Temperature dependence of methyl-coenzyme M reductase activity and of the formation of the methyl-coenzyme M reductase red2 state induced by coenzyme B

  • Meike Goenrich
  • Evert C. Duin
  • Felix Mahlert
  • Rudolf K. Thauer
Original Article

Abstract

Methyl-coenzyme M reductase (MCR) catalyses the formation of methane from methyl-coenzyme M (CH3-S-CoM) and coenzyme B (HS-CoB) in methanogenic archaea. The enzyme has an α2β2γ2 subunit structure forming two structurally interlinked active sites each with a molecule F430 as a prosthetic group. The nickel porphinoid must be in the Ni(I) oxidation state for the enzyme to be active. The active enzyme exhibits an axial Ni(I)-based electron paramagnetic resonance (EPR) signal and a UV–vis spectrum with an absorption maximum at 385 nm. This state is called the MCR-red1 state. In the presence of coenzyme M (HS-CoM) and coenzyme B the MCR-red1 state is in part converted reversibly into the MCR-red2 state, which shows a rhombic Ni(I)-based EPR signal and a UV–vis spectrum with an absorption maximum at 420 nm. We report here for MCR from Methanothermobacter marburgensis that the MCR-red2 state is also induced by several coenzyme B analogues and that the degree of induction by coenzyme B is temperature-dependent. When the temperature was lowered below 20°C the percentage of MCR in the red2 state decreased and that in the red1 state increased. These changes with temperature were fully reversible. It was found that at most 50% of the enzyme was converted to the MCR-red2 state under all experimental conditions. These findings indicate that in the presence of both coenzyme M and coenzyme B only one of the two active sites of MCR can be in the red2 state (half-of-the-sites reactivity). On the basis of this interpretation a two-stroke engine mechanism for MCR is proposed.

Keywords

Methyl-coenzyme M reductase Nickel enzymes Factor F430 Electron paramagnetic resonance spectroscopy Half-of-the-sites reactivity Mechanism of methane formation 

Abbreviations

EPR

Electron paramagnetic resonance

MCR

Methyl-coenzyme M reductase

CH3-S-CoM

Methyl-coenzyme M

HS-CoM

Coenzyme M

HS-CoB

Coenzyme B

MCR-red1

Active MCR exhibiting the EPR red1 signals

MCR-red1c

MCR-red1 in the presence of 10 mM coenzyme M

MCR-red2

MCR exhibiting the EPR red2 signal

MCR-red1/2

MCR exhibiting both the EPR red1 and red2 signal

MCR-ox

MCR exhibiting the EPR signals ox1, ox2 or ox3

Tris

Tris(hydroxymethyl)aminomethane

Introduction

Globally approximately 1 billion tons of methane per year is generated by the metabolic activity of methanogenic archaea in anoxic environments such as freshwater sediments, swamps, the hindgut of termites and the rumen of sheep and cows [1, 2]. In all methanogens methane is formed from methyl-coenzyme M (CH3-S-CoM) and coenzyme B (HS-CoB):
$$ \begin{array}{*{20}c} {{{\text{CH}}_{3} - {\text S} - {\text{CoM}} + {\text{HS}} - {\text{CoB}} = {\text{CH}}_{4} + {\text{CoM}} - {\text{S}} - {\text{S}} - {\text{CoB}}}} & {{\Delta G{^\circ }' = - {\text{30 }}{{\text{kJ}}} \mathord{\left/ {\vphantom {{{\text{kJ}}} {{\text{mol}}{\text{.}}}}} \right. \kern-\nulldelimiterspace} {{\text{mol}}{\text{.}}}}} \\ \end{array} $$
ΔGo′ was calculated from ΔGfo=−112.5 kJ mol−1 for methanol reduction with H2 to methane and water [3], from ΔGo′=−27.5 kJ mol−1 for methyl-coenzyme M formation from methanol and coenzyme M [4] and Eo′=−140 mV for the CoM-S-S-CoB/HS-CoM+HS-CoB couple [5]. In previous calculations [1] Eo′ was assumed to be −200 mV . The reverse reaction is most probably involved in the anaerobic oxidation of methane [6, 7].

Methane formation is catalysed by methyl-coenzyme M reductase (MCR). The 300-kDa enzyme is composed of three different subunits of molecular mass 66 kDa (α subunit), 48 kDa (β subunit) and 37 kDa (γ subunit) and each subunit is present twice (the values given are for MCR I from Methanothermobacter marburgensis). Per mole the enzyme contains 2 mol of the nickel porphinoid F430 tightly but not covalently bound. The prosthetic group must be in the Ni(I) oxidation state for the enzyme to be active. The redox potential Eo′ of the F430Ni(II)/F430Ni(I) couple has been determined to be between −600 and −700 mV [8]. Owing to the negative redox potential MCR is a very labile enzyme rapidly inactivated in the presence of trace amounts of O2 or of other electron acceptors [1].

The crystal structure of inactive MCR from M. marburgensis has been determined to 1.16-Å resolution [9, 10, 11, 12]. The structure revealed the presence of two active sites with F430Ni(II) deeply buried within the protein and accessible from the outside only via a long narrow channel. The distance between the two nickels in MCR is 50 Å . Methyl-coenzyme M can be positioned in the cavity above F430 such that either its methyl group or the thioether sulfur interact with the Ni(I). Methyl-coenzyme M must enter the channel before coenzyme B because after coenzyme B binding the substrate channel is completely locked. Coenzyme B binds in a manner such that its thioheptanoyl group points towards F430 and the phosphate moiety towards the entrance of the channel. The sulfur of coenzyme B gets positioned above the Ni(I) of F430 at a distance of 8 Å, which is too far for the thiol group to directly interact with the Ni(I). The channel and the coenzyme binding sites are formed by residues of subunits α′, α, β and γ and equivalently α, α′, β′ and γ′. Whereas the porphinoid ligand system of F430 is thus tightly attached to one α subunit, the distal axial ligand to nickel is contributed by a glutamine residue of the second α′ subunit, indicating that the two active sites at a distance of 50 Å are structurally interlinked. This is quite unique for two active sites at such a distance and indicates that the two active sites could also be functionally interlinked. A conformational change in one active site can thus be directly transferred via the α subunits to the other site. The α subunit contains five modified amino acids with still unknown function [13]. One of them is a highly conserved thioglycine forming a thiopeptide bond, which is susceptible to reduction-induced transcis isomerisation and which could therefore play a key role in coupling of the two active sites [12].

Active MCR has a greenish colour with an absorbance maximum at 385 nm [14, 15, 16]. It exhibits a Ni(I)-based axial electron paramagnetic resonance (EPR) signal designated MCR-red1 (gz=2.25; gy=2.07; gx=2.06). Double integration of the signal of the fully active enzyme revealed that both active sites contain F430 in the reduced form, the spin concentration per Ni being approximately 0.9. The red1 signal shows a superhyperfine splitting due to the interaction of the electron of Ni(I) with the nuclear spin of the four nitrogens of the tetrapyrrolic ring system. The superhyperfine splitting is clearly resolved when the active enzyme is in the absence or presence of its substrates. It is much less resolved when the enzyme is in the presence of coenzyme M (MCR-red1c) [14, 16]. Coenzyme M inhibits MCR competitively to methyl-coenzyme M [14, 16, 17].

When MCR-red1c is supplemented with coenzyme B a novel Ni(I)-based rhombic EPR signal designated MCR-red2 (gz=2.29; gy=2.24; gx=2.18) is induced at the expense of the red1 signal. MCR with a fully induced red2 signal shows the red1 and red2 signals at almost equal intensity, each with a spin concentration per Ni of approximately 0.4 [16]. Concomitantly with the change in the EPR spectrum the absorption maximum at 385 nm decreased and the absorption at 420 nm increased [14, 16]. In the presence of coenzyme M and coenzyme B MCR thus appears to be present at 50% in a MCR-red1 state and at 50% in the MCR-red2 state [14, 16].

The conversion of the MCR-red1 state into the MCR-red2 state upon addition of coenzyme B occurs only in the presence of coenzyme M and is associated with the reversible coordination of the thiol group of coenzyme M to the active-site Ni(I) as revealed by EPR and electron–nuclear double resonance spectroscopic data with unlabelled and 33S-labelled coenzyme M [18, 19]. Apparently, the addition of coenzyme B induces a conformational change in MCR-red1c, bringing the thiol group of coenzyme M into binding distance of the Ni(I). Such a conformational change is probably also required in the reaction of methyl-coenzyme M with the Ni(I), which has been proposed to be the first step in the catalytic cycle of methyl-coenzyme M reduction to methane in all but one mechanism discussed [17, 20, 21, 22, 23, 24, 25]. The finding that in the presence of methyl-coenzyme M the addition of coenzyme B to MCR-red1 does not induce a change in the UV–vis and the EPR spectra [16] can be explained assuming that the coenzyme B triggered conformational change is the rate-limiting step in methane formation from methyl-coenzyme M.

Most of the biochemical properties reported for MCR in the literature were obtained for MCR isoenzyme I from M. marburgensis, which contains two MCR isoenzymes [26, 27, 28] and which has an optimum growth temperature of 65°C. The following study was therefore also performed with purified MCR I from the hydrogenotrophic archaeon. We report here that the conformational change induced in MCR by coenzyme B in the presence of coenzyme M becomes thermodynamically unfavourable at temperatures below 20°C and that this correlates with enzyme activity. We further provide evidence that the conformational change induced by coenzyme B is restricted at the time to only one of the two active sites of MCR.

Materials and methods

M. marburgensis (M. thermoautotrophicum, strain Marburg [29]) is the strain deposited under DSM 2133 in the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig). Coenzyme M (2-mercaptoethanesulfonate) was obtained from Merck (Darmstadt); methyl-coenzyme M was synthesized from coenzyme M by methylation with methyl iodide (Fluka) [16, 30]. Coenzyme B (N-7-mercaptoheptanoylthreonine phosphate) was prepared from the symmetric disulfide CoB-S-S-CoB by reduction with NaBH4 [31, 32]. N-6-mercaptohexanoylthreonine phosphate (HS-CoB6) and N-8-mercaptooctanoylthreonine phosphate (HS-CoB8) were synthesized and purified as previously described [32, 33].

Purification of active MCR

M. marburgensis was grown at 65°C in a 13-L glass fermenter (New Brunswick) containing 10 L mineral medium stirred at 1,200 rpm and gassed with 80% H2/20% CO2/0.1% H2S at a rate of 1,200 mL min−1 [16]. When a ΔOD578 of 4.5 was reached, the gas supply was switched to 100% H2 for 30 min to induce the EPR signals of MCR-red1 and MCR-red2 in the cells. After 30 min the cells were cooled to 10°C within 10 min under continuous gassing and harvested anaerobically by centrifugation using a flow-through centrifuge (Hettich, centrifuge 17 RS). Approximately 70 g of wet cells was obtained. From these cells only the MCR isoenzyme I was purified [26, 28]. All steps of the purification were performed in the presence of 10 mM coenzyme M and in an anaerobic chamber (Coy Instruments) filled with 95% N2/5% H2 as described previously [16]. During purification the enzyme lost its MCR-red2 signal owing to the removal of coenzyme B. In one purification generally 150 mg active MCR in the red1c state (in 10 mL) was obtained. The purified enzyme exhibited a greenish colour and showed an UV–vis spectrum at room temperature with a maximum at 385 nm (ɛ=54,000 M−1 cm−1), and an axial EPR signal with gz=2.25, gy=2.07 and gx=2.06 characteristic for MCR-red1c [14, 16, 34]. The spin concentration per mole of F430 was approximately 0.9.

The protein concentration was determined using the method of Bradford [35] with bovine serum albumin (Serva) as a standard or by measuring the absorbance difference of oxidized enzyme (MCR-silent) at 420 nm using ɛ=44,000 M−1 cm−1 for a molecular mass of 280,000 Da. Both methods yielded almost the same results.

MCR activity determination

MCR activity was determined by following methane formation at temperatures between 0 and 75°C gas-chromatographically. The assays were performed in 8-mL serum bottles containing 0.4 mL assay solution and closed with a rubber stopper. The assay solution was composed of 50 mM tris(hydroxymethyl)aminomethane (Tris)/HCl, pH 7.6, 10 mM methyl-coenzyme M, 0.5 mM CoB-S-S-CoB, 10 mM Ti(III)citrate, 0.3 mM hydroxycobalamin and 20–200 μg MCR. The gas phase was 95% N2/5% H2. The reaction was started by the addition of MCR. At intervals of 2 min, 0.2-mL gas samples were withdrawn and analysed for methane by gas chromatography [15, 28].

EPR spectroscopy

Samples (0.35 mL) were routinely analysed by EPR spectroscopy at −196°C in 0.3-cm (inner diameter) quartz tubes with 95% N2/5% H2 as the gas phase and closed with a closed-off rubber tube. The EPR tubes were frozen in liquid nitrogen or, where indicated, in a viscous liquid nitrogen/ethanol mixture. For room temperature EPR measurements, the samples were analysed in quartz flat cell tubes with 95% N2/5% H2 as the gas phase and closed with parafilm. The samples contained 2.2–31 mg MCR (7.9–113 nmol) in 10 Tris/HCl, pH 7.6. EPR spectra at the X-band (9.4 GHz) were obtained with a Bruker EMX-6/1 EPR spectrometer composed of the EMX 1/3 console, an ER 041 X6 bridge with a built-in ER-0410-116 microwave frequency counter, an ER-070 magnet and an ER-4102st standard universal rectangular cavity. All spectra were recorded with a field modulation frequency of 100 kHz. Cooling of the sample was performed either with an Oxford Instruments ESR 900 cryostat with an ITC4 temperature controller or with liquid nitrogen in a finger Dewar at −196°C.

The EPR spin quantitations were carried out under nonsaturating conditions using 10 mM copper perchlorate as the standard (10 mM CuSO4; 2M NaClO4; 10 mM HCl). All signal intensities are expressed as spin concentrations per mole of F430. Note that the accuracy of the method is not very high, so the spin concentrations obtained have an error of at least 10%.

Results

As already indicated in the “Introduction”, active MCR (MCR-red1) is in part converted into the MCR-red2 state in the presence of both coenzyme M and coenzyme B. We now found that below 20°C the extent of conversion is temperature-dependent.

Temperature dependence of MCR-red1 activity

Purified MCR-red1c from M. marburgensis had a specific activity of approximately 0.1 units per milligram at 0°C and one of approximately 30 units per milligram at 65°C, the optimum activity of this enzyme (Fig. 1). From 0 to 65°C the Q10 (VT+10°C/VT) continuously decreased from approximately 4 between 0 and 10°C to below 2 between 55 and 65°C.
Fig. 1

Temperature dependence of methane formation from methyl-coenzyme M and coenzyme B as catalysed by methyl-coenzyme M reductase (MCR) from Methanothermobacter marburgensis. The 0.4-mL assay mixture contained 50 mM tris(hydroxymethyl)aminomethane (Tris)/HCl pH 7.6, 1 mM coenzyme B, 10 mM methyl-coenzyme M, 10 mM Ti(III)citrate, 0.3 mM hydroxycobalamin and purified MCR-red1c: 200 μg at temperatures between 0 and 15°C and 20 μg at temperatures above 15°C. The specific activity was approximately 50 mU mg−1 at 1°C and 220 mU mg−1 at 10°C.

Induction of the MCR-red2 state by coenzyme B and coenzyme B analogues

When MCR-red1c solutions at room temperature (spin concentration of 0.9 mol−1 Ni), which contained 10 mM coenzyme M, were supplemented with coenzyme B at 5 mM approximately 40% of MCR-red1 was converted into the MCR-red2 state (spin concentration of 0.3–0.4) and 60% remained in the MCR-red1 state (spin concentration of 0.5–0.6) as indicated by the EPR spectrum measured at −196°C (Table 1). The percentage converted was in the same range when MCR-red1c samples with lower spin concentrations were analysed. The conversion was reversible (not shown). The spin concentration of the red2 signal did not increase significantly when the coenzyme B concentration (5 mM) and/or the coenzyme M concentration (10 mM) in the enzyme solution were/was increased. The MCR-red2 state was not induced when coenzyme M was omitted or substituted by methyl-coenzyme M (not shown).
Table 1

Effect of coenzyme B (HS-CoB) and coenzyme B analogues on the activity and electron paramagnetic resonance spectroscopic properties of active methyl-coenzyme M reductase (MCR) from Methanothermobacter marburgensis

The coenzyme B induced MCR-red2 signal was irreversibly converted into the MCR-ox3 signal (gz=2.22; gy=2.14; gx=2.13) and the MCR-red1 signal disappeared upon exposure of the enzyme to air. The spin concentration of the ox3 signal was 0.45 per mole of F430 (Table 1) and thus 50% greater than the spin concentration of the red2 signal, which was 0.3 per mole of F430. The conversion of the red2 signal into the ox3 signal upon exposure of active MCR to air is a means to probe for the MCR-red2 state [34].

The coenzyme B analogue N-6-mercaptohexanoylthreonine phosphate (HS-CoB6) has been reported to be both a substrate and an inhibitor of coenzyme B (for the structures see Table 1) [23, 32]. With HS-CoB6 as a substrate, MCR has less than 1% of the activity shown with HS-CoB (see also Ref. [23]). We therefore tested whether this compound could also induce the MCR-red2 state, which was found to be the case (not shown). However, even at relatively high concentrations of HS-CoB6 (5 mM) only approximately 10% of the MCR-red1 state was converted to the MCR-red2 state, which was reflected in the low intensity of the ox3 signal exhibited by the enzyme after exposure to air (Table 1).

Of interest is that CH3-S-CoB can also induce the MCR-red2 state although this coenzyme B analogue is not a substrate and that HS-CoB8 and CH3-CH2-CoB cannot induce the MCR-red2 state although these compounds are potent inhibitors (Table 1) [34]. The finding that the red2 signal exhibited by active MCR in the presence of coenzyme M and methyl-coenzyme B was quenched by O2 rather than converted to the ox3 signal (Table 1) indicates that for the formation of the ox3 signal the free thiol group of coenzyme B is required.

Temperature-dependent equilibrium between the MCR-red1 and MCR-red2 states

Figure 2 shows the EPR spectrum measured at −196°C of a MCR-red1c sample (10 mM coenzyme M) to which at room temperature (18°C) coenzyme B had been added and which was then frozen in liquid nitrogen. Double integration revealed the presence of 40% MCR-red2 and 60% MCR-red1 ([MCR-red2]/[MCR-red1]≈1). When before freezing the temperature of the sample was lowered to 0°C, the percentage of MCR-red2 in the frozen EPR sample decreased to nearly 10% and that of MCR-red1 increased to 90% ([MCR-red2]/[MCR-red1]≈0.1) (Fig. 2). Almost identical results were obtained when the EPR tubes with the samples were frozen in liquid nitrogen/ethanol mixtures, in which cooling of the samples is much more rapid than in liquid nitrogen alone (not shown).
Fig. 2

Electron paramagnetic resonance (EPR) spectra at −196°C of MCR in the MCR-red1/2 state prepared at 18 and at 0°C. MCR-red1/2 is active MCR in the presence of 10 mM coenzyme M and 5 mM coenzyme B and exhibits the axial MCR-red1 signal (gz=2.25; gy=2.07; gx=2.06) and the rhombic MCR-red2 signal (gz=2.29; gy=2.24; gx=2.18). The sample at 18°C and that at 0°C were frozen by immersion of the EPR tubes in liquid nitrogen. The concentration of purified enzyme in both samples was 2.2 mg (7.9 nmol) in 0.35 mL 10 mM Tris/HCl, pH 7.6. The spin concentration in the samples was approximately 0.9 per mole of F430. Spectra were recorded under the following conditions: microwave frequency, 9,439 MHz; microwave power incident to the cavity, 2.01 mW; temperature, −196°C; modulation amplitude, 0.6 mT.

This temperature-dependent change was also seen in the UV–vis spectrum (Fig. 3). At 0°C the spectrum of active MCR in the presence of both coenzyme M and coenzyme B was very similar to that of MCR-red1c in the absence of coenzyme B (Fig. 3, dashed line). Upon increase of the temperature to 25°C the absorption at 385 nm, reflecting the concentration of MCR-red1, decreased. These changes were reversible. Apparently under the experimental conditions the MCR-red1 and MCR-red2 states were in a temperature-dependent equilibrium. As indicated from the plot of the absorbance at 385 nm versus the temperature, maximal conversion of the MCR-red1 state into the MCR-red2 state was reached at 20°C (Fig. 3, inset). Above this temperature the percentage of conversion remained constant.
Fig. 3

UV–vis spectra of MCR in the MCR-red1/2 state at temperatures between 25 and 0°C. For comparison the spectrum of MCR in the red1c state at room temperature is given (dashed line). MCR-red1c is active MCR in the presence of 10 mM coenzyme M. The inset shows the absorbance changes at 385 nm versus the temperature. The concentration of purified enzyme was 1.4 mg (5.1 nmol) in 1 mL 10 mM Tris/HCl pH 7.6. The spectra were not corrected for the 10% inactive MCR present in the samples.

The lowest temperature investigated was 0°C, at which approximately 10% of the MCR was present in the red2 state. At temperatures below 0°C the samples froze. When the samples were supplemented with ethylene glycol (10%) to prevent freezing maximal conversion (50%) of the MCR-red1 state into the MCR-red2 state was reached at about 5°C (not shown), indicating that in the presence of ethylene glycol the enzyme stayed flexible to lower temperatures.

Room temperature EPR spectra of the MCR-red1 and MCR-red2 states

MCR-red1c also exhibited the characteristic red1c signal when the EPR spectrum was measured at room temperature. The signals measured at −196 (Fig. 4a, spectrum 1) and 18°C (Fig. 4b, spectrum 1) were almost identical in line shape. The signal at 18°C had, however, only 3.3% of the intensity of that at −196°C. For MCR-red1c supplemented with coenzyme B the EPR spectra measured at −196 (Fig. 4a, spectrum 2) and 18°C (Fig. 4b, spectrum 2) were different. The spectrum at 18°C lacked the red2 signal and the red1 signal had only 1.8% of the intensity of that at −196°C. Comparison of the EPR spectra determined at room temperature revealed that the red1 signal exhibited by MCR-red1c (Fig. 4b, spectrum 1) had almost twice the intensity of the red1 signal shown by the enzyme in the presence of coenzyme B (Fig. 4b, spectrum 2).
Fig. 4

EPR spectra of MCR measured a at −196°C and b at room temperature (18°C). 1 MCR-red1c. 2 MCR-red1/2. 3 MCR-ox3 samples as for spectra 2 but after exposure to air. Before measurement all samples were at room temperature. For measurement of the EPR spectra at −196°C the samples were frozen in liquid nitrogen a and for the measurements at 18°C the samples were left at room temperature b. The concentration of purified enzyme in the samples in a was 8.9 mg (32 nmol) and in the samples in b 31 mg (113 nmol) in 0.35 mL 10 mM Tris/HCl, pH 7.6. Spectra were recorded under the following conditions: microwave frequency, (a) 9,434 MHz, (b) 9,746 MHz; microwave power incident to the cavity, (a) 2.00 mW, (b) 6.35 mW; temperature, (a) −196°C, (b) 18°C; modulation amplitude, 0.6 mT. The double integral of the EPR signals of spectra 1 and 2 in a were almost identical and that of the signal of spectrum 3 in a was 50% of that of the signals of spectra 1 and 2 in a. The double integral of the signal of spectrum 2 in b was 56% of that of spectrum 1 in b and that of spectrum 3 in b was 45% of that of spectrum 1 in b. When corrected for the different protein concentrations the signals of spectra 1, 2 and 3 in b had 3.3, 1.8 and 3.1% of the intensity of the signals of spectra 1, 2 and 3 in a, respectively.

As a control the samples at room temperature were exposed to air and then their EPR spectrum was measured at −196 (Fig. 4a, spectrum 3) and 18°C (Fig. 4b, spectrum 3). At both temperatures the ox3 signal was seen. At 18°C the intensity of the ox3 signal was 3.1% of that at −196°C. Apparently only the red2 signal does not show up at room temperature.

Temperature dependence of the MCR-red1c and MCR-red2 EPR signal intensity

To understand why the red1 signal rather than the red2 signal is visible at room temperature, the temperature dependence of the red1 and red2 signal intensities of MCR in the frozen state was determined (Fig. 5). For experimental reasons the temperature dependence could not be determined up to 0°C. From the slopes of the signal intensity decrease it can be estimated, however, that at 0°C the intensity of the red2 signal should be too low to be detectable.
Fig. 5

Intensities of the EPR signals of MCR-red1/2 measured at different temperatures. Active MCR in the presence of 10 mM coenzyme M was supplemented at room temperature with 5 mM coenzyme B to induce the MCR-red1/2 state and subsequently frozen in liquid nitrogen. In the measurement, the temperature of the frozen sample was varied from −268.5 to −53°C with an Oxford Instruments ESR 900 cryostat with an ITC4 temperature controller. The normalized signal amplitudes of the red1 and red2 signals were determined by measuring the height of the gx,y=2.066 peak for the red1 component and of the gz=2.288 peak for the red2 component at different temperatures. These amplitudes were normalized for power (nonsaturating), gain and temperature, and were plotted against the temperature. The concentration of purified enzyme was 30 mg (108 nmol) in 0.35 mL 50 mM Tris/HCl, pH 7.6. EPR conditions: microwave frequency, 9,458 MHz; microwave power, 2.01 μW (from −268.5 to −223°C) or 0.201 mW (from −223 to −53°C)

Both EPR signals exhibited a very short spin lattice relaxation time (T1). Owing to the Heisenberg uncertainty principle this causes the signals to be broad and the broadening to be temperature-dependent. As a result at room temperature the signals are often too broad to be detectable. In Fig. 5 the broadening can be detected as a decrease in the signal amplitude of the gxy peak (g=2.07/2.06) of the MCR-red1 signal and the gz=2.29 peak of the MCR-red2 signal at higher temperatures. The little jumps in the curves were due to artefacts in the temperature measurements and have not been corrected for since these changed from measurement to measurement. The figure shows that both signals are power-saturated below −253°C. Above −148°C the red2 signal started to broaden first. The differences in the Curie behaviour of the red1 and red2 signals is probably due to the fact that the nickel in the red1 state is five-coordinate with four equatorial N ligands and one axial O ligand, whereas the reduced nickel in the red2 form is six-coordinate with four equatorial ligands, one lower axial O ligand and one upper S ligand [18, 19].

Discussion

It was found that active MCR in the presence of both coenzyme M and coenzyme B is in a temperature-dependent equilibrium between the MCR-red1 and MCR-red2 states. The equilibrium constant (Keq=[MCR-red2]/[MCR-red1]) was 0.1 at 0°C and increased to approximately 1 at 20°C. Above 20°C Keq remained constant. This was shown both by EPR spectroscopy and by UV–vis spectroscopy.

Thermodynamics predicts for an endothermic reaction that the equilibrium constant increases with temperature until Keq=∞. The finding that the increase in MCR-red2 with increasing temperature levelled off, when approximately 50% of the red1 signal present initially was converted to the red2 signal (Keq=1) was therefore unexpected. It can be explained, however, considering that each MCR molecule harbours two structurally interacting active sites and postulating that in the presence of coenzyme M and coenzyme B only one of the two active sites can be converted from the MCR-red1 state into the MCR-red2 state.

$$ {\text{MCR - red}}{\text{1}} \mathord{\left/ {\vphantom {{\text{1}} {\text{1}}}} \right. \kern-\nulldelimiterspace} {\text{1}} + {\text{HS - CoM}} + {\text{HS - CoB}} \rightleftharpoons {\text{MCR - red}}{\text{1}} \mathord{\left/ {\vphantom {{\text{1}} {\text{2}}}} \right. \kern-\nulldelimiterspace} {\text{2}}, $$
(1)
where MCR-red1/1 denotes a MCR molecule with both active sites in the red1 state and MCR-red1/2 a MCR molecule with one active site in the red1 state and the other in the red2 state. In the case of reaction 1, Keq=∞ when in the sample 50% of the active sites are in the red2 state. The observed temperature dependence is therefore compatible with the formation of MCR-red1/2 from MCR-red1/1 in the presence of coenzyme M and coenzyme B and not with the formation of MCR-red2/2 in substantial amounts, at least not in the temperature range tested.

The temperature dependence thus indicates that in the presence of coenzyme M and coenzyme B the two active sites in one MCR molecule are in two different states, suggesting that active MCR shows “half-of-the-sites reactivity” [36]. Half-of-the-sites reactivity has been reported for many multimeric enzymes, examples being CTP synthase, phosphoribosylpyrophosphate aminotransferase and glutamine synthase [37], the pyruvate dehydrogenase complex [38] and aldehyde dehydrogenase [39, 40].

Interestingly, the red1 states in MCR-red1/1 and MCR-red1/2 do not appear to be identical, as is to be expected from a half-of-the-sites reactivity mechanism in which the binding of substrates in one active site affects the reactivity in the second active site. Thus the g values of the red1 signal exhibited by MCR-red1/2 (gz=2.27; gy=2.08; gx=2.07) and by MCR-red1/1 (gz=2.25; gy=2.07; gx=2.06) are slightly but significantly different. In addition magnetic circular dichroism measurements show that the 800-nm band characteristics for the MCR-red1/1 spectrum is absent or is much less intense in the MCR-red1/2 spectrum [14]. It has to be considered, however, that the differences in the red1 states could also be due to isosteric rather than allosteric effects.

The reactivity of MCR-red1/1 and of MCR-red1/2 towards oxidants was also found to be different. Whereas MCR-red1/1 is rendered completely EPR-silent in the presence of polysulfide, sulfite and O2, the EPR signal of the enzyme in the red1/2 state is converted to the EPR signals ox1 by polysulfide, ox2 by sulfite and ox3 by O2 [34] (for the mechanism see Refs. [14, 41, 42]). The spin concentrations per mole of F430 of the ox signals of the inactive MCR-ox states are generally higher than 0.5, in some cases as high as 0.8 [15, 34, 43], indicating that both the red1 and the red2 signals of MCR-red1/2 are converted to an ox form.

The reduction of methyl-coenzyme M with coenzyme B catalysed by MCR takes place in a hydrophobic pocket from which water is excluded [9, 12]. The two substrates thus have to be stripped off water when entering the active site and after reacting the product CoM-S-S-CoB has to be expelled into the water phase. The latter is most probably achieved by a conformational change of the enzyme, which is driven by one of the exergonic steps in the catalytic cycle [24, 25]. This conformational change could be restricted to the active site, in which the exergonic step occurs, or could be extended to the second active site. The finding of half-of-the-sites reactivity for MCR is in favour of the extension to the second site. We therefore propose that MCR operates similarly to a two-stroke engine as outlined in the cartoon shown in Fig. 6. The intertwined hexameric structure of MCR is optimally suited for such a mechanism. Two-stroke mechanisms have been postulated previously for the chaperone system GroEL/GroES [44] and for the 20S proteasome [45].
Fig. 6

Cartoon of the two-stroke engine mechanism proposed for MCR. The scheme shows a MCR molecule containing the two active sites 1 and 2. The binding of methyl-coenzyme M (square) and coenzyme B (circle) to one active site induces a conformational change, which is required to expel the product heterodisulfide (circle-square) from the second site into the water phase.

The proposed two-stroke mechanism predicts that MCR molecules with one active site in the red1 state and the other one in a silent state (MCR-red1/silent) should be inactive. This could explain why different MCR-red1 preparations exhibit different activity per spin. Thus, MCR-red1 obtained by reduction with Ti(III) from MCR-ox1 always has approximately twice the specific activity (corrected for spin concentration) than MCR-red1 purified from cells in the presence of coenzyme M [16]. Consistent with MCR-red1/silent being inactive is the finding that MCR-red1c/silent apparently cannot be converted to MCR-red2/silent in the presence of coenzyme B since the percentage of MCR-red1c converted to MCR-red 2 does not increase with decreasing spin concentration of the initial MCR-red1c signal and thus with increasing MCR-red1c/silent concentrations.

How could one obtain experimental evidence for the proposed two-stroke catalytic mechanism of MCR? One would have to show in stopped-flow experiments that intermediates in the catalytic cycle oscillate in their concentrations with a frequency twice that of the turnover number of the enzyme. Attempts to identify intermediates have failed until now. An explanation for this could be that under the experimental conditions employed (65°C and HS-CoB as a substrate) the rate-limiting step in methane formation is the conformational change associated with ternary complex formation from MCR, methyl-coenzyme M and coenzyme B as outlined in the “Introduction”. Our results reported here on the temperature dependence of MCR and on the induction of the MCR-red2 state by HS-CoB6, with which MCR shows less than 1% of the activity observed with HS-CoB, may help in the future to find conditions where intermediates in the catalytic cycle of MCR can be observed.

Notes

Acknowledgements

This work was supported by the Max Planck Society, the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie and by a fellowship from the Claussen-Simon-Stiftung (M.G.). We thank Antonio Pierik for his help in measuring the room temperature EPR spectra.

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Copyright information

© SBIC 2005

Authors and Affiliations

  • Meike Goenrich
    • 1
  • Evert C. Duin
    • 2
  • Felix Mahlert
    • 1
  • Rudolf K. Thauer
    • 1
  1. 1.Max-Planck-Institut für terrestrische Mikrobiologie and Laboratorium für Mikrobiologie, Fachbereich BiologiePhilipps-UniversitätMarburgGermany
  2. 2.Department of Chemistry and BiochemistryAuburn UniversityUSA

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