Publications

• Isolation and characterization of methanophenazine and function of phenazines in membrane-bound electron transport of Methanosarcina mazei Goe1.

  Abken H.J., Tietze M., Brodersen J., Baeumer S., Beifuss U., Deppenmeier U.

  A hydrophobic, redox-active component with a molecular mass of 538 Da was isolated from lyophilized membranes of Methanosarcina mazei Gö1 by extraction with isooctane. After purification on a high-performance liquid chromatography column, the chemical structure was analyzed by mass spectroscopy an ... >> More

  A hydrophobic, redox-active component with a molecular mass of 538 Da was isolated from lyophilized membranes of Methanosarcina mazei Gö1 by extraction with isooctane. After purification on a high-performance liquid chromatography column, the chemical structure was analyzed by mass spectroscopy and nuclear magnetic resonance studies. The component was called methanophenazine and represents a 2-hydroxyphenazine derivative which is connected via an ether bridge to a polyisoprenoid side chain. Since methanophenazine was almost insoluble in aqueous buffers, water-soluble phenazine derivatives were tested for their ability to interact with membrane-bound enzymes involved in electron transport and energy conservation. The purified F42OH2 dehydrogenase from M. mazei Gö1 showed highest activity with 2-hydroxyphenazine and 2-bromophenazine as electron acceptors when F420H2 was added. Phenazine-1-carboxylic acid and phenazine proved to be less effective. The Km values for 2-hydroxyphenazine and phenazine were 35 and 250 microM, respectively. 2-Hydroxyphenazine was also reduced by molecular hydrogen catalyzed by an F420-nonreactive hydrogenase which is present in washed membrane preparations. Furthermore, the membrane-bound heterodisulfide reductase was able to use reduced 2-hydroxyphenazine as an electron donor for the reduction of CoB-S-S-CoM. Considering all these results, it is reasonable to assume that methanophenazine plays an important role in vivo in membrane-bound electron transport of M. mazei Gö1. << Less

  J. Bacteriol. 180:2027-2032(1998) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Purification and properties of the heme- and iron-sulfur-containing heterodisulfide reductase from Methanosarcina thermophila.

  Simianu M., Murakami E., Brewer J.M., Ragsdale S.W.

  The heterodisulfide reductase (HDR) from Methanosarcina thermophila was purified to homogeneity from acetate-grown cells. In the absence of detergents, HDR consisted of an eight-protein complex with hydrogenase activity. However, when HDR was purified in the presence of 0.6% Triton X-100, a two-su ... >> More

  The heterodisulfide reductase (HDR) from Methanosarcina thermophila was purified to homogeneity from acetate-grown cells. In the absence of detergents, HDR consisted of an eight-protein complex with hydrogenase activity. However, when HDR was purified in the presence of 0.6% Triton X-100, a two-subunit (53 and 27 kDa) high specific activity ( approximately 200 units mg-1) enzyme was obtained that lacked hydrogenase activity. Sedimentation equilibrium experiments demonstrated that HDR has a molecular mass of 206 kDa and a high partial specific volume (0.9 cm3/g), indicating that the purified protein contains a significant amount of bound lipid. Like the HDR from Methanosarcina barkeri [Kunkel, A., Vaupel, M., Heim, S., Thauer, R. K., and Hedderich, R. (1997) Eur. J. Biochem. 244, 226-234], it was found to contain two discrete b-type hemes in the small subunit and two distinct [Fe4S4]2+/1+ clusters in the large subunit. One heme is high-spin and has a high midpoint potential (-23 mV), whereas the other heme apparently is low-spin and exhibits a relatively low midpoint potential (-180 mV). Only the high-spin heme binds CO. The midpoint potentials for the two clusters are -100 and -400 mV. In the fully reduced state, a complicated EPR spectrum with g values of 2.03, 1.97, 1.92, and 1.88 was observed. This spectrum resembles that of 8Fe ferredoxins in the fully reduced state, indicating that the two clusters in HDR are near enough to experience relatively strong dipolar interactions. Kinetic studies in which CO oxidation is coupled to heterodisulfide reduction strongly indicate that a membrane-associated compound is the direct electron donor to HDR. An electron-transfer pathway is presented that postulates a mechanism for coupling electron transport to proton translocation. << Less

  Biochemistry 37:10027-10039(1998) [PubMed] [EuropePMC]

• Purification and properties of heterodisulfide reductase from Methanobacterium thermoautotrophicum (strain Marburg).

  Hedderich R., Berkessel A., Thauer R.K.

  The reduction of the heterodisulfide of coenzyme M (H-S-CoM) and 7-mercaptoheptanoyl-L-threonine phosphate (H-S-HTP) is a key reaction in the metabolism of methanogenic bacteria. The heterodisulfide reductase catalyzing this step was purified 80-fold to apparent homogeneity from Methanobacterium t ... >> More

  The reduction of the heterodisulfide of coenzyme M (H-S-CoM) and 7-mercaptoheptanoyl-L-threonine phosphate (H-S-HTP) is a key reaction in the metabolism of methanogenic bacteria. The heterodisulfide reductase catalyzing this step was purified 80-fold to apparent homogeneity from Methanobacterium thermoautotrophicum. The native enzyme showed an apparent molecular mass of 550 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of three different subunits of apparent molecular masses 80 kDa, 36 kDa, and 21 kDa. The enzyme, which was brownish yellow, contained per mg protein 7 +/- 1 nmol FAD, 130 +/- 10 nmol non-heme iron and 130 +/-10 nmol acid-labile sulfur, corresponding to 4 mol FAD and 72 mol FeS/mol native enzyme. The purified heterodisulfide reductase catalyzed the reduction of CoM-S-S-HTP (app. Km = 0.1 mM) with reduced benzylviologen at a specific rate of 30 mumol.min-1.mg protein-1 (kcat = 68 s-1) and the reduction of methylene blue with H-S-CoM (app. Km = 0.2 mM) plus H-S-HTP (app. Km less than 0.05 mM) at a specific rate of 15 mumol.min-1.mg-1. The enzyme was highly specific for CoM-S-S-HTP and H-S-CoM plus H-S-HTP. The physiological electron donor/acceptor remains to be identified. << Less

  Eur. J. Biochem. 193:255-261(1990) [PubMed] [EuropePMC]

• Characterization of the intramolecular electron transfer pathway from 2-hydroxyphenazine to the heterodisulfide reductase from Methanosarcina thermophila.

  Murakami E., Deppenmeier U., Ragsdale S.W.

  Heterodisulfide reductase (HDR) is a component of the energy-conserving electron transfer system in methanogens. HDR catalyzes the two-electron reduction of coenzyme B-S-S-coenzyme M (CoB-S-S-CoM), the heterodisulfide product of the methyl-CoM reductase reaction, to free thiols, HS-CoB and HS-CoM. ... >> More

  Heterodisulfide reductase (HDR) is a component of the energy-conserving electron transfer system in methanogens. HDR catalyzes the two-electron reduction of coenzyme B-S-S-coenzyme M (CoB-S-S-CoM), the heterodisulfide product of the methyl-CoM reductase reaction, to free thiols, HS-CoB and HS-CoM. HDR from Methanosarcina thermophila contains two b-hemes and two [Fe(4)S(4)] clusters. The physiological electron donor for HDR appears to be methanophenazine (MPhen), a membrane-bound cofactor, which can be replaced by a water-soluble analog, 2-hydroxyphenazine (HPhen). This report describes the electron transfer pathway from reduced HPhen (HPhenH(2)) to CoB-S-S-CoM. Steady-state kinetic studies indicate a ping-pong mechanism for heterodisulfide reduction by HPhenH(2) with the following values: k(cat) = 74 s(-1) at 25 degrees C, K(m) (HPhenH(2)) = 92 microm, K(m) (CoB-S-S-CoM) = 144 microm. Rapid freeze-quench EPR and stopped-flow kinetic studies and inhibition experiments using CO and diphenylene iodonium indicate that only the low spin heme and the high potential FeS cluster are involved in CoB-S-S-CoM reduction by HPhenH(2). Fe-S cluster disruption by mersalyl acid inhibits heme reduction by HPhenH(2), suggesting that a 4Fe cluster is the initial electron acceptor from HPhenH(2). We propose the following electron transfer pathway: HPhenH(2) to the high potential 4Fe cluster, to the low potential heme, and finally, to CoB-S-S-CoM. << Less

  J. Biol. Chem. 276:2432-2439(2001) [PubMed] [EuropePMC]