The missing enzymatic link in syntrophic methane formation from fatty acids.

The microbial production of methane from organic matter is an essential process in the global carbon cycle and an important source of renewable energy. It involves the syntrophic interaction between methanogenic archaea and bacteria that convert primary fermentation products such as fatty acids to the methanogenic substrates acetate, H2, CO2, or formate. While the concept of syntrophic methane formation was developed half a century ago, the highly endergonic reduction of CO2 to methane by electrons derived from ß-oxidation of saturated fatty acids has remained hypothetical. Here, we studied a previously noncharacterized membrane-bound oxidoreductase (EMO) from Syntrophus aciditrophicus containing two heme b cofactors and 8-methylmenaquinone as key redox components of the redox loop-driven reduction of CO2 by acyl-coenzyme A (CoA). Using solubilized EMO and proteoliposomes, we reconstituted the entire electron transfer chain from acyl-CoA to CO2 and identified the transfer from a high- to a low-potential heme b with perfectly adjusted midpoint potentials as key steps in syntrophic fatty acid oxidation. The results close our gap of knowledge in the conversion of biomass into methane and identify EMOs as key players of ß-oxidation in (methyl)menaquinone-containing organisms.

Enoyl-Coenzyme A Respiration via Formate Cycling in Syntrophic Bacteria.

Syntrophic bacteria play a key role in the anaerobic conversion of biological matter to methane. They convert short-chain fatty acids or alcohols to H2, formate, and acetate that serve as substrates for methanogenic archaea. Many syntrophic bacteria can also grow with unsaturated fatty acids such as crotonate without a syntrophic partner, and the reducing equivalents derived from the oxidation of one crotonate to two acetate are regenerated by the reduction of a second crotonate. However, it has remained unresolved how the oxidative and reductive catabolic branches are interconnected and how energy may be conserved in the reductive branch. Here, we provide evidence that during axenic growth of the syntrophic model organism Syntrophus aciditrophicus with crotonate, the NAD+-dependent oxidation of 3-hydroxybutyryl-CoA to acetoacetyl-CoA is coupled to the reduction of crotonyl-CoA via formate cycling. In this process, the intracellular formate generated by a NAD+-regenerating CO2 reductase is taken up by a periplasmic, membrane-bound formate dehydrogenase that in concert with a membrane-bound electron-transferring flavoprotein (ETF):methylmenaquinone oxidoreductase, ETF, and an acyl-CoA dehydrogenase reduces intracellular enoyl-CoA to acyl-CoA. This novel type of energy metabolism, referred to as enoyl-CoA respiration, generates a proton motive force via a methylmenaquinone-dependent redox-loop. As a result, the beneficial syntrophic cooperation of fermenting bacteria and methanogenic archaea during growth with saturated fatty acids appears to turn into a competition for formate and/or H2 during growth with unsaturated fatty acids. IMPORTANCE The syntrophic interaction of fermenting bacteria and methanogenic archaea is important for the global carbon cycle. As an example, it accomplishes the conversion of biomass-derived saturated fatty acid fermentation intermediates into methane. In contrast, unsaturated fatty acid intermediates such as crotonate may serve as growth substrate for the fermenting partner alone. Thereby, the reducing equivalents generated during the oxidation of one crotonate to two acetate are regenerated by reduction of a second crotonate to butyrate. Here, we show that the oxidative and reductive branches of this pathway are connected via formate cycling involving an energy-conserving redox-loop. We refer to this previously unknown type of energy metabolism as to enoyl-CoA respiration with acyl-CoA dehydrogenases serving as cytoplasmic terminal reductases.

The PmoB subunit of particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath): The CuI sponge and its function.

In this study, we describe efforts to clarify the role of the copper cofactors associated with subunit B (PmoB) of the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) (M. capsulatus). This subunit exhibits strong affinity toward CuI ions. To elucidate the high copper affinity of the subunit, the full-length PmoB, and the N-terminal truncated mutants PmoB33-414 and PmoB55-414, each fused to the maltose-binding protein (MBP), are cloned and over-expressed into Escherichia coli (E. coli) K12 TB1 cells. The Y374F, Y374S and M300L mutants of these protein constructs are also studied. When this E. coli is grown with the pmoB gene in 1.0 mM CuII, it behaves like M. capsulatus (Bath) cultured under high copper stress with abundant membrane accumulation and high CuI content. The recombinant PmoB proteins are verified by Western blotting of antibodies directed against the MBP sub-domain in each of the copper-enriched PmoB proteins. Cu K-edge X-ray absorption near edge spectroscopy (XANES) of the copper ions confirms that all the PmoB recombinants are CuI proteins. All the PmoB proteins show evidence of a "dicopper site" according to analysis of the Cu extended X-ray absorption edge fine structure (EXAFS) of the membranes. No specific activities toward methane and propene oxidation are observed with the recombinant membrane-bound PmoB proteins. However, significant production of hydrogen peroxide is observed in the case of the PmoB33-414 mutant. Reaction of the dicopper site with dioxygen produces hydrogen peroxide and leads to oxidation of the CuI ions residing in the C-terminal sub-domain of the PmoB subunit.

Bioenergetic aspects of archaeal and bacterial hydrogen metabolism.

Hydrogenases are metal-containing biocatalysts that reversibly convert protons and electrons to hydrogen gas. This reaction can contribute in different ways to the generation of the proton motive force (PMF) of a cell. One means of PMF generation involves reduction of protons on the inside of the cytoplasmic membrane, releasing H2 gas, which being without charge is freely diffusible across the cytoplasmic membrane, where it can be re-oxidized to release protons. A second route of PMF generation couples transfer of electrons derived from H2 oxidation to quinone reduction and concomitant proton uptake at the membrane-bound heme cofactor. This redox-loop mechanism, as originally formulated by Mitchell, requires a second, catalytically distinct, enzyme complex to re-oxidize quinol and release the protons outside the cell. A third way of generating PMF is also by electron transfer to quinones but on the outside of the membrane while directly drawing protons through the entire membrane. The cofactor-less membrane subunits involved are proposed to operate by a conformational mechanism (redox-linked proton pump). Finally, PMF can be generated through an electron bifurcation mechanism, whereby an exergonic reaction is tightly coupled with an endergonic reaction. In all cases the protons can be channelled back inside through a F1F0-ATPase to convert the 'energy' stored in the PMF into the universal cellular energy currency, ATP. New and exciting discoveries employing these mechanisms have recently been made on the bioenergetics of hydrogenases, which will be discussed here and placed in the context of their contribution to energy conservation.