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In the hydrogenotrophic methanogenic pathway, formylmethanofuran dehydrogenase (Fmd) catalyzes the formation of formylmethanofuran through reducing CO2. Heterodisulfide reductase (Hdr) provides two low potential electrons for the Fmd reaction using a flavin‐based electron‐bifurcating mechanism. [NiFe]‐hydrogenase (Mvh) or formate dehydrogenase (Fdh) complexes with Hdr and provides electrons to Hdr from H2 and formate, or the reduced form of F420, respectively. Recently, an Fdh‐Hdr complex was purified as a 3‐MDa megacomplex that contained Fmd, and its three‐dimensional structure was elucidated by cryo‐electron microscopy. In contrast, the Mvh‐Hdr complex has been characterized only as a complex without Fmd. Here, we report the isolation and characterization of a 1‐MDa Mvh‐Hdr‐Fmd megacomplex from Methanothermobacter marburgensis. After anion‐exchange and hydrophobic chromatography was performed, the proteins with Hdr activity eluted in the 1‐ and 0.5‐MDa fractions during size exclusion chromatography. Considering the apparent molecular mass and the protein profile in the fractions, the 1‐MDa megacomplex was determined to be a dimeric Mvh‐Hdr‐Fmd complex. The megacomplex fraction contained a polyferredoxin subunit MvhB, which contains 12 [4Fe‐4S]‐clusters. MvhB polyferredoxin has never been identified in the previously purified Mvh‐Hdr and Fmd preparations, suggesting that MvhB polyferredoxin is stabilized by the binding between Mvh‐Hdr and Fmd in the Mvh‐Hdr‐Fmd complex. The purified Mvh‐Hdr‐Fmd megacomplex catalyzed electron‐bifurcating reduction of [13C]‐CO2 to form [13C]‐formylmethanofuran in the absence of extrinsic ferredoxin. These results demonstrated that the subunits in the Mvh‐Hdr‐Fmd megacomplex are electronically connected for the reduction of CO2, which likely involves MvhB polyferredoxin as an electron relay. [ABSTRACT FROM AUTHOR]

Methylene‐tetrahydropterin reductases catalyze the reduction of a methylene to a methyl group bound to a reduced pterin as C1 carrier in various one‐carbon (C1) metabolisms. F420‐dependent methylene‐tetrahydromethanopterin (methylene‐H4MPT) reductase (Mer) and the flavin‐independent methylene‐tetrahydrofolate (methylene‐H4F) reductase (Mfr) use a ternary complex mechanism for the direct transfer of a hydride from F420H2 and NAD(P)H to the respective methylene group, whereas FAD‐dependent methylene‐H4F reductase (MTHFR) uses FAD as prosthetic group and a ping–pong mechanism to catalyze the reduction of methylene‐H4F. A ternary complex structure and a thereof derived catalytic mechanism of MTHFR is available, while no ternary complex structures of Mfr or Mer are reported. Here, Mer from Methanocaldococcus jannaschii (jMer) was heterologously produced and the crystal structures of the enzyme with and without F420 were determined. A ternary complex of jMer was modeled on the basis of the jMer‐F420 structure and the ternary complex structure of MTHFR by superimposing the polypeptide after fixing hydride‐transferring atoms of the flavins on each other, and by the subsequent transfer of the methyl‐tetrahydropterin from MTHFR to jMer. Mutational analysis of four functional amino acids, which are similarly positioned in the three reductase structures, indicated despite the insignificant sequence identity, a common catalytic mechanism with a 5‐iminium cation of methylene‐tetrahydropterin as intermediate protonated by a shared glutamate. According to structural, mutational and phylogenetic analysis, the evolution of the three reductases most likely proceeds via a convergent development although a divergent scenario requiring drastic structural changes of the common ancestor cannot be completely ruled out. [ABSTRACT FROM AUTHOR]

[Fe]‐hydrogenase harbors the iron‐guanylylpyridinol (FeGP) cofactor, in which the Fe(II) complex contains acyl‐carbon, pyridinol‐nitrogen, cysteine‐thiolate and two CO as ligands. Irradiation with UV‐A/blue light decomposes the FeGP cofactor to a 6‐carboxymethyl‐4‐guanylyl‐2‐pyridone (GP) and other components. Previous in vitro biosynthesis experiments indicated that the acyl‐ and CO‐ligands in the FeGP cofactor can scramble, but whether scrambling occurred during biosynthesis or photolysis was unclear. Here, we demonstrate that the [18O1‐carboxy]‐group of GP is incorporated into the FeGP cofactor by in vitro biosynthesis. MS/MS analysis of the 18O‐labeled FeGP cofactor revealed that the produced [18O1]‐acyl group is not exchanged with a CO ligand of the cofactor, indicating that the acyl and CO ligands are scrambled during photolysis rather than biosynthesis, which ruled out any biosynthesis mechanisms allowing acyl/CO ligands scrambling. Time‐resolved infrared spectroscopy indicated that an acyl‐Fe(CO)3 intermediate is formed during photolysis, in which scrambling of the CO and acyl ligands can occur. This finding also suggests that the light‐excited FeGP cofactor has a higher affinity for external CO. These results contribute to our understanding of the biosynthesis and photosensitive properties of this unique H2‐activating natural complex. [ABSTRACT FROM AUTHOR]

The anaerobic oxidation of methane (AOM) with sulphate, an area currently generating great interest in microbiology, is accomplished by consortia of methanotrophic archaea (ANME) and sulphate-reducing bacteria (1,2). The enzyme activating methane in methanotrophic archaea has tentatively been identified as a homologue of methyl-coenzyme M reductase (MCR) that catalyses the methane-forming step in methanogenic archaea (3,4).Here we report an X-ray structure of the 280 kDa heterohexameric ANME-1MCR complex. It was crystallized uniquely from a protein ensemble purified from consortia of microorganisms collected with a submersible from a Black Sea mat catalysing AOM with sulphate (4). Crystals grown from the heterogeneous sample diffract to 2.1 A resolution and consist of a single ANME-1 MCR population, demonstrating the strong selective power of crystallization. The structure revealed ANME-1 MCR in complex with coenzyme M and coenzyme B, indicating the same substrates for MCR from methanotrophic and methanogenic archaea. Differences between the highly similar structures of ANME-1 MCR and methanogenic MCR include a [F.sub.430] modification, a cysteine-rich patch and an altered post-translational amino acid modification pattern, which may tune the enzymes for their functions in different biological contexts., Large amounts (up to 300 Tg per year) of the potent greenhouse gas, methane, are oxidized to C[O.sub.2] (reaction 1) in marine sediments by consortia of methanotrophic archaea (ANME-1, ANME-2 [...]