Carbon Fixation in the Chemolithoautotrophic Bacterium Aquifex aeolicus Involves Two Low-Potential Ferredoxins as Partners of the PFOR and OGOR Enzymes

Abstract

Aquifex aeolicus is a microaerophilic hydrogen- and sulfur -oxidizing bacterium that assimilates CO2 via the reverse tricarboxylic acid cycle (rTCA). Key enzymes of this pathway are pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (OGOR), which are responsible, respectively, for the reductive carboxylation of acetyl-CoA to pyruvate and of succinyl-CoA to 2-oxoglutarate, two energetically unfavorable reactions that require a strong reduction potential. We have confirmed, by biochemistry and proteomics, that A. aeolicus possesses a pentameric version of these enzyme complexes ((αβγδε)2) and that they are highly abundant in the cell. In addition, we have purified and characterized, from the soluble fraction of A. aeolicus, two low redox potential and oxygen-stable [4Fe-4S] ferredoxins (Fd6 and Fd7, E0 = −440 and −460 mV, respectively) and shown that they can physically interact and exchange electrons with both PFOR and OGOR, suggesting that they could be the physiological electron donors of the system in vivo. Shotgun proteomics indicated that all the enzymes assumed to be involved in the rTCA cycle are produced in the A. aeolicus cells. A number of additional enzymes, previously suggested to be part of a putative partial Wood-Ljungdahl pathway used for the synthesis of serine and glycine from CO2 were identified by mass spectrometry, but their abundance in the cell seems to be much lower than that of the rTCA cycle. Their possible involvement in carbon assimilation is discussed.

A cascade of sulfur transferases delivers sulfur to the sulfur‐oxidizing heterodisulfide reductase‐like complex

“…aeolicus is an excellent model organism for identifying interactions between sHdr‐associated sulfur transferases and other proteins. Membrane fractions, cell extracts, and partially purified proteins from this hyperthermophile have repeatedly served as the basis for cross‐linking, co‐purification and co‐migration approaches, which have enabled the identification of physiological protein partners (Aussignargues et al, 2012 ; Boughanemi et al, 2016 ; Prioretti et al, 2023 ). Here, we incubated pure recombinant His‐tagged AqTusA with Aq.…”

“…Resulting bands were cut out of the gel and analyzed by LC–MS as described previously (Boughanemi et al, 2016 ). For the second experiment, total proteins in the elution fraction of the column were identified after the proteins were introduced in a 5% acrylamide stacking gel, as described in Prioretti et al ( 2023 ) under the name “stacking method”. Protein concentrations were determined with the BCA protein assay kit from Sigma–Aldrich.…”

The potential of CO2-based production cycles in biotechnology to fight the climate crisis

“…The lowest MDFs are found in the Wood Ljungdahl pathway and the rTCA cycle, ranging between 0.53–0.67 kJ per C-mol, and could only be achieved by increasing the maximum CO 2 concentration. While the rTCA in anaerobic bacteria involves ferredoxins as electron carriers 55 we have based the calculation on NADH as ferredoxins are not generally accessible in eQuilibrator due to their wide range of reduction potentials.…”

A cascade of sulfur transferases delivers sulfur to the sulfur-oxidizing heterodisulfide reductase-like complex

“…Membrane fractions, cell extracts and partially purified proteins from this hyperthermophile have repeatedly served as the basis for cross-linking, co-purification and co-migration approaches, which have enabled the identification of physiological protein partners. (16,34,44) Here, we incubated pure recombinant His-tagged AqTusA with Aq. aeolicus soluble extracts, prepared from cells that had been grown in the presence of various ratios of hydrogen and thiosulfate, (16) at room temperature as specified in the Material and methods section.…”

“…(54) Resulting bands were cut out of the gel and analyzed by LC-MSMS as described previously. (16) For the second experiment, total proteins in the elution fraction of the column were identified after the proteins were introduced in a 5% acrylamide stacking gel, as described in Prioretti et al (44) under the name "stacking method". Protein concentrations were determined with the BCA protein assay kit from Sigma-Aldrich.…”