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Biochemistry of carbon monoxide dehydrogenase/acetyl -CoA synthase complex and heterodisulfide reductase from Methanosarcina thermophila: Key enzymes involved in aceticlastic methanogenesis

Eisuke Murakami, University of Nebraska - Lincoln

Abstract

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex from Methanosarcina thermophila catalyzes, (1) reversible oxidation of CO to CO2, (2) cleavage or synthesis of acetyl-CoA to or from a methyl group, CO, and coenzyme A, and (3) methyl transfer of tetrahydromethanopterin. The enzyme complex consists of three components and they can be separated by a treatment with a cationic detergent or limited proteolysis. By using electron paramagnetic resonance (EPR) spectroscopy, SDS polyacrylamide gel electrophoresis (SDS-PAGE), and kinetic methods, the location of the site for the acetyl-CoA cleavage was determined to be in the beta subunit of the five-subunit complex. Furthermore, it was shown that the other component(s) is (are) required for acetyl-CoA cleavage or synthesis indicating that there is intersubunit communication during catalysis. ^ Heterodisulfide reductase (HDR) from M. thermophila catalyzes the reduction of the heterodisulfide, CoB-S-S-CoM, to the corresponding free thiols. The enzyme contains two hemes and two [Fe4S4 ] clusters. The mechanism of electron transfer from 2-hydroxyphenazine, which is a water-soluble analog of the physiological electron donor methanophenazine, to HDR were studied by steady-state and presteady-state kinetics, and spectroscopic methods. It was shown that the high potential heme and the low potential [Fe 4S4] cluster are not involved in the electron transfer pathway from 2-hydroxyphenazine to CoB-S-S-CoM. Based on the results, the electron transfer pathway was proposed to be from 2-hydroxyphenazine → [Fe 4S4]high → hemelow → CoB-S-S-CoM. ^ The HDR reaction is known as an energy conservation step since HDR transfers protons across the cytoplasmic membrane during the reduction of CoB-S-S-CoM and generates a proton gradient that leads to ATP synthesis. To study this proton translocation, the purified HDR from M. thermophila was reconstituted in liposomes. The proton translocation activity was measured by following the pH change in the reaction mixture during catalysis by the proteoliposomes. The optimal conditions for preparation of proteoliposomes were obtained, and proton translocation activity was observed, although these results were not reproducible. ^

Subject Area

Chemistry, Biochemistry|Chemistry, Organic

Recommended Citation

Murakami, Eisuke, "Biochemistry of carbon monoxide dehydrogenase/acetyl -CoA synthase complex and heterodisulfide reductase from Methanosarcina thermophila: Key enzymes involved in aceticlastic methanogenesis" (2000). ETD collection for University of Nebraska - Lincoln. AAI9967395.
http://digitalcommons.unl.edu/dissertations/AAI9967395

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