Microbiology (2005), 151, 1697–1705 DOI 10.1099/mic.0.27679-0 Hydrogen concentrations in methane-forming cells probed by the ratios of reduced and oxidized coenzyme F420 Linda M. I. de Poorter,3 Wim J. Geerts and Jan T. Keltjens Department of Microbiology, Faculty of Science, Radboud University Nijmegen, Toernooiveld 1, NL-6525 ED, Nijmegen, The Netherlands Correspondence Jan T. Keltjens J.Keltjens@science.ru.nl Received 6 October 2004 Revised 10 February 2005 Accepted 14 February 2005 Coenzyme F420 is the central low-redox-potential electron carrier in methanogenic metabolism. The coenzyme is reduced under hydrogen by the action of F420-dependent hydrogenase. The standard free-energy change at pH 7 of F420 reduction was determined to be ”15 kJ mol”1, irrespective of the temperature (25–65 6C). Experiments performed with methane-forming cell suspensions of Methanothermobacter thermautotrophicus incubated under various conditions demonstrated that the ratios of reduced and oxidized F420 were in thermodynamic equilibrium with the gas-phase hydrogen partial pressures. During growth in a fed-batch fermenter, ratios changed in connection with the decrease in dissolved hydrogen. For most of the time, the changes were as expected for thermodynamic equilibrium between the oxidation state of F420 inside the cells and extracellular hydrogen. Also, methanol-metabolizing, but not acetate-converting, cells of Methanosarcina barkeri maintained the ratios of reduced and oxidized coenzyme F420 in thermodynamic equilibrium with external hydrogen. The results of the study demonstrate that F420 is a useful probe to assess in situ hydrogen concentrations in H2-metabolizing methanogens. INTRODUCTION Most methanogenic archaea derive their energy for growth from the hydrogen-dependent reduction of CO2 into methane (reaction 1). The amount of energy that can be gained in the process depends on the in situ hydrogen concentration, which may vary by orders of magnitude in natural habitats and during growth under laboratory conditions. 4 H2 zCO2 ?CH4 z2 H2 O ð1Þ F420 zH2 'F420 H2 ð2Þ F420 H2 zN 5 ,N 10 -methenyl-H4 MPT' F420 zN 5 ,N 10 -methylene-H4 MPT F420 H2 zN 5 ,N 10 -methylene-H4 MPT' F420 zN 5 -methyl-H4 MPT ð3Þ ð4Þ 3Present address: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands. Abbreviations: pH2 , hydrogen partial pressure; pHi, intracellular pH. 0002-7679 G 2005 SGM A central electron carrier in methane metabolism is the 8-OH-5-deazaflavin derivative coenzyme F420. The compound is present in high concentrations. Oxidized F420 shows an intense blue fluorescence when excited at 420 nm (DiMarco et al., 1990; Eirich et al., 1978, 1979). UV–visible light and fluorescence spectral properties are pHdependent, making F420 a useful probe to measure the pH inside the cell (intracellular pH or pHi) (de Poorter & Keltjens, 2001; von Felten & Bachofen, 2000). F420 is reduced to the non-fluorescent species (F420H2) by the action of F420-reducing hydrogenase (reaction 2) (Fox et al., 1987; Thauer, 1998). F420H2 is the substrate in two consecutive reactions in the methanogenic pathway, viz. the reduction of N5,N10-methenyl-tetrahydromethanopterin (H4MPT) and N5,N10-methylene-H4MPT (reactions 3 and 4). The reactions are catalysed by F420-dependent methylene-H4MPT dehydrogenase and methylene-H4MPT reductase, respectively. Reactions (2–4) are reversible (Thauer, 1998). The enzymes involved display high turnover numbers (kcat) and each represents as much as 0?5–1 % of the total cellular protein (Enßle et al., 1991; Ma & Thauer, 1990; Schwörer & Thauer, 1991; te Brömmelstroet et al., 1990, 1991a, b). Thus, the catalytic capacities of the hydrogenase, dehydrogenase and reductase substantially exceed the specific rate of methane formation. Under these conditions, the concentration ratios of reduced and oxidized coenzyme F420 are Downloaded from www.microbiologyresearch.org by IP: 54.224.135.207 On: Fri, 22 Apr 2016 09:53:58 Printed in Great Britain 1697 L. M. I. de Poorter, W. J. Geerts and J. T. Keltjens predicted to be in thermodynamic equilibrium with the hydrogen partial pressure (pH2 ). Taking advantage of the fluorescent properties of F420, ratios of reduced and oxidized species were measured in H2–CO2-metabolizing cells of Methanothermobacter thermautotrophicus and in methanol- and acetate-utilizing Methanosarcina barkeri. It was found that the ratios were, indeed, in close thermodynamic equilibrium with the hydrogen concentrations applied (0–2 %). For reasons discussed, this did not hold for acetate-converting Methanosarcina barkeri. The results of the study indicate that coenzyme F420 is not only a useful probe to measure pHi, but also to determine the in situ hydrogen concentration in H2-metabolizing methanogens. METHODS Materials. Coenzyme F420 was purified from whole cells of Methano- thermobacter thermautotrophicus and cell extracts of the organism were prepared by using established procedures (te Brömmelstroet et al., 1991b). Gases were supplied by Hoek-Loos. To remove traces of oxygen, hydrogen-containing gases were passed over a BASF RO-20 catalyst at room temperature and nitrogen-containing gases over a pre-reduced BASF R3-11 catalyst at 150 uC. The catalysts were a gift from BASF Aktiengesellschaft. All other chemicals used were of the highest grade available. methods. Methanothermobacter thermautotrophicus (formerly Methanobacterium thermautotrophicum) strain DHT= DSM 1053T was grown at 65 uC and pH 7?0 in a 3?5 l fermenter (MBR) containing 2?5 l mineral medium and gassed with H2/CO2 (80 : 20 %, v/v) at 1500 r.p.m. Mineral medium contained the following constituents (g l21): KH2PO4 (6?8), Na2CO3 (3?3), NH4Cl (2?1), trace elements as described by Schönheit et al. (1979) and sodium resazurin (0?1 mg l21), and cysteine hydrochloride (0?6 g l21) and Na2S2O3 (0?5 g l21) as reducing agents. At regular time intervals, samples were collected anoxically for the determination of OD600, F420 measurement, pHi determination and for cellsuspension incubations. The dissolved pH2 and medium pH were monitored online with an amperometric (Ag/Ag2O) H2 probe (de Poorter et al., 2003; Schill et al., 1996) and a pH electrode (Ingold, Elscolab Nederlands BV), respectively. Culturing Alternatively, Methanothermobacter thermautotrophicus was cultured in 115 ml serum bottles containing 50 ml mineral medium supplemented with 0?6 g Na2S.2H2O l21. Growth was performed at various temperatures (50–65 uC) and pH values (6?0–7?5) to an OD600 of 0?2–0?3. Incubation took place in a rotary-shaking water bath operating at 150 r.p.m. After inoculation, cultures were pressured daily with H2/CO2 (80 : 20 %, v/v; 200 kPa). Methanosarcina barkeri strain Fusaro (=DSM 804) was cultured in 50 ml amounts in 115 ml serum bottles. Media were prepared as described previously (Hutten et al., 1981) and contained 10 g sodium acetate l21 (122 mM) or 10 ml methanol l21 (200 mM) as a carbon and energy source. Cells were grown without shaking at 35 uC under an N2/CO2 (80 : 20 %, v/v; 120 kPa) atmosphere to an OD600 of 0?1–0?2. Reduction of coenzyme F420. Purified coenzyme F420 was reduced enzymically by using cell extract of Methanothermobacter thermautotrophicus as described previously (Vermeij et al., 1997). Reaction mixtures (3 ml) were incubated in 25 ml serum bottles under 0–80 % H2, 20 % 1698 CO2, complemented with N2 (80–0 %). After reactions had come to equilibrium, anoxic acetone was added and fluorescence spectra were recorded immediately as described below. Cell-suspension incubations. Cells were collected from 3?5 l fed- batch cultures or were obtained from serum-bottle cultures. Inside an anaerobic glove box, 2 ml portions of cells were divided over a series of 115 ml serum bottles. Cell suspensions with an OD600 of >1 were diluted with anoxic mineral medium. After filling, bottles were closed with butyl rubber stoppers and aluminium-crimped seals, evacuated and pressured with mixtures of H2/CO2 (80 : 20 %, v/v) and N2/CO2 (80 : 20 %, v/v) to obtain the pH2 values specified in the text. Hereafter, titanium citrate (1 mM) was added to remove traces of oxygen (Zehnder & Wuhrmann, 1976). Ethane (1 ml) was added as an internal standard for methane measurements (Gijzen et al., 1991). Serum bottles were subsequently placed in a water bath without shaking at the specified temperatures. At regular times, headspace samples were withdrawn to follow methane formation. As soon as methanogenesis had started, incubations were continued for 30 min at 150 r.p.m. (Methanothermobacter thermautotrophicus) or 100 r.p.m. (Methanosarcina barkeri) rotation. Reactions were then stopped by cooling the serum bottles rapidly in ice-cold water and samples were immediately withdrawn with a gas-tight syringe for F420 fluorescence analysis. Coenzyme F420 fluorescence measurements. A known volume of cells from the fermenter (1–5 ml) or from cell-suspension incubations (1 ml) was injected under anoxic conditions into a serum bottle closed with a bromobutyl rubber stopper and containing icecold anoxic acetone kept under N2/CO2 (80 : 20 %, v/v). Before use, acetone was stored overnight in an anaerobic glove box to remove traces of oxygen. Immediately afterwards, cell–acetone mixtures were pipetted into cuvettes placed inside the glove box. Cuvettes were closed with bromobutyl stoppers and the contents were analysed by anaerobic fluorescence spectroscopy. This gave the fluorescence intensities of oxidized F420 present in the samples (Fox). To determine the fluorescence of total coenzyme F420 (Ftot), cell samples were mixed, after brief exposure to air, with oxic acetone and spectra were measured under aerobic conditions. To correct for background fluorescence (Fb), cell samples were incubated under (H2/ CO2) 80 : 20 % at 65 uC, added to cold anoxic acetone and measured anaerobically. Fluorescence emission was recorded at room temperature on an Aminco SPF-500 fluorimeter with excitation wavelength at 427 nm (band pass, 4 nm) and emission wavelength at 471 nm (band pass, 2 nm). Alternatively, excitation spectra (340–470 nm) were recorded at an emission wavelength of 471 nm. The concentration ratios of F420H2 and F420 were calculated as (Ftot2Fox)/Fox. The experimental values (Ftot, Fox) were corrected for background fluorescence (Fb) measured for the fully (80 % H2) reduced cell samples. Acetone extracts were alkaline (pH 9–10). Under these conditions, oxidized F420 is measured exclusively as the phenolate–quinoid anionic species (see Appendix). Other analytical methods. Methane-production rates during the fermenter culturing were calculated from the flow rate and methane content of the outflow gas, which were measured by use of a soapfilm meter and by GC, respectively. GC was performed on an HP 5890 gas chromatograph equipped with a Poropak Q column and a flame-ionization detector. Cellular dry weights (DW) to determine specific methane-forming activities were derived from the OD600 value of the culture. Previous research established the linear relation between both parameters, at which 1 l culture showing an OD600 of 1 equalled 425 mg dry cells (unpublished results). pHi values were measured by a previously described method, using the pH-dependent fluorescence properties of oxidized coenzyme F420 (de Poorter & Keltjens, 2001). Downloaded from www.microbiologyresearch.org by IP: 54.224.135.207 On: Fri, 22 Apr 2016 09:53:58 Microbiology 151 Hydrogen metabolism and F420 reduction Coenzyme F420 reduction in methane-forming cell suspensions of Methanothermobacter thermautotrophicus Fig. 1. Hydrogen-dependent reduction of coenzyme F420. F420 (5 mM) was reduced at the indicated pH2 values (%, v/v), using cell-free extract (15 mg protein) as described in Methods. Reactions were performed at 60 6C and pH 7?0. Excitation spectra were recorded at 471 nm emission. In the inset, concentration ratios of reduced (F420H2) and oxidized F420 are plotted against the applied hydrogen partial pressures ( pH2 ). a.u., Arbitrary units. RESULTS Hydrogen-dependent reduction of coenzyme F420 F420 was incubated in the presence of cell extract in a series of serum bottles under different pH2 (0–80 %; 0–0?8 bar), and fluorescence-excitation spectra were recorded after reactions had come to equilibrium (Fig. 1). F420 incubated under an N2/CO2 atmosphere (80 : 20 %, v/v) showed maximal fluorescence emission at 427 nm excitation. The same fluorescence intensities of H2-incubated reaction mixtures were found after exposure to air or after mixing with aerobic acetone. Incubations at increased hydrogen concentrations resulted in the concomitant decrease of the excitation spectra, characteristic of F420 reduction. Under 80 % H2, the spectrum was bleached almost completely. Concentration ratios of F420H2 and F420, determined as described in Methods, were related linearly to the pH2 applied (Fig. 1, inset). From the slope of the plot and by using equation (A.8) from the Appendix, a DG09of 215 kJ mol21 was calculated at the experimental conditions (pH 7?0, 60 uC). Remarkably, the same value of DG09 was found under standard conditions (pH 7?0, 25 uC). Whole cells incubated under hydrogen revealed excitation and emission spectra that were indistinguishable from those obtained for purified F420 (data not shown). This demonstrated that other cellular components did not interfere with F420 fluorescence measurements. The fluorescence characteristics were subsequently used to determine the concentration ratios of reduced and oxidized F420 in metabolizing cells. http://mic.sgmjournals.org To investigate the effect of the applied pH2 on coenzyme F420 reduction in methane-producing cells, cell suspensions of Methanothermobacter thermautotrophicus were incubated under a variety of conditions and at 0–2 % (v/v) hydrogen in the gas phase (pH2 , 0–0?02 bar). Cell suspensions were obtained from different growth stages in the fed-batch fermenter (see below) or from serum-bottle cultures. At low pH2 , the specific rates of methanogenesis in the suspension incubations were linearly dependent on the pH2 applied. Specific activities at a pH2 of 0?02 bar were 5–50 % of the maximal values measured at 80 % H2 [1–3 mmol CH4 min21 (mg DW)21]. The former percentages depended on the hydrogen concentration at which growth had occurred and reflect changes in the affinities (Km) of the cells for hydrogen. It is known that Methanothermobacter thermautotrophicus cells derived from cultures grown under lowhydrogen conditions display a higher hydrogen affinity (Km approx. 2 % H2) than cells grown at a high hydrogen concentration (Km approx. 20 % H2) (Pennings et al., 2000). In addition, maximal specific activities of the cultured cells varied in a growth phase- and growth condition-related way (Pennings et al., 2000; L. M. I. de Poorter & J. T. Keltjens, unpublished observations). This explains the differences in values measured at 80 % H2 during the suspension incubations. When cell suspensions collected from different growth stages in the fed-batch fermenter were incubated at 60 uC and pH 7, a linear relationship was found between the [F420H2]/[F420] ratios and the pH2 values applied (Fig. 2). Slopes of the graphs measured with cells from different growth stages were identical. The mass–action ratio was associated with RT ln qr at +15 kJ mol21. Above data established a DGr09 of 215 kJ mol21 at 60 uC and pH 7. From the resulting DGr9 of 0 kJ mol21 (equation A.1), it is inferred that the concentrations of reduced and oxidized coenzyme F420 within the cells are in thermodynamic equilibrium with the pH2 in the gas phase. To investigate the effect of temperature and pH on the hydrogen-dependent reduction of coenzyme F420, Methanothermobacter thermautotrophicus was cultured in serum bottles at a range of temperatures (50–65 uC) and pH values (6?0–7?5). Cells were subsequently incubated under various pH2 , using medium pH values and temperatures at which culturing had occurred. Separate incubations were performed to measure the pHi after incubation. At the experimental conditions, pHi was found to be equal to the medium pH. As before, [F420H2]/[F420] ratios were related linearly to the pH2 values applied (Figs 3a and 4a). Slopes were pH-dependent and an approximately tenfold decrease in the mass–action ratio was observed when medium pH increased by 1 unit (Fig. 3a). This indicates that coenzyme F420 reduction is described by equation (5), in which F4202 refers to (deprotonated) phenolate anion (Fig. 7): Downloaded from www.microbiologyresearch.org by IP: 54.224.135.207 On: Fri, 22 Apr 2016 09:53:58 1699 L. M. I. de Poorter, W. J. Geerts and J. T. Keltjens Fig. 2. Effect of pH2 on coenzyme F420 reduction in Methanothermobacter thermautotrophicus. Cell suspensions were incubated under 20 % CO2 and the indicated gas-phase pH2 values. Incubations took place at 60 6C and pH 7?0 as described in Methods. Suspensions were collected from fedbatch fermenter cultures at the different growth phases. Symbols: X, early-exponential phase (OD600=0?2); %, exponential phase (OD600=0?6); m, linear phase (OD600=2); #, stationary phase (OD600=6). H2 zHz zF420 { ' F420 H2 ð5Þ The plot of RT ln qr versus pH gave a straight line (Fig. 3b). The slope (26?4 kJ mol21 pH21) at the incubation temperature (60 uC) was in full agreement with the net uptake of one proton. By use of equation (A.6) and the experimental DG09 of 215 kJ mol21at pH 7, DG600 could be calculated for the different pH values (Fig. 3b). Again considering that the Gibbs free-energy change at 60 uC (DG60) sums as DG600+RT ln qr (equation A.1), a DG60 of 0 kJ mol21 was derived for all pH values tested, indicative of thermodynamic equilibrium (Fig. 3b). When incubated at pH 7, mass–action ratios varied with the incubation temperatures (50–65 uC), but the RT ln qr term was constant (+15 kJ mol21) and exactly opposite to the (temperatureindependent) DG09 of 215 kJ mol21, again demonstrating thermodynamic equilibrium (DG9=0) (Figs 4a and b). By routine, cell-suspension incubations were performed at relatively low pH2 values (0–0?02 bar). When incubated at higher headspace-hydrogen concentrations, large variations in [F420H2]/[F420] ratios were found among repeated experiments and the ratios were generally lower than expected. At the higher pH2 values, methane production and, in direct connection, hydrogen uptake took place at correspondingly enhanced rates. The consumption of dissolved hydrogen during the brief but variable period between rotary incubation and cooling of the samples (5–15 s) probably caused the variation in and underestimation of the [F420H2]/[F420] ratios. 1700 Fig. 3. Effect of pH on the thermodynamics of F420 reduction in Methanothermobacter thermautotrophicus. (a) Cell suspensions were incubated at 60 6C under 20 % CO2 and the indicated gas-phase pH2 values as described in Methods. Reactions took place at the following pH values of the medium: 6?3 (X), 6?5 (&), 6?8 (%), 7?1 (m) or 7?2 ($). Data represent the means and errors of triplicate experiments. (b) Plots of mass–action ratio terms (RT ln q) (n) derived from the slopes presented in (a), calculated pH-dependent DG0 values ($) and net Gibbs free-energy changes (DG) (m). Coenzyme F420 reduction in methanol- and acetate-metabolizing cell suspensions of Methanosarcina barkeri Methanosarcina barkeri was grown in serum bottles on methanol (200 mM) or acetate (122 mM) as substrates to an OD600 of 0?1–0?2. At this time, cultures still contained approximately 150 mM methanol and 80 mM acetate, whilst methane was formed with specific activities of 0?4 and 0?1 mmol min21 (mg DW)21, respectively. Portions (2 ml) of the cultures were subsequently incubated under 0–80 % hydrogen at 35 uC. Determination of the [F420H2]/ [F420] ratios revealed a linear relationship between the ratios and the pH2 values applied in the case of methanol-grown cells (Fig. 5). From the slope of the curve, an RT ln qr of +15 kJ mol21 could be calculated, which equals the abovedetermined values. From this, we conclude that methanolmetabolizing Methanosarcina barkeri cells maintain their [F420H2]/[F420] ratios in thermodynamic equilibrium with the pH2 in the environment. In acetate-grown cells, the situation was different. Although clearly detectable by the sensitive fluorescence method used, the F420 content was lower by Downloaded from www.microbiologyresearch.org by IP: 54.224.135.207 On: Fri, 22 Apr 2016 09:53:58 Microbiology 151 Hydrogen metabolism and F420 reduction Fig. 5. Effect of pH2 on coenzyme F420 reduction in Methanosarcina barkeri. Methanol (X) and acetate (&)-metabolizing cell suspensions were incubated under 20 % CO2 and the indicated headspace pH2 values. Reactions took place at 37 6C and pH 7?0 as described in Methods. Fig. 4. Effect of temperature on the thermodynamics of F420 reduction in Methanothermobacter thermautotrophicus. (a) Cell suspensions were incubated at pH 7?0 under 20 % CO2 and the indicated headspace pH2 values as described in Methods. Reactions took place at the following temperatures: 50 6C (&), 55 6C (m) and 65 6C (#). Data represent the means and errors of triplicate experiments. (b) Plots of mass–action ratio terms (RT ln q) (n) derived from the slopes presented in (a), calculated DG0 values ($) and net Gibbs free-energy changes (DG) (m). The mass–action ratio at 60 6C was obtained from data presented in Fig. 2. more than a factor of ten than that in methanol-grown cells. Moreover, coenzyme F420 was only present in the oxidized state [(F420H2)/(F420)=0] (Fig. 5), even if incubations were performed under high hydrogen concentrations (up to 80 %). Changes in the ratios of reduced and oxidized coenzyme F420 during growth of Methanothermobacter thermautotrophicus in a fed-batch fermenter Methanothermobacter thermautotrophicus was cultured in a fed-batch fermenter at constant gassing with 80 : 20 % H2/ http://mic.sgmjournals.org CO2 (Fig. 6). Growth was characterized by an exponential increase of cell density up to an OD600 of 1?7 [specific growth rate, 0?24 h21; doubling time (td), 2?9 h]. Hereafter, cell density increased linearly with time. During exponential growth, methane was formed with a specific activity of 1?5–2?5 mol min21 (mg DW)21. Considering that 4 mol hydrogen is used (mol methane formed)21 (equation 1), the specific hydrogen-consumption rate amounted to 6–10 mol min21 mg21. Together with the increase in biomass, the overall hydrogen-consumption rate increased tenfold (0?6–6 mmol min21). The increase in hydrogen consumption was accompanied by the decline in the dissolved pH2 from 70 to 3 % (0?7 to 0?03 bar). Remarkably, the intracellular pH of the cells decreased as well, in particular during the mid-exponential phase (Fig. 6). During the linear-growth phase (10–12 h), the hydrogenconsumption rate and pH2 became constant at 6 mmol min21 and 0?03 bar, respectively. Now, pHi was about equal to the medium pH of 7?0. At regular time intervals, cells were collected anoxically from the fermenter and analysed for the [F420H2]/[F420] ratios (Fig. 6). The apparent ratios tended to decrease, but became somewhat higher during the linear-growth phase. From the recorded pH2 and pHi values, [F420H2]/[F420] ratios were calculated theoretically, assuming thermodynamic equilibrium. It can be seen that experimental and theoretic ratios were about equal during the early-exponential (0–3 h) and linear (10–12 h) phases, where pH2 was as high as 0?70 bar and as low as 0?03 bar, respectively. During the intermediary-exponential phase, however, experimental [F420H2]/[F420] ratios were five- to 15-fold lower than the theoretical values. It is conceivable that, during this stage, the hydrogen concentration inside the cells was lower than that in the medium. However, at least part of the difference Downloaded from www.microbiologyresearch.org by IP: 54.224.135.207 On: Fri, 22 Apr 2016 09:53:58 1701 L. M. I. de Poorter, W. J. Geerts and J. T. Keltjens Fig. 6. Changes in the concentration ratios of reduced and oxidized coenzyme F420 during growth of Methanothermobacter thermautotrophicus in a fed-batch fermenter. The organism was grown under 80 : 20 % H2/CO2 at a constant gassing rate of 218 ml min”1. Culturing took place at 65 6C and pH 7?0 as described in Methods. Measurements started (t=0) 12 h after inoculation. Symbols: X, OD600; $, pH2 in growth medium; #, intracellular pH (pHi); n, experimental concentration ratios of reduced and oxidized F420 (means and errors of triplicate fluorescence measurements); m, [F420H2]/[F420] ratios (assuming thermodynamic equilibrium). could be due to an underestimation of the [F420H2]/[F420] ratios as a result of the sampling procedure. Sampling included the passage of the culture liquid through the device interconnecting the fermenter and the acetone-containing sample bottle, which took about 5 s. During the passage, a substantial part of the dissolved hydrogen could have been utilized, especially at high cellular hydrogen-uptake rates and at high medium pH2 , conditions that typically apply to the exponential phase. Indeed, when acetone mixtures were analysed by GC for dissolved hydrogen, levels in samples collected during the intermediary-exponential phase were lower by a factor of 5–15 than measured with the hydrogen probe. In contrast, GC determinations on liquids from early-exponential and linear-phase cells agreed well with those recorded in the fermenter (data not shown). DISCUSSION Hydrogen-metabolizing cells of Methanothermobacter thermautotrophicus consistently maintained the concentration ratios of reduced and oxidized coenzyme F420 in thermodynamic equilibrium with the pH2 , if below approximately 0?02 bar. However, equilibrium was also observed at pH2 values as high as 0?7 bar and at high specific hydrogenconsumption rates (see Fig. 6, early-exponential phase). Therefore, the relationship could be valid for all conditions, but this could not be substantiated by the method applied, due to the time delay in our sampling procedure. Online in situ fluorescence measurements might clarify this issue. In the temperature range tested (25–65 uC), the standard free-energy change at pH 7 related to the hydrogendependent reduction of coenzyme F420 was constant (DG09, 215 kJ mol21). As the midpoint potential of the H+/H2 couple varies with temperature, Em,F for the F420/ F420H2 couple has to show the same temperature dependency. On the basis of the experimental DG09 values, the H+/H2 midpoint potentials and by using equation (A.7), Em values of 2340 and 2385 mV are then calculated for the F420/F420H2 couple at 25 and 60 uC, respectively, by 1702 the biochemical assay described here. The former value equals reported data (2340 to 2350 mV) determined at ambient temperature by electrochemical methods (Jacobson & Walsh, 1984; Pol et al., 1980). Thermodynamic equilibrium was also found in methanolutilizing Methanosarcina barkeri cells. This is remarkable, as the conversion of methanol into methane and CO2 does not involve hydrogen (equation 6). 4 CH3 OH?3 CH4 zCO2 z2 H2 O ð6Þ However, methanol-grown cells contain high levels of F420-reducing hydrogenase (Michel et al., 1995), whilst F420 serves as the electron carrier in two reactions of the methyl group-oxidation pathway, notably N5-methyl-H4MPT and N5,N10-methylene-H4MPT oxidation (reversed reactions 3 and 4) (Enßle et al., 1991; Schwörer & Thauer, 1991; te Brömmelstroet et al., 1991a; Thauer, 1998). During growth on methanol, the compound serves as both the energy and carbon source. As cell carbon is formally more oxidized than that in methanol, anabolism is associated with a net electron production. It is conceivable that the generation (or consumption) of hydrogen gas is required to balance electron flows in catabolic and anabolic reactions at which F420-hydrogenase could act as a redox valve. Indeed, it is known that Methanosarcina growing on methanol accumulates small concentrations of hydrogen gas in the gas atmosphere (Lovley & Ferry, 1985). In contrast, acetate catabolism does not involve F420-dependent reactions. Under these conditions, F420-reducing hydrogenase, as well as F420-dependent N5,N10-methylene-H4MPT dehydrogenase and reductase, are repressed (Schwörer & Thauer, 1991; Vaupel & Thauer, 1998). As expected for a limited role in cellular metabolism, F420 is present at only low levels (Heine-Dobbernack et al., 1988; this study). Furthermore, it was found here that hydrogen had no effect on the F420 reduction state during acetate metabolism. Apparently, hydrogen does not equilibrate with the intermediary F420 metabolism, serving now only some specific anabolic steps. Downloaded from www.microbiologyresearch.org by IP: 54.224.135.207 On: Fri, 22 Apr 2016 09:53:58 Microbiology 151 Hydrogen metabolism and F420 reduction Fig. 7. Structure of coenzyme F420 and its (de)protonation and redox reactions. In nature, methanogenic archaea form part of densely packed, complex microbial consortia that degrade organic matter into methane and CO2 (Zinder, 1993). Hydrogen is a central intermediate in the degradation and the gas is presumably present as steep spatial-concentration gradients. Detailed understanding of the processes will require methods to measure in situ hydrogen concentrations within the microsystems. By taking advantage of its fluorescent properties, coenzyme F420 could serve as a probe to assess hydrogen concentrations by using, for example, noninvasive laser techniques. absolute temperature (K) and qr is the mass–action ratio: qr ~½F420 H2
=½F420
pH2 (A:2) APPENDIX qr equals the slope in the experimental [F420H2]/[F420] versus pH2 plots. It should be noted that [F420] and [F420H2] represent total concentrations of the oxidized and reduced species, respectively. In the physiological pH range, the 5-deazaflavin chromophore of oxidized coenzyme F420 contains one ionizable group, viz. 8-OH (pKa1 6?18– 6?47, depending on the temperature) (Jacobson & Walsh, 1984; Purwantini et al., 1992). Deprotonation of 8-OH results in the phenolate anion, which tautomerizes into the conjugated paraquinoid anion (Fig. 7). In (non-fluorescent) reduced F420, NH(1) (pKa2 6?9) and the 8-hydroxyl group (pKa19 9?7) are of relevance. Thus, oxidized and reduced F420 are composed of a mixture of species that will affect the redox potential of the F420/F420H2 couple in a pH-dependent fashion. Theory ½F420
tot ~½F420
(1zK a1 =½Hz
) (A:3) Equation (2) in the Introduction formally describes the reduction of coenzyme F420 into 1,5-dihydro-F420 (F420H2) with hydrogen. The (Gibbs) free-energy change, DGr (kJ mol21), at specified reaction conditions (suffix r; temperature, pH) of the reaction is: ½F420 H2
tot ~½F420 H2
(1zK a2 =½Hz
zK a2 :K a10 =½Hz
2 ) (A:4) In addition, the free-energy changes of coenzyme F420 reduction with hydrogen will vary with the pH: DG r ~DG 0r zRT ln qr H2 + m Hz zF420 m{ ' F420 H2 (A:1) 23 in which R is the gas constant (8?314?10 http://mic.sgmjournals.org kJ mol 21 21 K ), T is the Defining DGr09 (kJ (A:5) 21 mol ) as the free-energy change at pH 7 and at the Downloaded from www.microbiologyresearch.org by IP: 54.224.135.207 On: Fri, 22 Apr 2016 09:53:58 1703 L. M. I. de Poorter, W. J. Geerts and J. T. Keltjens temperature at which the reaction is followed and m as the net number of protons that are consumed or produced per reaction, the following relations hold: DG 0r ~DG 0r + 2:303 mRT (7-pH) (A:6) DG 0r ~nFDE m,7 (A:7) In equation (A.6), the sign of the term is minus in a proton-consuming reaction. In equation (A.7), n is the number (2) of electrons involved, F is the Faraday constant (96?49 kJ V21 mol21) and DEm,7 is the difference between the midpoint potentials (V) of the H+/H2 (Em,H) and F420/F420H2 (Em,F) redox couples, respectively, at pH 7 and the specified temperature. Em,H is derived for each given temperature from the Nernst equation: Em,H=22?303(7RT/F). Em,F should be measured, or it can be calculated if DGr09 (at pH 7) is known. The latter can be determined from the reaction at equilibrium. Considering that, under these conditions, DGr=0 and that the mass–action ratio (qr) equals the equilibrium constant Kr, it follows from equation (A.1): Heine-Dobbernack, E., Schoberth, S. M. & Sahm, H. (1988). Relationship of intracellular coenzyme F420 content to growth and metabolic activity of Methanobacterium bryantii and Methanosarcina barkeri. Appl Environ Microbiol 54, 454–459. Hutten, T. J., de Jong, M. H., Peeters, B. P. H., van der Drift, C. & Vogels, G. D. (1981). Coenzyme M derivatives and their effects on methane formation from carbon dioxide and methanol by cell extracts of Methanosarcina barkeri. J Bacteriol 145, 27–34. Jacobson, F. & Walsh, C. (1984). Properties of 7,8-didemethyl-8- hydroxy-5-deazaflavins relevant to redox coenzyme function in methanogen metabolism. Biochemistry 23, 979–988. Lovley, D. R. & Ferry, J. G. (1985). Production and consumption of H2 during growth of Methanosarcina spp. on acetate. Appl Environ Microbiol 49, 247–249. Ma, K. & Thauer, R. K. (1990). Purification and properties of N5,N10-methylenetetrahydromethanopterin reductase from Methanobacterium thermoautotrophicum (strain Marburg). Eur J Biochem 191, 187–193. Michel, R., Massanz, C., Kostka, S., Richter, M. & Fiebig, K. (1995). 0 DG 0r ~{RT ln K 0r (A:8) Biochemical characterization of the 8-hydroxy-5-deazaflavin-reactive hydrogenase from Methanosarcina barkeri Fusaro. Eur J Biochem 233, 727–735. ACKNOWLEDGEMENTS Pennings, J. L. 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