A di-heme FeS oxidoreductase involved in syntrophic methane formation and enoyl-CoA respiration via formate cycling

Methane is a potent greenhouse gas and a valuable energy source. Its production from biomass is crucial for the global carbon cycle and involves a syntrophic interaction between methanogenic archaea and fermenting bacteria. The latter convert primary fermentation products such as saturated fatty acids into the methanogenic substrates acetate, H2, CO2 or formate. It has remained unresolved for many decades how β-oxidation of saturated fatty acids is coupled to the reduction of CO2 during this process. Recent studies identified a di-heme and FeS-cluster dependent methylmenaquinone:ETF oxidoreductase (EMO) as missing link in the endergonic reduction of CO2 by electrons derived from the fermenting partner. Together with a membrane-bound formate dehydrogenase, it drives electron transfer from reduced ETF to CO2 by a reverse redox-loop. This energetic coupling appears to be crucial for all (methyl)menaquinone-dependent microorganisms during β-oxidation of fatty acids.The model organism Syntrophus aciditrophicus and many other syntrophic bacteria can also grow with unsaturated fatty acids like crotonate without a syntrophic partner. Thereby, oxidation of crotonate to acetate is coupled to the reduction of crotonate to butyrate or elongated reduced products [2]. We demonstrate that these oxidative and reductive branches are connected via formate cycling that generates a proton motive force via a methylmenaquinone-dependent redox-loop involving EMO. The formate formed intracellularly by NADH-dependent CO2 reduction is taken up by a periplasmic membrane-bound formate dehydrogenase. Electrons are transferred back to cytoplasmic enoyl-CoA via methylmenaquinone, EMO, ETF and acyl-CoA dehydrogenases, thereby a membrane potential is generated via a redox-loop. We refer to this mode of energy metabolism as enoyl-CoA respiration with acyl-CoA dehydrogenases serving as terminal reductases [3]

The missing enzymatic link in syntrophic methane formation from fatty acids

Significance The syntrophic interaction of fermenting bacteria with methanogenic archaea is crucial for the globally relevant conversion of biomass into methane. Fifty years after the discovery of syntrophy, it has remained enigmatic how the oxidation of saturated fatty acid fermentation intermediates can be coupled to the thermodynamically extremely unfavorable reduction of CO 2 to methane and how such a process can sustain growth of both syntrophic partners. Here, we provide biochemical evidence that heme b cofactors of a membrane-bound oxidoreductase and a modified quinone with perfectly fine-tuned redox potentials are the key players in this microbial process. Bioinformatics analyses suggest that the oxidoreductase plays a crucial role in lipid catabolism of the majority of prokaryotes. </jats:p

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 β-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 β-oxidation in (methyl)menaquinone-containing organisms

Serine 363 of a Hydrophobic Region of Archaeal Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Archaeoglobus fulgidus and Thermococcus kodakaraensis Affects CO2/O2 Substrate Specificity and Oxygen Sensitivity

Archaeal ribulose 1, 5-bisphospate carboxylase/oxygenase (RubisCO) is differentiated from other RubisCO enzymes and is classified as a form III enzyme, as opposed to the form I and form II RubisCOs typical of chemoautotrophic bacteria and prokaryotic and eukaryotic phototrophs. The form III enzyme from archaea is particularly interesting as several of these proteins exhibit unusual and reversible sensitivity to molecular oxygen, including the enzyme from Archaeoglobus fulgidus. Previous studies with A. fulgidus RbcL2 had shown the importance of Met-295 in oxygen sensitivity and pointed towards the potential significance of another residue (Ser-363) found in a hydrophobic pocket that is conserved in all RubisCO proteins. In the current study, further structure/function studies have been performed focusing on Ser-363 of A. fulgidus RbcL2; various changes in this and other residues of the hydrophobic pocket point to and definitively establish the importance of Ser-363 with respect to interactions with oxygen. In addition, previous findings had indicated discrepant CO2/O2 specificity determinations of the Thermococcus kodakaraensis RubisCO, a close homolog of A. fulgidus RbcL2. It is shown here that the T. kodakaraensis enzyme exhibits a similar substrate specificity as the A. fulgidus enzyme and is also oxygen sensitive, with equivalent residues involved in oxygen interactions