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Abstract

The greenhouse gas and energy carrier methane is produced on Earth mainly by methanogenic archaea. In the hydrogenotrophic methanogenic pathway the reduction of one CO₂ to one methane molecule requires four molecules of H₂ containing eight electrons. Four of the electrons from two H₂ are supplied for reduction of an electron carrier F₄₂₀, which is catalyzed by F₄₂₀-reducing [NiFe]-hydrogenase under nickel-sufficient conditions. The same reaction is catalysed under nickel-limiting conditions by [Fe]-hydrogenase coupled with a reaction catalyzed by F₄₂₀-dependent methylene tetrahydromethanopterin dehydrogenase. [Fe]-hydrogenase contains an iron-guanylylpyridinol (FeGP) cofactor for H₂ activation at the active site. Fe^II of FeGP is coordinated to a pyridinol-nitrogen, an acyl-carbon, two CO and a cysteine-thiolate. We report here on comparative genomic analyses of biosynthetic genes of the FeGP cofactor, which are primarily located in a hmd-co-occurring (hcg) gene cluster. One of the gene products is HcgB which transfers the guanosine monophosphate (GMP) moiety from guanosine triphosphate (GTP) to a pyridinol precursor. Crystal structure analysis of HcgB from Methanococcus maripaludis and its complex with 6-carboxymethyl-3,5-dimethyl-4-hydroxy-2-pyridinol confirmed the physiological guanylyltransferase reaction. Furthermore, we tested the properties of semi-synthetic [Fe]-hydrogenases using the [Fe]-hydrogenase apoenzyme from several methanogenic archaea and a mimic of the FeGP cofactor. On the basis of the enzymatic reactions involved in the methanogenic pathway, we came up with an idea how the methanogenic pathway could be simplified to develop an artificial methanogenesis system.