesellschaft, the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie for financial support. Fig. 10. Redox titrations of Ech in the presence of dyes and at different pH values. The amplitudes of the g ¼ 1.92 (left panel) and g ¼ 1.89 signals (right panel) were plotted against the redox potential. Solid curves represent Nernst curves with n ¼ 2 fitting to the data points at pH 6, 6.5 and pH 7. At pH 8 the best fit was obtained with an n ¼ 1curve.TheE 0 ¢ values are listed in Table 1. Fig. 11. Plots of the midpoint potentials (E 0 ¢) for both signals (g = 1.92 and g = 1.89) against the pH from the titration in the absence of dyes (left panel) and from the titration in the presence of dyes (right panel). ThevaluesusedarethoselistedinTable1. Ó FEBS 2002 The membrane-bound [NiFe]-hydrogenase (Ech) from M. barkeri (Eur. J. Biochem. 269) 6109 REFERENCES 1. Adams, M.W.W. & Mortenson, L.E. (1984) The physical and catalytic properties of hydrogenase II of Clostridium pasteurianum. A comparison with hydrogenase I. J. Biol. Chem. 259, 7045–7055. 2. Adams, M.W.W. 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