Untersuchungen zum Katalysemechanismus von Methyl-Coenzym M Reduktase (MCR) aus methanogenen Archaea

Die 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-Mechani...

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Bibliographic Details
Main Author: Goenrich, Meike
Contributors: Thauer, Rudolf (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2004
Online Access:PDF Full Text
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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.