Microbial metabolism of methane formation and oxidation:
In addition to being a potent greenhouse gas, methane is also a cheap, renewable fuel that has great potential for manufacturing value-added chemicals. Acetate-utilizing methane-producing anaerobes (acetotrophic methanogens) account for two-thirds of the methane produced in anaerobic microbial food chains converting complex renewable biomass to methane (biomethanation). Although much is known regarding one-carbon transformations leading from the methyl group of acetate to methane, there is a rudimentary understanding of electron transport processes coupled to energy conservation and oxidative stress. The mechanism of energy conservation and oxidative stress in acetotrophic methanogens needs to be studied in order to gain a deeper understanding of acetate metabolism and harness energy from methane. Overall, the understanding of energy conservation and stress response in methanogens will lead to better methods for modulating biological methane production. Our aim is to advance understanding by determining the role and mechanism of only two known [4Fe-4S] constituting disulfide reductases: heterodisulfide reductases (HDR) and ferredoxin disulfide reductases (FDR), which are important for energy conservation and oxidative stress in methanogens, respectively.
Heterodisulfide reductases (Hdr):
We aim to advance an understanding by employing biochemical, biophysical, and molecular approaches to determine the mechanism of heterodisulfide reductases essential for energy conservation in methanogens. The project will investigate two heterodisulfide reductase complexes (HdrED and HdrA2B2C2), essential for acetotrophic growth of the model acetotroph Methanosarcina acetivorans and homologs essential in other diverse methanogenic pathways. All catalyze reduction of the heterodisulfide of coenzyme M and coenzyme B (CoMS-SCoB), yielding HS-CoM and HSCoB. HdrA2B2C2 is an electron bifurcating complex representing a new mechanism of energy conservation. The genomes of diverse species in the domains Bacteria and Archaea are annotated with genes encoding homologs which suggests roles in diverse energy-conserving metabolisms including the anaerobic oxidation of CH4.
Mechanistic and physiological role of ferredoxin: disulfide reductase:
Thioredoxins and thioredoxin reductases (TrxR) are important group of enzymes responding to oxidative stress in all domains of life. Acetotrophic methanogens, M. acetivorans produces the Fdx:disulfide reductase (FDR), and a homolog Archael Ferreodxin: Thioredoxin Reductase (AFTR). Both FDR and AFTR are upregulated during extracellular electron transfer suggesting a role in respiratory metabolism and methane oxidation. Although FDR and AFTR are widely distributed in organisms from the domain Archaea and Bacteria, substantially less is understood of the biochemistry and physiology. The extensively characterized plant-type Fdx:thioredoxin reductase (FTR) and FDRs (FDR and AFTR) belong to a distinct class of disulfide reductases that contain a non-canonical CPC*XnCPCXnCY/HC* [4Fe4S] motif for which the ‘asterisked’ cysteines form the redox-active disulfide, and the remaining cysteines ligate the unusual [4Fe4S] cluster. Not reported for either FTR or FDR, reduction of AFTR yields a transient [4Fe-4S]1+ cluster with features attributed to an S=7/2 spin state that accompany a classical S=1/2 signal of the [4Fe-4S]1+ cluster. We propose to further investigate the catalytic mechanism that includes this unprecedented S=7/2 intermediate. Overall, the findings will provide a more in-depth understanding of oxidative stress in methanogens and the broader field of anaerobes.