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Hydrogenase from the thermophilic bacterium Thermococcus stetteri : isolation and characterisation of EPR-detectable redox centres

Hydrogenase from the thermophilic bacterium Thermococcus stetteri : isolation and characterisation of EPR-detectable redox centres
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  ELSEVIER FEMS Microbiology Letters 142 (1996) 71-76 MICROBIOLOGY LETTERS Hydrogenase from the thermophilic bacterium Thermococcus stetteri: isolation and characterisation of EPR-detectable redox centres Nikolay A. Zorin a, Milagros Medina b, Margarita A. Pusheva c, Ivan N. Gogotov a, Richard Cammack by* ’ Institute of Soil Science and Photosynthesis, Russian Academy of Sciences, Pushchino, Moscow Region 142292, Russian Fe&ration b Centre for the Study of etals n Biology and Medicine, Division of Life Sciences, Kings College, London W 7AH, UK ’ Institute of Microbiology, Russian Academy of Sciences, Moscow 117811, Russian Fe&ration Received 25 March 1996; revised 4 June 1996; accepted 8 June 1996 bstract The hydrogenase of the extremely thermophilic archaebacterium Thermococcus stetteri has been isolated. The procedure involved four steps, the last stage being preparative electrophoresis, with an overall yield of 40%. The molecular mass was estimated to be approx. 106 kDa. Analysis for metal content found iron and nickel in the ratio 13: 1. Flavin was also detected in the enzyme and identified as FMN. The hydrogenase activity was relatively insensitive to inhibition by carbon monoxide, Ki 67 PM. This behaviour is indicative of a nickel-iron type hydrogenase. Electron paramagnetic resonance spectroscopy of the enzyme showed signals characteristic of iron-sulfur clusters. The spectra of the hydrogen-reduced T. stetteri hydrogenase, recorded at 70 K, indicated the presence of a single type of [2Fe-2S] cluster. Below 10 K, the spectra were complex, and indicated the presence of other iron-sulfur clusters with extremely fast electron-spin relaxation rates, interpreted as [4Fe-4s) clusters, and minor amounts of [3Fe-4S] clusters. These properties indicate that T. stetteri hydrogenase is related to the hydrogenases of Pyrococcw furiosus, a hyperthermophilic archaebacterium, and Alcaligenes eutrophus. Keywords: Thermococcus stetteri; Extreme thermophile; Hydrogenase; EPR spectroscopy; Iron-sulfur protein 1 Introduction The discovery of hyperthermophilic bacteria has stimulated considerable interest in applications of their enzymes [ 11. Thermococcus stetteri, srcinally isolated from hot springs in Kamchatka, is an anae- robic thermophilic archaebacterium which grows at * Corresponding author. Fax: +44 (171) 333 4500; E-mail: temperatures up to 100°C. This organism utilises peptides as carbon and energy source, and liberates excess reductant by reduction of elemental sulfur to H or protons to hydrogen [2]. It does not use hydrogen as an energy source. Hydrogenases are a class of enzymes that catalyse the reversible activation of molecular hydrogen. They have been isolated from different organisms, including methanogens, photosynthetic, sulfate-re- ducing, fermentative, aerobic hydrogen-oxidising 0378-1097/961 12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PZZSO378-1097(96)00245-5  72 N.A. Zorin et d. I FEMS Microbiology Letters 142 (1996) 71.-76 and anaerobic nitrogen-fixing bacteria [3,4]. Hydro- genases may be divided into two different classes on the basis of their metal contents. One group, [Fe]- hydrogenases, consists of enzymes possessing only iron-sulfur clusters, one of them being the ‘H’ clus- ter, the hydrogen-binding site [3]. Other hydrogen- ases (the [NiFe]-hydrogenases) contain nickel in ad- dition to iron. They are the most commonly found hydrogenases in many prokaryotic organisms [5]. The structure of the [NiFe]-hydrogenase from Desul- fovibrio gigas has been determined by crystallogra- phy [6]. It indicates the presence of a dinuclear centre of nickel and iron, believed to be the site at which hydrogen binds [7], Also present in the enzyme are three iron-sulfur clusters, (two [4Fe-4S] and one [3Fe-4S]), which serve as secondary electron carriers. A comparison of sequence homologies of other nick- el-containing hydrogenases with D. gigas hydrogen- ase indicates that the minimum common features are the nickel site and one [4Fe-4S] cluster [8]. Hydro- genases having different electron acceptors or donors may contain other iron-sulfur clusters and, in some cases, flavin. Thermophilic hydrogenases may find applications as components in solar energy conversion systems, as catalysts in production of fine chemicals or, in hy- drogen enzyme electrodes of biofuel cells, in energy transformations based on hydrogen [9]. Hydrogen- ases have been isolated from the extremely thermo- philic archaebacteria, Pyrococcus furiosus, Methano- coccus jannaschii and Pyrodictium brockii [lo-l 21. T. stetteri differs from P. iiriosus in that it uses peptides as carbon and energy source, producing hydrogen, carbon dioxide and organic acids as products, whereas P. furiosus uses carbohydrates [ 131. The hy- drogenase of T. stetteri has previously been shown to be a highly active soluble enzyme which catalysed the reduction of elemental sulfur, benzyl viologen, methyl viologen and methylene blue, in the presence of hydrogen [2]. The reduction of sulfur has also been shown in the hydrogenase of P. furiosus [14]. In T. stetteri the reaction was maximal at pH values between 6 and 9.2, with a specific activity of 408 umol min-’ (mg protein)-i at 85°C. The enzyme is catalytically active and stable up to 100°C [2]. This paper describes the purification and preliminary mo- lecular characterisation of hydrogenase from T. stet- teri. 2. Materials and methods 2 1 Cell growth T stetteri K-15 was grown at 85°C in a mineral medium containing elemental sulfur (1 g/l), peptone (5 g/l) and yeast extract (1 g/l) [2]. Cells were har- vested by centrifugation after 24 h of growth and stored at -80°C. 2.2. PuriJication procedure Cells (5 g wet weight) were thawed and suspended in 50 ml 20 mM Tris-HCl buffer, pH 8.0. The buffer was thoroughly degassed and flushed with oxygen- free argon. Hydrogenase was released from the cells by sonication at 4°C (22 kHz, 3x30 s). All subse- quent steps were carried out at room temperature. The homogenate was centrifuged for 30 min at 20 000 X g. The precipitate, containing unbroken cells and cell fragments, was discarded and the superna- tant, which is referred to as the cell-free extract, was loaded into a DEAE-cellulose (Whatman DE-52) column (1 x 10 cm), previously equilibrated with 50 mM Tris-HCl buffer, pH 8.0. The retained proteins, including hydrogenase, were eluted with a linear gra- dient (&0.5 M) of NaCl. The fractions containing hydrogenase activity were pooled and applied to a phenyl-Sepharose CL4B column (1 X 20 cm), pre- viously equilibrated with 0.8 M (NH&S04 in buffer. The column was washed with 0.4 M (NH&S04 and hydrogenase was eluted with a reverse gradient (0.4 0 M) of (NH4)sS04. The hydrogenase-containing fraction was further purified by preparative electro- phoresis under non-denaturing conditions on a 10% polyacrylamide gel. The band containing hydroge- nase activity was cut out from the gel, and the en- zyme was eluted in 20 mM Tris-HCl buffer, pH 8.0 and concentrated by ultrafiltration through Centri- con 30 microconcentrators (Amicon). The purified protein was stored either in the frozen state at -20°C or at room temperature in the presence of 0.02% azide. 2.3. General methods The hydrogen uptake activity of the purified en- zyme was measured spectrophotometrically by fol-  N.A. Zorin et al. IFEMS Microbiology Letters 142 1996) 71-76 73 lowing the reduction of methyl viologen (1 mM) in 50 mM Tris-HCl buffer, pH 8.0 [15]. The serum- stoppered cuvettes were flushed with hydrogen gas, or mixtures of hydrogen with carbon monoxide (CO) prior to sample addition. One unit of hydrogenase activity is the amount of enzyme which catalyses the oxidation of 1 umol of hydrogen per minute at 40°C. Quantitative protein determinations were performed by the Lowry procedure using bovine serum albumin as standard. The relative molecular mass was esti- mated from the protein mobility on a lO-15% gra- dient native gel relative to the molecular mass mark- ers from Pharmacia. 2.4. Identljkation of lavin In order to study the possible presence of a flavin group, the protein was precipitated with trichloro- acetic acid as previously described [16]. The presence of a flavin group in the supernatant was detected by optical absorption and by fluorescence. For the iden- tification of the flavin, neutralised trichloroacetic acid supernatants were separated by HPLC with a Kontron system liquid chromatograph using an Aquapore AX-300 Cl8 7 u column (250 mmX 7 mm) in 0.1 M ammonium acetate:methanol. Detec- tion was made with excitation at 450 nm and emis- sion of fluorescence at 530 nm. Riboflavin, FMN and FAD standards (Sigma) were used to calibrate the column. 2.5. Metal analysis Analysis of iron and nickel was carried out on a Varian Zeeman atomic absorption spectrophoto- meter with an HGA-400 graphite tube analyser. Table 1 Purification of T. stetteri hydrogenase 2.6. Electron paramagnetic resonance EPR) measurements The enzyme was transferred to 50 mM MES buf- fer, pH 6.5 by diluting oxidised enzyme in the buffer and reconcentrating on Centricon 30 microconcen- trators (Amicon), at 4°C. Hydrogenase was activated in an EPR tube by reduction under oxygen-free, water-saturated hydrogen gas in a water bath at 60°C and pH 6.5 (50 mM MES) for 2 h. The EPR tubes were frozen and stored in liquid nitrogen until use. EPR spectra were recorded on a Bruker ESP300 spectrometer with an Oxford Instruments ESR900 helium flow cryostat. The spin concentration was determined by double integration, using 1 mM Cu”-EDTA as standard. EPR spectra were simu- lated by the program ‘EPR’ written by Dr F. Neese, University of Konstanz. 3. Results 3.1. Activity and molecular properties of T. stetteri hydrogenase Hydrogenase was purified from the soluble frac- tion of the T. stetteri cells by the method described above, which employed hydrophobic interaction chromatography on phenyl-Sepharose CL4B. The overall purification was 35-fold with a 40% yield of enzyme activity (Table 1). The purified enzyme proved difficult to denature and so SDS-electropho- resis for estimation of the &Z, was not feasible. The approximate molecular mass was estimated by com- parison with protein standards on a polyacrylamide gradient gel as 106 kDa. Purification step Protein (mg) Specific activity Total activity (U) Yield, % Purification Wmg) (fold) Cell-free extract 140 12 1736 100 1 DEAE-cellulose 18.4 74 1370 79 6 Phenyl-sepharose 4.2 282 1184 68 23 CL-4B Preparative electro- 1.6 430 688 40 35 phoresis Activity was measured by hydrogen uptake with methyl viologen as electron acceptor.  74 N. A. Zorin et al. I FEMS Microbiology Letters 142 (1996) 71-76 3.2. Metal andflavin content Analysis of a sample of the enzyme by atomic absorption spectrophotometry yielded iron and nick- el. The ratio was 13 g atom Fe:1 g atom Ni. The absorption spectrum was typical of iron-sulfur pro- teins having [4Fe-4S] clusters, with a maximum at 280 nm, and a shoulder in the 400-470 nm region. Upon sodium dithionite addition, a 25% decrease in A4c0 was observed. The possible presence of flavins linked non-covalently to the protein was investi- gated. Because of the strong, broad absorption bands of the clusters the spectrum of any flavin would be obscured. Non-covalently bound groups were extracted by precipitating the apoprotein frac- tion with trichloroacetic acid. The presence of a fla- vin cofactor was detected by its characteristic ab- sorption peaks at 370 and 450 nm and fluorescence maximum in emission at 530 nm when excited at 450 nm. The flavin group was identified as FMN by HPLC. 3.3. Kinetic properties T. stetteri hydrogenase showed activity at tem- peratures between 30 and 110°C. The specific activity of the purified enzyme was relatively low at tempera- tures below 50°C and increased 50 times upon ele- vation of the temperature from 40°C to lOO”C, reaching 20000 umol Hz consumed min-’ (mg protein)-l at 98°C with methyl viologen as electron acceptor. No activity was detected with NAD or NADP as electron acceptor. At 90°C the time re- quired for the enzyme to lose 50% of its activity was 115 min. By varying the concentration in the gas mixture with which the assay solution was equili- brated the K,, for hydrogen was estimated to be 23 uM. The inhibition of T. stetteri hydrogenase by carbon monoxide was determined from the rate of methyl viologen reduction as a function of carbon monoxide and hydrogen concentrations. The concen- tration of gases was adjusted by adding calculated volumes of carbon monoxide through a gas-tight syringe. The rates were extrapolated to infinite hy- drogen concentration, and plotted as a Dixon plot, l/V vs CO concentration (not shown), from which the Ki for carbon monoxide was estimated to be 67 PM. This value indicates that T. stetteri hydrogenase is relatively insensitive to this inhibitor. The effect of carbon monoxide on hydrogenase activity was fully reversible. This behaviour is untypical of the [Fe]- hydrogenases, which require 0.1-2 uM CO for 50% inhibition [3]. The [NiFe]-hydrogenases are less sen- sitive to CO, some of them not being significantly inhibited even at saturating concentrations of carbon monoxide [ 171. 3.4. EPR characterisation of hydrogenase from T. stetteri EPR spectroscopy has proved to be a discriminat- ing technique for the characterisation of the metal centres in hydrogenases [3,5,18]. The iron-sulfur and nickel centres in hydrogenases have distinct EPR spectra, which are useful in distinguishing be- tween the different types of enzymes. As isolated, T. stetteri hydrogenase exhibited a nearly isotropic EPR signal in the g = 2.02 region (Fig. 1). Upon reduction under hydrogen atmosphere, or with sodium dithio- nite, this signal disappeared. The shape of this signal is characterised by g factors centred around 2.02 and a particularly broad line width in the high field re- gion. This is typical of oxidised [3Fe-4S] clusters of the type known to occur in D. gigas hydrogenase [6,18]. However it is also known that [4Fe-4S] clus- ters can be converted into [3Fe-4S] clusters under oxidising conditions [19]. In T. stetteri hydrogenase the spin concentration of the g = 2.02 signal was ap- proximately 10% of the intensity of the signals ob- served in the reduced state, indicating that it is a minority species. The intensity doubled after reduc- tion under hydrogen atmosphere and reoxidation with air. Similar signals, in variable and substoichio- metric amounts, have been reported for a number of different hydrogenases, including the [NiFe]-hydro- genase from Alcaligenes eutrophus [17]. It is probable that the signal represents a degradative artefact due to the oxidation of a [4Fe-4S] cluster. After incubation under hydrogen at 60°C a sharp rhombic EPR signal with resonances at g= 2.03, 1.936 and 1.92 appeared (Fig. lb). This signal could be observed at temperatures below 80 K. The g-fac- tors, line shape, and temperature dependence of the observed EPR signal indicate the presence of a sin- gle, reduced [2Fe-2S] cluster. A much more complex spectrum was observed below 15 K, conditions un-  N.A. Zorin t al. FEMS Microbiology Letters 142 1996) 71-76 75 g factor 2.2 2.1 2.0 1.9 1.8 { / 1 I I I I I I 300 320 340 360 380 MAGNETIC FIELD mT) Fig. 1. EPR spectra of T. stetteri hydrogenase, in 20 mM MES, pH 6.5. The dotted lines are simulations. (a) Oxidized state, as prepared, recorded at temperature 10 K; (b) reduced under hy- drogen gas, and then flushed with argon for 40 mm at 2YC, re- corded at 30 K; (c) as (b), spectrum recorded at 10 K. Other EPR conditions: microwave power 2 mW; microwave frequency 9.36 MHz; magnetic field modulation amplitude 1.0 mT, modu- lation frequency 100 kHz. der which EPR absorption from reduced [4Fe-4S] clusters can be seen. This spectrum had features at g= 1.88, 1.965, 1.99, 2.056 and 2.14 (Fig. lc). The temperature dependence and complexity of the EPR spectrum at 10 K suggest the presence of at least two iron-sulfur clusters with very fast spin re- laxation rates. Reduction by dithionite produced spectra similar to Fig. 1 b, but more intense. Spectra of this type, with similar relaxation properties, have also been reported for the iron-sulfur clusters of hy- drogenase from P. furiosus [lo]. Some of the hydro- gen-activating sites of hydrogenases may be EPR-de- tectable, but not in all cases. WiFeI-hydrogenases yield a number of different EPR signals due to nick- el, either their oxidised states (Ni-A, N&B) or upon reduction with hydrogen (Ni-C) [5]. [Fel-hydrogen- ases yield a signal from the ‘H-cluster’, with g-fac- tors 2.00-2.10 [3]. For T. stetteri hydrogenase, upon progressive incubation of the reduced enzyme under argon atmosphere, the EPR signals from the reduced iron-sulfur clusters disappeared, giving an EPR-silent state. Under these conditions, H+ was presumably acting as oxidant, being converted to Hz. No evi- dence was detected of EPR signals either from nickel or from an H-cluster, in any of the oxidised and reduced states examined. A number of other [NiFe]-hydrogenases have been reported which do not exhibit EPR signals from nickel, including the NAD-reducing hydrogenase of Nocardia opaca [20], and, most significantly, the sulfur-reducing hydroge- nase of P. furiosus [lo]. Upon treatment under carbon monoxide atmos- phere, no changes were observed in the EPR spectra of the either of the oxidised or reduced states. Pro- longed incubation of reduced sample under CO gave an EPR silent state, probably by reoxidation of the iron-sulfur clusters on removal of the hydrogen at- mosphere. 4. Discussion Different types of iron-sulfur clusters may be dis- tinguished by their g-factors and the temperature dependence of their EPR signals. The axial signal detected in T. stetteri hydrogenase at temperatures above 30 K (Fig. 1 b) is typical of a [2Fe-2S] cluster, while those detected below 10 K probably represent [4Fe-4S] clusters (Fig. lc); the complexity of the sig- nal derives from spin-spin interactions between the clusters. These signals do not resemble the spectra of the so-called H cluster of the [Fe]-hydrogenases, which are characteristically sharp signals in the re- gion g = 2.0-2.1, detected at temperatures up to 77 K, [3]. This evidence, taken with the observation of nickel in the enzyme, and the relatively low sensitiv- ity of the enzyme to carbon monoxide, suggests that the hydrogenase from T. stetteri is a [NiFe]-hydro- genase. Moreover, we have also found evidence for
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