Two sub-states of the red2 state of methyl-coenzyme M reductase revealed by high-field EPR spectroscopy

Methyl-coenzyme M reductase (MCR) catalyzes the formation of methane from methyl-coenzyme M and coenzyme B in methanogenic archaea. The enzyme has two structurally interlinked active sites embedded in an alpha(2)beta(2)gamma(2) subunit structure. Each active site has the nickel porphyrinoid F(430) as a prosthetic group. In the active state, F(430) contains the transition metal in the Ni(I) oxidation state. The active enzyme exhibits an axial Ni(I)-based continuous wave (CW) electron paramagnetic resonance (EPR) signal, called red1a in the absence of substrates or red1c in the presence of coenzyme M. Addition of coenzyme B to the MCR-red1 state can partially and reversibly convert it into the MCR-red2 form, which shows a rhombic Ni(I)-based EPR signal (at X-band microwave frequencies of approximately 9.4 GHz). In this report we present evidence from high-field/high-frequency CW EPR spectroscopy (W-band, microwave frequency of approximately 94 GHz) that the red2 state consists of two substates that could not be resolved by EPR spectroscopy at X-band frequencies. At W-band it becomes apparent that upon addition of coenzyme B to MCR in the red1c state, two red2 EPR signals are induced, not one as was previously believed. The first signal is the well-characterized (ortho)rhombic EPR signal, thus far called red2, while the second previously unidentified signal is axial. We have named the two substates MCR-red2r and MCR-red2a after their rhombic and axial signals, respectively

The key nickel enzyme of methanogenesis catalyses the anaerobic oxidation of methane

Large amounts (estimates range from 70 Tg per year to 300 Tg per year) of the potent greenhouse gas methane are oxidized to carbon dioxide in marine sediments by communities of methanotrophic archaea and sulphate-reducing bacteria1, 2, 3, and thus are prevented from escaping into the atmosphere. Indirect evidence indicates that the anaerobic oxidation of methane might proceed as the reverse of archaeal methanogenesis from carbon dioxide with the nickel-containing methyl-coenzyme M reductase (MCR) as the methane-activating enzyme4, 5. However, experiments showing that MCR can catalyse the endergonic back reaction have been lacking. Here we report that purified MCR from Methanothermobacter marburgensis converts methane into methyl-coenzyme M under equilibrium conditions with apparent Vmax (maximum rate) and Km (Michaelis constant) values consistent with the observed in vivo kinetics of the anaerobic oxidation of methane with sulphate6, 7, 8. This result supports the hypothesis of ‘reverse methanogenesis’4, 9 and is paramount to understanding the still-unknown mechanism of the last step of methanogenesis. The ability of MCR to cleave the particularly strong C–H bond of methane without the involvement of highly reactive oxygen-derived intermediates is directly relevant to catalytic C–H activation, currently an area of great interest in chemistry10, 11, 12, 13

Experimental methods for screening parameters influencing the growth to product yield (Y_(x/CH4)) of a biological methane production (BMP) process performed with <em>Methanothermobacter marburgensis</em>

1. Specht M, Brellochs J, Frick V, et al. (2010) Storage of renewable energy in the natural gas grid. Erdoel, Erdgas, Kohle 126: 342-345.2. Thauer RK, Kaster AK, Goenrich M, et al. (2010) Hydrogenases from methanogenic archaea, nickel, a novel cofactor, and H2 storage. Annu Rev Biochem 79: 507-536.3. Liu Y, Whitman WB (2008) Metabolic, phylogenetic, and ecological diversity of the methanogenic archaea. Ann N Y Acad Sci 1125: 171-189.4. Kaster AK, Goenrich M, Seedorf H, et al. (2011) More than 200 genes required for methane formation from H2 and CO2 and energy conservation are present in Methanothermobacter marburgensis and Methanothermobacter thermautotrophicus. Archaea ID 973848: 1-23.5. Seifert AH, Rittmann S, Herwig C (2014) Analysis of process related factors to increase volumetric productivity and quality of biomethane with Methanothermobacter marburgensis Appl Energ 132: 155-162.6. Bernacchi S, Weissgram M, Wukovits W, et al. (2014) Process efficiency simulation for key process parameters in biological methanogenesis. AIMS bioengineering 1: 53-71.7. Thauer RK, Kaster AK, Seedorf H, et al. (2008) Methanogenic archaea: ecologically relevant differences in energy conservation. Nat Rev Microbiol 6: 579-591.8. Schill N, van Gulik WM, Voisard D, et al. (1996) Continuous cultures limited by a gaseous substrate: development of a simple, unstructure mathematical model and experimental verification with Methanobacterium thermoautotrophicum. Biotechnol Bioeng 51: 645-658.9. Jud G, Schneider K, Bachofen R (1997) The role of hydrogen mass transfer for the growth kinetics of Methanobacterium thermoautotrophicum in batch and chemostat cultures. J Ind Microbiol Biotechnol 19: 246-251.10. Tsao JH, Kaneshiro SM, Yu SS, et al. (1994) Continuous culture of Methanococcus jannaschii, an extremely thermophilic methanogen. Biotechnol Bioeng 43: 258-261.11. Schill N, van Gulik WM, Voisard D, et al. (1996) Continuous cultures limited by a gaseous substrate: development of a simple, unstructured mathematical model and experimental verification with Methanobacterium thermoautotrophicum. Biotechnol Bioeng 51: 645-658.12. Peillex JP, Fardeau ML, Belaich JP (1990) Growth of Methanobacterium thermoautotrophicum on hydrogen-carbon dioxide: high methane productivities in continuous culture. Biomass 21:315-321.13. Nishimura N, Kitaura S, Mimura A, et al. (1992) Cultivation of thermophilic methanogen KN-15 on hydrogen-carbon dioxide under pressurized conditions. J Ferment Bioeng 73: 477-480.14. Morii H, Koga Y, Nagai S (1987) Energetic analysis of the growth of Methanobrevibacter arboriphilus A2 in hydrogen-limited continuous cultures. Biotechnol Bioeng 29: 310-315.15. de Poorter LMI, Geerts WJ, Keltjens JT (2007) Coupling of Methanothermobacter thermautotrophicus methane formation and growth in fed-batch and continuous cultures under different H2 gassing regimens. Appl Environ Microbiol 73: 740-749.16. Schoenheit P, Moll J, Thauer RK (1980) Growth parameters (Ks, μmax, Ys) of Methanobacterium thermoautotrophicum. Arch Microbiol 127: 59-65.17. Gerhard E, Butsch BM, Marison IW, et al. (1993) Improved growth and methane production conditions for Methanobacterium thermoautotrophicum. Appl Microbiol Biotechnol 40: 432-437.18. Seifert AH, Rittmann S, Bernacchi S, et al. (2013) Method for assessing the impact of emission gasses on physiology and productivity in biological methanogenesis. Bioresour Technol 136:747-751.19. Fardeau ML, Peillex JP, Belaich JP (1987) Energetics of the growth of Methanobacterium thermoautotrophicum and Methanococcus thermolithotrophicus on ammonium chloride and dinitrogen. Arch Microbiol 148: 128-131.20. Fardeau ML, Belaich JP (1986) Energetics of the growth of Methanococcus thermolithotrophicus. Arch Microbiol 144: 381-385.21. Morgan RM, Pihl TD, Nolling J (1997) Hydrogen regulation of growth, growth yields, and methane gene transcription in Methanobacterium thermoautotrophicum Delta H. J Bacteriol 179:889-898.22. Archer DB (1985) Uncoupling of methanogenesis from growth of Methanosarcina barkeri by phosphate limitation. Appl Environ Microbiol 50: 1233-1237.23. Rittmann S, Seifert A, Herwig C (2012) Quantitative analysis of media dilution rate effects on Methanothermobacter marburgensis grown in continuous culture on H2 and CO2. Biomass Bioenerg 36: 293-301.24. Fuchs G, Stupperich E, Thauer RK (1978) Acetate assimilation and the synthesis of alanine, aspartate and glutamate in Methanobacterium thermoautotrophicum. Arch Microbiol 117: 61-66.25. Schoenheit P, Moll J, Thauer RK (1979) Nickel, cobalt, and molybdenum requirement for growth of Methanobacterium thermoautotrophicum. Arch Microbiol 123: 105-107.26. Bonacker LG, Baudner S, Thauer RK (1992) Differential expression of the two methyl-coenzyme M reductases in Methanobacterium thermoautotrophicum as determined immunochemically via isoenzyme-specific antisera. Eur J Biochem 206: 87-92.27. Bonacker LG, Baudner S, Moerschel E, et al. (1993) Properties of the two isoenzymes of methyl-coenzyme M reductase in Methanobacterium thermoautotrophicum. Eur J Biochem 217:587-595.28. Hallenbeck PC, Ghosh D (2009) Advances in fermentative biohydrogen production: the way forward? Trends Biotechnol 27: 287-297.29. Wang J, Wan W (2008) Optimization of fermentative hydrogen production process by response surface methodology. Int J Hydrogen Energy 33: 6976-6984.30. Rittmann S, Herwig C (2012) A comprehensive and quantitative review of dark fermentative biohydrogen production. Microbial Cell Factories 11:115.31. Spadiut O, Rittmann S, Dietzsch C, et al. (2013) Dynamic process conditions in bioprocess development. Eng Life Sci 13: 88-101.32. Rittmann S, Seifert A, Herwig C (2013) Essential prerequisites for successful bioprocess development of biological CH4 production from CO2 and H2. Crit Rev Biotechnol 1-11.33. Costa KC, Yoon SH, Pan M, et al. (2013) Effects of H2 and formate on growth yield and regulation of methanogenesis in Methanococcus maripaludis. J Bacteriol 195: 1456-1462.34. Haydock AK, Porat I, Whitman WB, et al. (2004) Continuous culture of Methanococcus maripaludis under defined nutrient conditions. FEMS Microbiol Lett 238: 85-91

Catabolic Pathways and Enzymes Involved in Anaerobic Methane Oxidation

Microbes use two distinct catabolic pathways for life with the fuel methane: aerobic methane oxidation carried out by bacteria and anaerobic methane oxidation carried out by archaea. The archaea capable of anaerobic oxidation of methane, anaerobic methanotrophs (ANME), are phylogenetically related to methanogens. While the carbon metabolism in ANME follows the pathway of reverse methanogenesis, the mode of electron transfer from methane oxidation to the terminal oxidant is remarkably versatile. This chapter discusses the catabolic pathways of methane oxidation coupled to the reduction of nitrate, sulfate, and metal oxides. Methane oxidation with sulfate and metal oxides are hypothesized to involve direct interspecies electron transfer and extracellular electron transfer. Cultivation of ANME, their mechanisms of energy conservation, and details about the electron transfer pathways to the ultimate oxidants are rather new and quickly developing research fields, which may reveal novel metabolisms and redox reactions. The second section focuses on the carbon catabolism from methane to CO2 and the biochemistry in ANME with its unique enzymes containing Fe, Ni, Co, Mo, and W that are compared with their homologues found in methanogens