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The Na+-translocating methyltransferase complex from methanogenic archaea
No full textAbstractMethanogenic archaea are dependent on sodium ions for methane formation. A sodium ion-dependent step has been shown to be methyl transfer from N5-methyltetrahydromethanopterin to coenzyme M. This exergonic reaction (ΔG°′=−30 kJ/mol) is catalyzed by a Na+-translocating membrane-associated multienzyme complex composed of eight different subunits, MtrA–H. Subunit MtrA harbors a cob(I)amide prosthetic group which is methylated and demethylated in the catalytic cycle, demethylation being sodium ion-dependent. Based on the finding that in the cob(II)amide oxidation state the corrinoid is bound in a base-off/His-on configuration it is proposed that methyl transfer from MtrA to coenzyme M is associated with a conformational change of the protein and that this change drives the electrogenic translocation of the sodium ions
H-2-FORMING N-5,N-10-METHYLENETETRAHYDROMETHANOPTERIN DEHYDROGENASE FROM METHANOBACTERIUM-THERMOAUTOTROPHICUM CATALYZES A STEREOSELECTIVE HYDRIDE TRANSFER AS DETERMINED BY 2-DIMENSIONAL NMR-SPECTROSCOPY
No full text5,6,7,8-Tetrahydromethanopterin is a coenzyme playing a key role in the energy metabolism of methanogenic archaea. In Methanobacterium thermoautotrophicum, the reduction of N5,N-10-methenyl-5,6,7,8-tetrahydromethanopterin at C(14a) with H-2 to N5,N-10-methylene-5,6,7,8-tetrahydromethanopterin can be catalyzed by H-2-forming methylenetetrahydromethanopterin dehydrogenase, a new hydrogenase present in most methanogenic archaea, which is unique because it does not contain nickel or iron/sulfur clusters. In this work, the stereochemistry of this enzymatic hydride-transfer reaction is elucidated by means of a series of heteronuclear two-dimensional NMR experiments. It is found that the hydride from H-2 is transferred by the enzyme into the rel-(pro-R) position of the C(14a) methylene group of the reaction product N5,N-10-methylene-5,6,7,8-tetrahydromethanopterin. NMR experiments are described that show that the hydrogen nucleus of the hydride transferred to the oxidized coenzyme partially originates from water. The stereochemical course of this reaction is the same as that for direct hydride transfer. It is demonstrated that the diastereotopic atoms at C(14a) of the reaction product epimerize in an uncatalyzed reaction under the conditions of operation of the enzyme (k = 0.01 s-1 at 58-degrees-C and pH 6.5). On the basis of the known relative configuration of the pterin moiety of 5,6,7,8-tetrahydromethanopterin [Schleucher, J., Schworer, B., Zirngibl, C., Koch, U., Weber, W., Egert, E., Thauer, R. K., & Griesinger, C. (1992) FEBS Lett. 314, 440-444], the absolute configuration of this moiety is tentatively assigned to be (6S,7S, 11R) on the basis of a comparison of the CD spectra of N5,N-10-methenyl-5,6,7,8-tetrahydromethanopterin and its analog N5,N-10-methenyl-5,6,7,8-tetrahydrofolate. Given this absolute configuration of the pterin moiety, the rel-(pro-R) stereochemistry of the C(14a) methylene proton corresponds to the absolute (pro-R) stereochemistry
N-Carboxymethanofuran (carbamate) formation from methanofuran and CO2 in methanogenic archaea - Thermodynamics and kinetics of the spontaneous reaction
No full textN-Carboxymethanofuran (carbamate) formation from unprotonated methanofuran (MFR) and CO2 is the first reaction in the reduction of CO2 to methane in methanogenic archaea. The reaction proceeds spontaneously. We address here the question whether the rate of spontaneous carbamate formation is high enough to account for the observed rate of methanogenesis from CO2. The rates of carbamate formation (v(1)) and cleavage (v(2)) were determined under equilibrium conditions via 2D proton exchange NMR spectroscopy (EXSY). At pH 7.0 and 300 K the second order rate constant k(1) of carbamate formation from 'MFR'(MFR + MFRH+) and 'CO2' (CO2 + H2CO3 + HCO3- + CO32-) was found to be 7 m(-1).s(-1) (v(1) = k(1) ['MFR'] ['CO2']) while the pseudo first order rate constant k(2) of carbamate cleavage was 12 s(-1) (v(2) = k(2) [carbamate]). The equilibrium constant K = k(1) /k(2) = [carbamate]/['MFR']['CO2'] was 0.6 m(-1) at pH 7.0 corresponding to a free energy change Delta G degrees' of + 1.3 kJ.mol(-1). The pH and temperature dependence of k(1) , of k(2) and of K were determined. From the second order rate constant k(1) it was calculated that under physiological conditions the rate of spontaneous carbamate formation is of the same order as the maximal rate of methane formation and as the rate of spontaneous CO2 formation from HCO3- in methanogenic archaea, the latter being important as CO2 is mainly present as HCO3- which has to be converted to CO2 before it can react with MFR. An enzyme catalyzed carbamate formation thus appears not to be required for methanogenesis from CO2. Consistent with this conclusion is our finding that the rate of carbamate formation was not enhanced by cell extracts of Methanosarcina barkeri and Methanobacterium thermoautotrophicum or by purified formylmethanofuran dehydrogenase which catalyzes the reduction of N-carboxymethanofuran to N-formylmethanofuran. From the concentrations of 'CO2' and of 'MFR' determined by 1D-NMR spectroscopy and the pK(a) of H2CO3 and of MFRH+ the concentrations of CO2 and of MFR were obtained, allowing to calculate k(1) (v(1) = k(1) [MFR] [CO2]). The second order rate constant k(1) was found to be approximately 1000 m(-1).s(-1) at 300 K and pH values between 7.0 and 8.0 which is in the order of k(1) values determined for other carbamate forming reactions by stopped flow
Re-citrate synthase from Clostridium kluyveri is phylogenetically related to homocitrate synthase and isopropylmalate synthase rather than to Si-citrate synthase.
No full text<div id="abstract-1" class="section abstract" style="text-align: justify; widows: 2; text-transform: none; background-color: #ffffff; text-indent: 0px; margin: 0px; outline-style: none; letter-spacing: normal; font: 13px/16px 'Lucida Sans Unicode', Arial, 'Lucida Grande', Tahoma, Verdana, Helvetica, sans-serif; white-space: normal; orphans: 2; color: #403838; clear: both; vertical-align: baseline; word-spacing: 0px; border-image: initial; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; border-width: 0px; padding: 0px"><p id="p-1" style="text-align: left; line-height: 1.5; margin: 15px 0px; outline-style: none; font-family: inherit; word-wrap: break-word; vertical-align: baseline; border-image: initial; border-width: 0px; padding: 0px">The synthesis of citrate from acetyl-coenzyme A and oxaloacetate is catalyzed in most organisms by a<span class="Apple-converted-space"> </span><em style="font-style: italic; margin: 0px; outline-style: none; font-family: inherit; vertical-align: baseline; border-image: initial; border-width: 0px; padding: 0px">Si</em>-citrate synthase, which is<span class="Apple-converted-space"> </span><em style="font-style: italic; margin: 0px; outline-style: none; font-family: inherit; vertical-align: baseline; border-image: initial; border-width: 0px; padding: 0px">Si</em>-face stereospecific with respect to C-2 of oxaloacetate. However, in<span class="Apple-converted-space"> </span><em style="font-style: italic; margin: 0px; outline-style: none; font-family: inherit; vertical-align: baseline; border-image: initial; border-width: 0px; padding: 0px">Clostridium kluyveri</em><span class="Apple-converted-space"> </span>and some other strictly anaerobic bacteria, the reaction is catalyzed by a<span class="Apple-converted-space"> </span><em style="font-style: italic; margin: 0px; outline-style: none; font-family: inherit; vertical-align: baseline; border-image: initial; border-width: 0px; padding: 0px">Re</em>-citrate synthase, whose primary structure has remained elusive. We report here that<span class="Apple-converted-space"> </span><em style="font-style: italic; margin: 0px; outline-style: none; font-family: inherit; vertical-align: baseline; border-image: initial; border-width: 0px; padding: 0px">Re</em>-citrate synthase from<span class="Apple-converted-space"> </span><em style="font-style: italic; margin: 0px; outline-style: none; font-family: inherit; vertical-align: baseline; border-image: initial; border-width: 0px; padding: 0px">C. kluyveri</em><span class="Apple-converted-space"> </span>is the product of a gene predicted to encode isopropylmalate synthase.<span class="Apple-converted-space"> </span><em style="font-style: italic; margin: 0px; outline-style: none; font-family: inherit; vertical-align: baseline; border-image: initial; border-width: 0px; padding: 0px">C. kluyveri</em><span class="Apple-converted-space"> </span>is also shown to contain a gene for<span class="Apple-converted-space"> </span><em style="font-style: italic; margin: 0px; outline-style: none; font-family: inherit; vertical-align: baseline; border-image: initial; border-width: 0px; padding: 0px">Si</em>-citrate synthase, which explains why cell extracts of the organism always exhibit some<span class="Apple-converted-space"> </span><em style="font-style: italic; margin: 0px; outline-style: none; font-family: inherit; vertical-align: baseline; border-image: initial; border-width: 0px; padding: 0px">Si</em>-citrate synthase activity.</p></div
More Than 200 Genes Required for Methane Formation from H(2) and CO(2) and Energy Conservation Are Present in Methanothermobacter marburgensis and Methanothermobacter thermautotrophicus
Get PDFThe hydrogenotrophic methanogensMethanothermobacter marburgensisandMethanothermobacter thermautotrophicuscan easily be mass cultured. They have therefore been used almost exclusively to study the biochemistry of methanogenesis from H2and CO2, and the genomes of these two model organisms have been sequenced. The close relationship of the two organisms is reflected in their genomic architecture and coding potential. Within the 1,607 protein coding sequences (CDS) in common, we identified approximately 200 CDS required for the synthesis of the enzymes, coenzymes, and prosthetic groups involved in CO2reduction to methane and in coupling this process with the phosphorylation of ADP. Approximately 20 additional genes, such as those for the biosynthesis of F430and methanofuran and for the posttranslational modifications of the two methyl-coenzyme M reductases, remain to be identified.</jats:p
Bioprocess development and quantitative analysis of biomethane production and dark fermentative biohydrogen production
No full textDie wissenschaftliche Bioprozessentwicklung zur Biomethanproduktion ist der Hauptfokus dieser Dissertation. Darüber hinaus werden jedoch auch die fermentativ basierte Bioprozessentwicklung zur Biowasserstoffproduktion und die Anwendung von dynamischen Bedingungen in der Bioprozessentwicklung behandelt. Die Voraussetzungen um die Biomethanproduktion quantifizieren zu können werden in dieser Dissertation ausführlich behandelt. Die Biomethanproduktion wird hauptsächlich durch den Gas-Flüssigphasentransfer und durch die komplexen Interaktionen zwischen der Gasphase, der Flüssigphase und der Zellen beeinflusst. Während der Überprüfung der Biomethanproduktion in größerem Maßstab konnte der Einfluß von Flüssigphasenkomponenten auf die Biomasseproduktivität der methanogenen Mikroben gezeigt werden. Die Quantifizierung der fermentativen Biowasserstoffproduktion seit dem Jahre 1902 wird in einer Abhandlung komplett analysiert und statistische Auswertungen zeigen die Vor- und Nachteile der mesophilen und thermophilen fermentativen Biowasserstoffproduktion sowie verschiedener Mikroben genau auf. Die kommerzielle Nutzung der fermentativen Biowasserstoffproduktion wird durch die ungewöhnlich komplizierte Verwendung von Einheiten in der wissenschaftlichen Literatur gehemmt. In dieser Dissertation wird aufgezeigt welche Einheiten für die industrielle Verwendung der fermentativen Biowasserstoffproduktion von Bedeutung sind. Des Weiteren kann eindeutig gezeigt werden, daß ein Vertreter der thermophilen Mikroben einen deutlichen Vorteil im Verhältnis der Produktion von Wasserstoff gegenüber der gleichzeitigen Produktion von Kohlendioxid aufweist. Die gegenwärtige Nutzung von biologisch erzeugten Kraftstoffen wird komplett Analysiert und eine Einteilung der Biokraftstoffgenerationen in fünf unterschiedliche Gruppen kann aufgezeigt werden. In dieser Dissertation wird die Biomethanproduktion als die fünfte Generation der biologischen erzeugten Kraftstoffe implementiert. Die Nutzung der Biomethanproduktion im Pilot-und im industriellen Maßstab kann durch die gezielte quantitative Analyse und parallel ablaufende Bioprozessentwicklung gewährleistet werden.Science-based bioprocess development for biological methane production (BMP) from gaseous substrates is the main focuses of this dissertation. However, also bioprocess development and quantitative analysis of dark fermentative biohydrogen production (BHP) is performed. The prerequisites for quantification of BMP by using closed batch, as well as fed-batch and chemostat culture cultivation are presented. Most crucial parameters influence the gas-limitation fundamentals or contribute to the complex effects that arise during medium development for scale-up of BMP. More than on century of dark fermentative BHP is reviewed. Statistically based evidence shows that thermophilic strains comprise high substrate conversion efficiency, but mesophilic strains achieve high volumetric productivity. Many shortcomings, which limit the utilisation of dark fermentative BHP in large-scale, emerge through a mingle-mangle of different entities and are lacking a clear vision for scale-up of this bioprocessing technology. A comparison of different biofuel bioprocessing applications shows that the 1st biofuel generation has been successfully carried into a commercial-scale, while the 2nd one still needs very much improvement. Biodiesel, biohydrogen and biomethane from the 3rd, 4th and 5th biofuel generation, respectively, are still in being studied in lab-scale to ensure high productivity and conversion efficiency. The utilisation of BMP for conversion and storage of biological and industrial waste gasses in the form of CH4 is just not any longer an illusion and the way forward to pilot- and industrial production scale for CH4 production is now feasible. Manufacturing BMP- based, decentralised or centralised, small- or large-scale power storage and H2/CO2 conversion plants, finally comes within our grasp
Energiestoffwechsel von Cytochrom-freien methanogenen Archaea bei Wachstum auf H2 und CO2
No full textMethanogene Archaea sind Organismen, die Methan als Endprodukt ihres anaeroben Energiestoffwechsels bilden. Es gibt zwei stoffwechselphysiologisch unterschiedliche Gruppen: Zum einen die Cytochrom-freien Methanogenen, welche nur H2 und CO2 und/oder Formiat zu Methan umsetzen und zum anderen die Cytochrom-haltigen Methanogenen, die auf Acetat, Methanol und/oder Methylamin spezialisiert sind und von denen nur einige wenige auf H2 und CO2 wachsen können. Da lediglich der Energiestoffwechsel Cytochrom-haltiger Methanogener bei Wachstum auf H2 und CO2 weitgehend verstanden ist, war es das Ziel der vorliegenden Arbeit, den Energiestoffwechsel Cytochrom-freier Methanogener aufzuklären. Dazu wurden zunächst die Wachstumserträge und H2-Schwellenkonzentrationen von zwei Cytochrom-freien Methanogenen bestimmt und mit denen eines Cytochrom-haltigen verglichen. Anschließend wurde die Kopplung des ersten und letzten Schrittes der Methanbildung aus H2 und CO2 auf Enzymebene untersucht.In methanogenic archaea growing on H2 and CO2 the first step in methanogenesis is the endergonic reduction of ferredoxin (Fd) with H2 and the last step is the exergonic reduction of the heterodisulfide CoM-S-S-CoB with H2 to coenzyme M (CoM-SH) and coenzyme B (CoB-SH). We recently proposed that in hydrogenotrophic methanogens the two reactions are energetically coupled via the cytoplasmic MvhADG/HdrABC complex. It is reported here that the purified complex from Methanothermobacter marburgensis catalyzes the CoM-S-S-CoB dependent reduction of Fd with H2. Per mol CoM-S-S-CoB added, which appeared to be completely reduced to CoM-S-H and CoB-SH, one mol of Fd was reduced indicating an electron bifurcation coupling mechanism:
2 H2 + Fdox + CoM-S-S-CoB → Fdred2- + CoM-SH + CoB-SH + 2 H+.
This stoichiometry of coupling is consistent with an ATP gain per mol methane from 4 H2 and CO2 of near 0.5 deduced from a H2-threshold concentration of 8 Pa and a growth yield of up to 3 g per mol methane which we have determined for two hydrogenotrophic methanogens
Probing the catalytic mechanism of methyl-coenzyme M reductase (MCR) from methanogenic archaea
No full textDie Bildung von Methan erfolgt in allen methanogenen Archeaen durch die Reduktion von Methyl-Coenzym M (CH3-S-CoM) mit Coenzym B (HS-CoB) zu CH4 und dem Heterodisulfid CoM-S-S-CoB. Diese Reaktion, die mit Umkehr der Stereokonfiguration der Methylgruppe erfolgt, wird in einem ternären Komplex-Mechanismus von Methyl-Coenzym M Reduktase (MCR) katalysiert. Das sauerstofflabile Enzym ist aus drei verschiedenen Untereinheiten zusammengesetzt, die in einem α2β2γ2 Hexamer angeordnet sind und zwei strukturell verknüpfte aktive Zentren ausbilden, in denen je ein Molekül des Nickelporphinoids Faktors F430 als prosthetische Gruppe wirkt. Im aktiven Enzym befindet sich F430 in der Oxidationsstufe Ni(I) und läßt sich durch seine paramagnetische Eigenschaft mittels Elektronenparamagnetischer Resonanz (EPR)-Spektroskopie detektieren. Derzeit lassen sich für MCR fünf EPR-aktive und zwei EPR-inaktive (silent) Zustände definieren: die enzymatisch aktiven Zustände MCR-red1 und MCR-red2, sowie die enzymatisch inaktiven Zustände MCR-ox1, MCR-ox2, MCR-ox3, MCR-ox1-silent und MCR-silent. Von den beiden Ni(II)-Formen ohne EPR Signal (MCR-ox1-silent und MCR-silent) liegen detaillierte Kristallstrukturen vor, die zusammen mit biochemischen Eigenschaften zur Formulierung von zwei alternativen Katalysemechanismen geführt haben: Mechanismus I favorisiert einen nukleophilen Angriff von Ni(I) auf die Methylgruppe von CH3-S-CoM, was zur Bildung einer Methyl-Ni(III)F430-Zwischenstufe führt. Dagegen postuliert Mechanismus II die Entstehung eines Methylradikals aufgrund eines Angriffs von Ni(I) auf den Thioetherschwefel von CH3-S-CoM.
In der vorliegenden Arbeit wurde die Wirkung von Methyl-Coenzym M- und Coenzym B-Substratanaloga auf die enzymatische Akivität und den Nickel-Redoxzustand von MCR untersucht, um tiefere Einblicke in den Katalysemechanismus dieses Enzyms zu erhalten. Neben Aktivitätsmessungen wurden dazu im wesentlichen EPR-spektroskopische Untersuchungen durchgeführt.
Analoga des Substrats CH3-S-CoM wurden aufgrund ihrer Wirkung in drei Gruppen unterteilt: (i) Reversible Inhibitoren wie Ethyl-Coenzym M, Propyl-Coenzym M, Allyl-Coenzym M und Coenzym M (HS-CoM), in deren Gegenwart der Ni(I)-Zustand erhalten blieb. Von den vier Inhibitoren wurde nur Ethyl-Coenzym M reduziert, allerdings mit einer katalytischen Effizienz, die geringer als 1% der Effizienz mit Methyl-Coenzym M war; (ii) Irreversible Inhibitoren wie 2-Bromoethansulfonat, 3-Bromopropionat, Cyano-Coenzym M, Seleno-Coenzym M und Trifluoromethyl-Coenzym M, die nach Zugabe zu aktiver MCR das Ni(I)-EPR-Signal auslöschten und bei Anwesenheit von HS-CoB zur Induktion eines isotropen Radikalsignals führten. Die Reaktivität des Ni(I)-Zustandes gegenüber dieser Gruppe von Inhibitoren wurde in Gegenwart von HS-CoB um das 10-fache gesteigert; und (iii) Irreversible Inhibitoren wie 3-Bromopropansulfonat, 3-Iodopropansulfonat und 4-Bromobutyrat, in deren Gegenwart das EPR Signal von aktiver MCR in das MCR-BPS-Signal umgewandelt wurde. Das MCR-BPS-Signal ist denen der MCRox-Signale ähnlich und wurde wie diese in Gegenwart von 2-Bromoethansulfonat nicht ausgelöscht. Messungen des magnetischen zirkularen Dichroismus (MCD) identifizierten Nickel im MCR-ox1-Zustand als High Spin Ni(II), welches axial mit einem Thiyl-Radikal koordiniert ist. Analog dazu könnte das MCR-BPS-Signal von einem Alkyl-Ni(III)-Zustand stammen.
Ein weiterer Schwerpunkt der Arbeit beschäftigt sich mit dem MCR-red2-Zustand, der im Enzym in Gegenwart von HS-CoM und HS-CoB induziert wird und durch ein rhombisches EPR-Signal charakterisiert ist. Eine solche Induktion wurde neben HS-CoB ebenfalls für die zwei HS-CoB-Analoga HS-CoB6 und Methyl-CoB beobachtet. Durch den Einsatz von 33S-markiertem Coenzym M konnte eindeutig gezeigt werden, daß im MCR-red2-Zustand der Thioetherschwefel von HS-CoM axial mit dem Ni(I) aus F430 koordiniert ist. Das Ausmaß der MCR-red2 Induktion durch HS-CoM und HS-CoB zeigte sich in den Untersuchungen abhängig von der Temperatur. Unterhalb von 20oC wandelte sich der red2-Zustand mit sinkender Temperatur mehr und mehr in den red1-Zustand um. Oberhalb von 20oC allerdings lagen nur maximal 50% des Enzyms im red2-Zustand vor, was u. a. dafür spricht, daß sich jeweils nur eines der beiden aktiven Zentren von MCR im red2-Zustand befindet. Dies weist auf eine Halbseitenreaktivität von MCR hin, was für eine phasenversetzte Kopplung der beiden aktiven Zentren, ähnlich wie in einem Zweitaktmotor, spricht.In methanogenic archaea methyl-coenzyme M reductase (MCR) catalyses the formation of methane from methyl-coenzyme M (CH3-S-CoM) and coenzyme B (HS-CoB). The enzyme has an α2β2γ2 subunit structure forming two structurally interlinked active sites each with a molecule F430 as 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 EPR signal and a UV-visible 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-visible spectrum with an absorption maximum at 420 nm.
One part of the work is concerned with methyl-coenzyme M analogues showing how they affect the activity and the MCR-red1 signal of MCR from Methanothermobacter marburgensis.
Ethyl-coenzyme M was the only methyl-coenzyme M analogue tested that was used by MCR as a substrate. Ethyl-coenzyme M was reduced to ethane (apparent KM = 20 mM; apparent Vmax = 0.1 U/mg) with a catalytic efficiency of less than 1% of that of methyl-coenzyme M reduction to methane (apparent KM = 5 mM; apparent Vmax = 30 U/mg). Propyl-coenzyme M (apparent Ki = 2 mM) and allyl-coenzyme M (apparent Ki = 0.1 mM) were reversible inhibitors.
2-Bromoethanesulfonate ([I]0.5V = 2 µM), cyano-coenzyme M ([I]0.5V = 0.2 mM), 3-bromopropionate ([I]0.5V = 3 mM), seleno-coenzyme M ([I]0.5V = 6 mM) and trifluoromethyl-coenzyme M ([I]0.5V = 6 mM) irreversibly inhibited the enzyme. In their presence the MRC-red1 signal was quenched indicating the oxidation of Ni(I) to Ni(II). The rate of oxidation in the presence of coenzyme B increased over 10 fold in the presence of coenzyme B indicating that the Ni(I) reactivity was increased. Enzyme inactivated in the presence of coenzyme B showed an isotropic signal characteristic of a radical, that is spin coupled with one hydrogen nucleus. The coupling was also observed in D2O. The signal was abolished upon exposure of the enzyme to O2.
3-Bromopropanesulfonate ([I]0.5V = 0.1 µM), 3-iodopropanesulfonate ([I]0.5V = 1 µM), and 4-bromobutyrate also inactivated MCR. In their presence the EPR signal of MCR-red1 was converted to a Ni based EPR signal MCR-BPS that resembles in line shape the MCR-ox1 signal. The signal was quenched by O2.
2-Bromoethanesulfonate and 3-bromopropanesulfonate, which both rapidly reacted with Ni(I) of MRC-red1, did not react with the Ni of MCR-ox1 and MCR-BPS: The Ni based EPR spectra of both inactive forms were not affected in the presence of high concentrations of these two potent inhibitors.
The second part reported 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 20oC 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). Based on this interpretation a two-stroke engine mechanism for MCR is proposed
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