Reiner Hedderich

Methanogenic archaea: ecologically relevant differences in energy conservation

Most methanogenic archaea can reduce CO2 with H2 to methane, and it is generally assumed that the reactions and mechanisms of energy conservation that are involved are largely the same in all methanogens. However, this does not take into account the fact that methanogens with cytochromes have considerably higher growth yields and threshold concentrations for H2 than methanogens without cytochromes. These and other differences can be explained by the proposal outlined in this Review that in methanogens with cytochromes, the first and last steps in methanogenesis from CO2 are coupled chemiosmotically, whereas in methanogens without cytochromes, these steps are energetically coupled by a cytoplasmic enzyme complex that mediates flavin-based electron bifurcation.

The Genome Sequence of Methanosphaera stadtmanae Reveals Why This Human Intestinal Archaeon Is Restricted to Methanol and H2 for Methane Formation and ATP Synthesis

Methanosphaera stadtmanae has the most restricted energy metabolism of all methanogenic archaea. This human intestinal inhabitant can generate methane only by reduction of methanol with H2 and is dependent on acetate as a carbon source. We report here the genome sequence of M. stadtmanae, which was found to be composed of 1,767,403 bp with an average G+C content of 28% and to harbor only 1,534 protein-encoding sequences (CDS). The genome lacks 37 CDS present in the genomes of all other methanogens. Among these are the CDS for synthesis of molybdopterin and for synthesis of the carbon monoxide dehydrogenase/acetyl-coenzyme A synthase complex, which explains why M. stadtmanae cannot reduce CO2 to methane or oxidize methanol to CO2 and why this archaeon is dependent on acetate for biosynthesis of cell components. Four sets of mtaABC genes coding for methanol:coenzyme M methyltransferases were found in the genome of M. stadtmanae. These genes exhibit homology to mta genes previously identified in Methanosarcina species. The M. stadtmanae genome also contains at least 323 CDS not present in the genomes of all other archaea. Seventy-three of these CDS exhibit high levels of homology to CDS in genomes of bacteria and eukaryotes. These 73 CDS include 12 CDS which are unusually long (>2,400 bp) with conspicuous repetitive sequence elements, 13 CDS which exhibit sequence similarity on the protein level to CDS encoding enzymes involved in the biosynthesis of cell surface antigens in bacteria, and 5 CDS which exhibit sequence similarity to the subunits of bacterial type I and III restriction-modification systems.

Energy-converting [NiFe] hydrogenases from archaea and extremophiles: ancestors of complex I.

Abstract[NiFe] hydrogenases are well-characterized enzymes that have a key function in the H2 metabolism of various microorganisms. In the recent years a subfamily of [NiFe] hydrogenases with unique properties has been identified. The members of this family form multisubunit membrane-bound enzyme complexes composed of at least four hydrophilic and two integral membrane proteins. These six conserved subunits, which built the core of these hydrogenases, have closely related counterparts in energy-conserving NADH:quinone oxidoreductases (complex I). However, the reaction catalyzed by these hydrogenases differs significantly from the reaction catalyzed by complex I. For some of these hydrogenases the physiological role is to catalyze the reduction of H+ with electrons derived from reduced ferredoxins or polyferredoxins. This exergonic reaction is coupled to energy conservation by means of electron-transport phosphorylation. Other members of this hydrogenase family mainly function to provide the cell with reduced ferredoxin with H2 as electron donor in a reaction driven by reverse electron transport. As complex I these hydrogenases function as ion pumps and have therefore been designated as energy-converting [NiFe] hydrogenases.

LytB protein catalyzes the terminal step of the 2‐C‐methyl‐D‐erythritol‐4‐phosphate pathway of isoprenoid biosynthesis

Recombinant LytB protein from the thermophilic eubacterium Aquifex aeolicus produced in Escherichia coli was purified to apparent homogeneity. The purified LytB protein catalyzed the reduction of (E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl diphosphate (HMBPP) in a defined in vitro system. The reaction products were identified as isopentenyl diphosphate and dimethylallyl diphosphate. A spectrophotometric assay was established to determine the steady‐state kinetic parameters of LytB protein. The maximal specific activity of 6.6±0.3 μmol min−1 mg−1 protein was determined at pH 7.5 and 60°C. The k cat value of the LytB protein was 3.7±0.2 s−1 and the K m value for HMBPP was 590±60 μM.

N5,N10-Methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum has hydrogenase activity

N5,N10‐Methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum (strain Marburg) was purified under anaerobic conditions to apparent homogeneity and a specific activity of approximately 750 μol/min/mg protein. Polyacrylamide gel electrophoresis under native and denaturing conditions revealed that the enzyme is composed of only one polypeptide with an apparent molecular mass of 43 kDa. The purified enzyme catalyzed the dehydrogenation of N5,N10‐methylenetetrahydromethanopterin (CH2=H4MPT) (apparent Km≡20 μM) to N5,N10‐methenyltetrahydromethanopterin (CH≡H4MPT) in the absence of any added electron acceptors. One mol of H2 was generated per mol CH≡H4MPT formed, indicating that protons served as electron acceptor. Coenzyme F420, NAD, NADP and viologen dyes were not reduced by CH2=H4MPT. The dehydrogenase also catalyzed the reverse reaction, the reduction of CH≡H4MPT to CH2=H4MPT with H2. The data indicate that CH2=H4MPT dehydrogenase from M. thermoautotrophicum is a novel type of hydrogenase.

Energy-Converting [NiFe] Hydrogenases: More than Just H2 Activation

The well-characterized [NiFe] hydrogenases have a key function in the H2 metabolism of various microorganisms. A subfamily of the [NiFe] hydrogenases with unique properties has recently been identified. The six conserved subunits that build the core of these membrane-bound hydrogenases share sequence similarity with subunits that form the catalytic core of energy-conserving NADH:quinone oxidoreductases (complex I). The physiological role of some of these hydrogenases is to catalyze the reduction of H+ with electrons derived from reduced ferredoxins or polyferredoxins. This exergonic reaction is coupled to energy conservation by means of electron-transport phosphorylation. Other members of this hydrogenase subfamily mainly function in providing the cell with reduced ferredoxin using H2 as electron donor in a reaction driven by reverse electron transport. These hydrogenases have therefore been designated as energy-converting [NiFe] hydrogenases.

Reactions and Enzymes Involved in Methanogenesis from CO2 and H2

This chapter concentrates on the reactions and enzymes involved in methanogenesis from CO2 and H2. The coenzymes and electron carriers involved are described only as far as necessary for the understanding of their functions; they are dealt with extensively in other chapters. The bioenergetics of CO2 reduction to CH4 are only briefly discussed. For details the reader is referred to Chapter 8. We have tried to cover the complete literature dealing with the enzymology of methanogenesis from CO2 and H2. However, mainly work is cited which was performed with purified or at least partially purified preparations following the famous dictum of Efraim Racker: don’t waste clean thinking on dirty enzymes.

Functional characterization of GcpE, an essential enzyme of the non‐mevalonate pathway of isoprenoid biosynthesis

The gcpE gene product controls one of the terminal steps of isoprenoid biosynthesis via the mevalonate independent 2‐C‐methyl‐D‐erythritol‐4‐phosphate (MEP) pathway. This pathway is utilized by a variety of eubacteria, the plastids of algae and higher plants, and the plastid‐like organelle of malaria parasites. Recombinant GcpE protein from the hyperthermophilic bacterium Thermus thermophilus was produced in Escherichia coli and purified under dioxygen‐free conditions. The protein was enzymatically active in converting 2‐C‐methyl‐D‐erythritol‐2,4‐cyclodiphosphate (MEcPP) into (E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl diphosphate (HMBPP) in the presence of dithionite as reductant. The maximal specific activity was 0.6 μmol min−1 mg−1 at pH 7.5 and 55°C. The k cat value was 0.4 s−1 and the K m value for HMBPP 0.42 mM.

Sodium Ion Pumps and Hydrogen Production in Glutamate Fermenting Anaerobic Bacteria

Anaerobic bacteria ferment glutamate via two different pathways to ammonia, carbon dioxide, acetate, butyrate and molecular hydrogen. The coenzyme B12-dependent pathway in Clostridium tetanomorphum via 3-methylaspartate involves pyruvate:ferredoxin oxidoreductase and a novel enzyme, a membrane-bound NADH:ferredoxin oxidoreductase. The flavin- and iron-sulfur-containing enzyme probably uses the energy difference between reduced ferredoxin and NADH to generate an electrochemical Na+ gradient, which drives transport processes. The other pathway via 2-hydroxyglutarate in Acidaminococcus fermentans and Fusobacterium nucleatum involves glutaconyl-CoA decarboxylase, which uses the free energy of decarboxylation to generate also an electrochemical Na+ gradient. In the latter two organisms, similar membrane-bound NADH:ferredoxin oxidoreductases have been characterized. We propose that in the hydroxyglutarate pathway these oxidoreductases work in the reverse direction, whereby the reduction of ferredoxin by NADH is driven by the Na+ gradient. The reduced ferredoxin is required for hydrogen production and the activation of radical enzymes. Further examples show that reduced ferredoxin is an agent, whose reducing energy is about 1 ATP ‘richer’ than that of NADH.

Learning from hydrogenases: location of a proton pump and of a second FMN in bovine NADH-ubiquinone oxidoreductase (Complex I).

Hydrogenases have clear evolutionary links to the much more complex NADH–ubiquinone oxidoreductases (Complex I). Certain membrane‐bound [NiFe]‐hydrogenases presumably pump protons. From a detailed comparison of hydrogenases and Complex I, it is concluded here that the TYKY subunit in these enzymes is a special 2[4Fe–4S] ferredoxin, which functions as the electrical driving unit for a proton pump. The comparison further revealed that the flavodoxin fold from [NiFe]‐hydrogenases is presumably conserved in the PSST subunit of Complex I. It is proposed that bovine Complex I and the soluble NAD+‐reducing hydrogenase from Ralstonia eutropha each contain a second FMN group.