Department of Microbiology, Faculty of Science, Radboud University Nijmegen, Toernooiveld 1, NL-6525 ED, Nijmegen, The Netherlands
Correspondence
Jan T. Keltjens
J.Keltjens{at}science.ru.nl
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Present address: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
![]() |
![]() |
![]() |
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
). UVvisible light and fluorescence spectral properties are pH-dependent, 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 (24) are reversible (Thauer, 1998
). The enzymes involved display high turnover numbers (kcat) and each represents as much as 0·51 % 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 predicted to be in thermodynamic equilibrium with the hydrogen partial pressure (
).
Taking advantage of the fluorescent properties of F420, ratios of reduced and oxidized species were measured in H2CO2-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 (02 %). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Culturing methods.
Methanothermobacter thermautotrophicus (formerly Methanobacterium thermautotrophicum) strain HT=DSM 1053T was grown at 65 °C 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 l1): KH2PO4 (6·8), Na2CO3 (3·3), NH4Cl (2·1), trace elements as described by Schönheit et al. (1979)
and sodium resazurin (0·1 mg l1), and cysteine hydrochloride (0·6 g l1) and Na2S2O3 (0·5 g l1) as reducing agents. At regular time intervals, samples were collected anoxically for the determination of OD600, F420 measurement, pHi determination and for cell-suspension incubations. The dissolved
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.
Alternatively, Methanothermobacter thermautotrophicus was cultured in 115 ml serum bottles containing 50 ml mineral medium supplemented with 0·6 g Na2S.2H2O l1. Growth was performed at various temperatures (5065 °C) and pH values (6·07·5) to an OD600 of 0·20·3. 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 l1 (122 mM) or 10 ml methanol l1 (200 mM) as a carbon and energy source. Cells were grown without shaking at 35 °C under an N2/CO2 (80 : 20 %, v/v; 120 kPa) atmosphere to an OD600 of 0·10·2.
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 080 % H2, 20 % CO2, complemented with N2 (800 %). 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 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 (15 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 ice-cold 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, cellacetone 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 °C, 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 (340470 nm) were recorded at an emission wavelength of 471 nm. The concentration ratios of F420H2 and F420 were calculated as (FtotFox)/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 910). Under these conditions, oxidized F420 is measured exclusively as the phenolatequinoid 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 soap-film 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).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Coenzyme F420 reduction in methane-forming cell suspensions of Methanothermobacter thermautotrophicus
To investigate the effect of the applied on coenzyme F420 reduction in methane-producing cells, cell suspensions of Methanothermobacter thermautotrophicus were incubated under a variety of conditions and at 02 % (v/v) hydrogen in the gas phase (
, 00·02 bar). Cell suspensions were obtained from different growth stages in the fed-batch fermenter (see below) or from serum-bottle cultures. At low
, the specific rates of methanogenesis in the suspension incubations were linearly dependent on the
applied. Specific activities at a
of 0·02 bar were 550 % of the maximal values measured at 80 % H2 [13 µmol CH4 min1 (mg DW)1]. 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 low-hydrogen 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 °C and pH 7, a linear relationship was found between the [F420H2]/[F420] ratios and the values applied (Fig. 2
). Slopes of the graphs measured with cells from different growth stages were identical. The massaction ratio was associated with RT ln qr at +15 kJ mol1. Above data established a
Gr0' of 15 kJ mol1 at 60 °C and pH 7. From the resulting
Gr' of 0 kJ mol1 (equation A.1), it is inferred that the concentrations of reduced and oxidized coenzyme F420 within the cells are in thermodynamic equilibrium with the
in the gas phase.
|
![]() |
|
|
|
By routine, cell-suspension incubations were performed at relatively low values (00·02 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
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 (515 s) probably caused the variation in and underestimation of the [F420H2]/[F420] ratios.
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 0·10·2. 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 µmol min1 (mg DW)1, respectively. Portions (2 ml) of the cultures were subsequently incubated under 080 % hydrogen at 35 °C. Determination of the [F420H2]/[F420] ratios revealed a linear relationship between the ratios and the values applied in the case of methanol-grown cells (Fig. 5
). From the slope of the curve, an RT ln qr of +15 kJ mol1 could be calculated, which equals the above-determined values. From this, we conclude that methanol-metabolizing Methanosarcina barkeri cells maintain their [F420H2]/[F420] ratios in thermodynamic equilibrium with the
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 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 %).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the temperature range tested (2565 °C), the standard free-energy change at pH 7 related to the hydrogen-dependent reduction of coenzyme F420 was constant (G0', 15 kJ mol1). 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
G0' values, the H+/H2 midpoint potentials and by using equation (A.7), Em values of 340 and 385 mV are then calculated for the F420/F420H2 couple at 25 and 60 °C, respectively, by the biochemical assay described here. The former value equals reported data (340 to 350 mV) determined at ambient temperature by electrochemical methods (Jacobson & Walsh, 1984
; Pol et al., 1980
).
Thermodynamic equilibrium was also found in methanol-utilizing Methanosarcina barkeri cells. This is remarkable, as the conversion of methanol into methane and CO2 does not involve hydrogen (equation 6).
![]() |
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.
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, non-invasive laser techniques.
![]() |
APPENDIX |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
![]() |
qr equals the slope in the experimental [F420H2]/[F420] versus 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 6·186·47, 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 (pKa1' 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.
![]() |
![]() |
![]() |
![]() |
![]() |
![]() |
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
de Poorter, L. M. I., Geerts, W. G., Theuvenet, A. P. R. & Keltjens, J. T. (2003). Bioenergetics of the formyl-methanofuran dehydrogenase and heterodisulfide reductase reactions in Methanothermobacter thermautotrophicus. Eur J Biochem 270, 6675.
DiMarco, A. A., Bobik, T. A. & Wolfe, R. S. (1990). Unusual coenzymes of methanogenesis. Annu Rev Biochem 59, 355394.[CrossRef][Medline]
Eirich, L. D., Vogels, G. D. & Wolfe, R. S. (1978). Proposed structure for coenzyme F420 from Methanobacterium. Biochemistry 17, 45834593.[CrossRef][Medline]
Eirich, L. D., Vogels, G. D. & Wolfe, R. S. (1979). Distribution of coenzyme F420 and properties of its hydrolytic fragments. J Bacteriol 140, 2027.[Medline]
Enßle, M., Zirngibl, C., Linder, D. & Thauer, R. K. (1991). Coenzyme F420 dependent N5,N10-methylenetetrahydromethanopterin dehydrogenase in methanol-grown Methanosarcina barkeri. Arch Microbiol 155, 483490.
Fox, J. A., Livingston, D. J., Orme-Johnson, W. H. & Walsh, C. T. (1987). 8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum. 1. Purification and characterization. Biochemistry 26, 42194227.[CrossRef][Medline]
Gijzen, H. J., Broers, C. A. M., Barughare, M. & Stumm, C. K. (1991). Methanogenic bacteria as endosymbionts of the ciliate Nyctotherus ovalis in the cockroach hindgut. Appl Environ Microbiol 57, 16301634.[Medline]
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, 454459.
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, 2734.[Medline]
Jacobson, F. & Walsh, C. (1984). Properties of 7,8-didemethyl-8-hydroxy-5-deazaflavins relevant to redox coenzyme function in methanogen metabolism. Biochemistry 23, 979988.[CrossRef]
Lovley, D. R. & Ferry, J. G. (1985). Production and consumption of H2 during growth of Methanosarcina spp. on acetate. Appl Environ Microbiol 49, 247249.
Ma, K. & Thauer, R. K. (1990). Purification and properties of N5,N10-methylenetetrahydromethanopterin reductase from Methanobacterium thermoautotrophicum (strain Marburg). Eur J Biochem 191, 187193.[Abstract]
Michel, R., Massanz, C., Kostka, S., Richter, M. & Fiebig, K. (1995). Biochemical characterization of the 8-hydroxy-5-deazaflavin-reactive hydrogenase from Methanosarcina barkeri Fusaro. Eur J Biochem 233, 727735.[Abstract]
Pennings, J. L. A., Vermeij, P., de Poorter, L. M. I., Keltjens, J. T. & Vogels, G. D. (2000). Adaptation of methane formation and enzyme contents during growth of Methanobacterium thermoautotrophicum (strain H) in a fed-batch fermentor. Antonie van Leeuwenhoek 77, 281291.[CrossRef][Medline]
Pol, A., van der Drift, C., Vogels, G. D., Cuppen, T. J. H. M. & Laarhoven, W. H. (1980). Comparison of coenzyme F420 from Methanobacterium bryantii with 7- and 8-hydroxyl-10-methyl-5-deazaisoalloxazine. Biochem Biophys Res Commun 92, 255260.[CrossRef][Medline]
Purwantini, E., Mukhopadhyay, B., Spencer, R. W. & Daniels, L. (1992). Effect of temperature on the spectral properties of coenzyme F420 and related compounds. Anal Biochem 205, 342350.[CrossRef][Medline]
Schill, N., van Gulik, W. M., Voisard, D. & von Stockar, U. (1996). Continuous cultures limited by a gaseous substrate: development of a simple, unstructured mathematical model and experimental verification with Methanobacterium thermoautotrophicum. Biotechnol Bioeng 51, 645658.[CrossRef]
Schönheit, P., Moll, J. & Thauer, R. K. (1979). Nickel, cobalt, and molybdenum requirement for growth of Methanobacterium thermoautotrophicum. Arch Microbiol 123, 105107.[CrossRef][Medline]
Schwörer, B. & Thauer, R. K. (1991). Activities of formylmethanofuran dehydrogenase, methylenetetrahydromethanopterin dehydrogenase, methylenetetrahydromethanopterin reductase, and heterodisulfide reductase in methanogenic bacteria. Arch Microbiol 155, 459465.
te Brömmelstroet, B. W., Hensgens, C. M. H., Keltjens, J. T., van der Drift, C. & Vogels, G. D. (1990). Purification and properties of 5,10-methylenetetrahydromethanopterin reductase, a coenzyme F420-dependent enzyme, from Methanobacterium thermoautotrophicum strain H. J Biol Chem 265, 18521857.
te Brömmelstroet, B. W., Geerts, W. J., Keltjens, J. T., van der Drift, C. & Vogels, G. D. (1991a). Purification and properties of 5,10-methylenetetrahydromethanopterin dehydrogenase and 5,10-methylenetetrahydromethanopterin reductase, two coenzyme F420-dependent enzymes, from Methanosarcina barkeri. Biochim Biophys Acta 1079, 293302.[Medline]
te Brömmelstroet, B. W., Hensgens, C. M. H., Keltjens, J. T., van der Drift, C. & Vogels, G. D. (1991b). Purification and characterization of coenzyme F420-dependent 5,10-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum strain H. Biochim Biophys Acta 1073, 7784.[Medline]
Thauer, R. K. (1998). Biochemistry of methanogenesis: a tribute to Marjory Stephenson. Microbiology 144, 23772406.[Medline]
Vaupel, M. & Thauer, R. K. (1998). Two F420-reducing hydrogenases in Methanosarcina barkeri. Arch Microbiol 169, 201205.[CrossRef][Medline]
Vermeij, P., Pennings, J. L. A., Maassen, S. M., Keltjens, J. T. & Vogels, G. D. (1997). Cellular levels of factor 390 and methanogenic enzymes during growth of Methanobacterium thermoautotrophicum strain H. J Bacteriol 179, 66406648.
von Felten, P. & Bachofen, R. (2000). Continuous monitoring of the cytoplasmic pH in Methanobacterium thermoautotrophicum using the intracellular factor F420 as indicator. Microbiology 146, 32453250.[Medline]
Zehnder, A. J. B. & Wuhrmann, K. (1976). Titanium (III) citrate as a nontoxic oxidation-reduction buffering system for the culture of obligate anaerobes. Science 194, 11651166.[Medline]
Zinder, S. H. (1993). Physiological ecology of methanogens. In Methanogenesis: Ecology, Physiology, Biochemistry and Genetics, pp. 128206. Edited by J. G. Ferry. New York: Chapman & Hall.
Received 6 October 2004;
revised 10 February 2005;
accepted 14 February 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |