Genetic analysis of the archaeon Methanosarcina barkeri Fusaro reveals a central role for Ech hydrogenase and ferredoxin in methanogenesis and carbon fixation -- Data Supplement - Supporting Information -- Proceedings of the National Academy of Sciences

Supporting information for Meuer et al. (April 2, 2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.072615499.

Detailed Methods for Enzyme Assays
Formation of CO₂ and H₂ from Formyl-Methanofuran by Membrane Fractions.
The assays contained 550 µg of membrane protein, 30 nmol of ferredoxin, and 400 µmol of formyl-methanofuran (CHO-MFR) in 1 ml of 50 mM MOPS, pH 7.0/2 mM DTT in sealed serum vials. The temperature was 37°C, and the gas phase (9 ml) was 100% N2 at 200 kPa. H2, CO₂, and CH₄ were measured by GC. Determination of Enzyme Activities.
All enzyme activities were measured under strictly anaerobic conditions in sealed gas-tight 1-ml cuvettes at 37°C. In hydrogenase assays, the gas phase was 100% H₂ at 130 kPa. All other assays were performed under 100% N₂ at 130 kPa. Ferredoxin-dependent hydrogenase (Ech) was assayed by using the ferredoxin isolated from acetate-grown Methanosarcina barkeri cells as described (1). The 0.8-ml assay mixture contained 50 mM MOPS (pH 7.0), 2 mM DTT, 2 mM dodecyl-b,d-maltoside, 20 µM ferredoxin, and 0.16 mM metronidazole (MTZ). The gas phase was 100% H₂ at 130 kPa. Ferredoxin reduction was measured by following the subsequent reduction of MTZ at 320 nm (e₃₂₀ = 9.3 mM^–1 cm^–1). F₄₂₀ reducing hydrogenase (Frh) was assayed by following the reduction of F₄₂₀ by H₂ photometrically at 420 nm (e₄₂₀ = 41 mM^–1 cm^–1). The 0.7-ml assay mixture contained 50 mM MOPS (pH 7.0), 40 µM F₄₂₀, and 2 mM DTT. Formyl-methanofuran dehydrogenase (Fmd) was assayed by following the reduction of benzyl viologen with formyl-methanofuran at 578 nm (e₅₇₈ = 8.6 mM^–1 cm^–1). The 0.8-ml assay mixture contained 50 mM MOPS (pH 7.0), 2 mM DTT, 2 mM benzyl viologen, and 40 µM formyl-methanofuran. Formyl-methanofuran was prepared as described (2). Methylene-H₄SPT dehydrogenase (Mtd) (H₄SPT, tetrahydrosarcinapterin) was assayed by following the formation of methenyl-H₄MPT (H₄MPT, tetrahydromethanopterin) from methylene-H₄MPT with F₄₂₀ as electron acceptor photometrically at 335 nm (e₃₃₅ = 21.6 mM^–1 cm^–1) (3). Methenyl-H₄SPT cyclohydrolase (Mch) was assayed as described (4) following the conversion of methenyl-H₄MPT to formyl-H₄MPT at 335 nm (e₃₃₅ = 21.6 mM^–1 cm^–1). Formyl-H₄SPT:methanofuran formyltransferase (Fth) was assayed by following the formation of formyl-H₄MPT from H₄MPT and formyl-methanofuran at 282 nm (e₂₈₂ = 5.9 mM^–1 cm^–1) as described (5). GC.
H₂ was measured by thermal conductivity with a Carlo Erba (Rodano, Italy) GC Series 6000 after separation over a molecular sieve (5 Å) column. CH₄, CO, and CO₂ were measured by using a Shimadzu GC (model 8A) with a connected methanizer and a flame ionization detector after separation over a Porapak A (50–80 mesh) column. A Hewlett–Packard 5890 series II (flame ionization detector) equipped with a Supelco 80/120 Carbopack B/3% SP-1500 column was used for quantification of CH₄in some experiments. The detection limit was 0.5 nmol for H₂, and below 0.1 nmol for CH₄, CO, and CO₂.

1. Meuer, J., Bartoschek, S., Koch, J., Künkel, A. & Hedderich, R. (1999) Eur. J. Biochem. 265, 325–335.

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3. Klein, A. R. & Thauer, R. K. (1997) Eur. J. Biochem. 245, 386–391.

4. Breitung, J., Schmitz, R. A., Stetter, K. O. & Thauer, R. K. (1991) Arch. Microbiol. 156, 517–524.

5. Breitung, J. & Thauer, R. K. (1990) FEBS Lett. 275, 226–230.