A Na⁺ A₁A_O ATP synthase with a V‐type c subunit in a mesophilic bacterium

Abstract

A 1 A O ATP synthases with a V-type c subunit have only been found in hyperthermophilic archaea which makes bioenergetic analyses impossible due to the instability of liposomes at high temperatures. A search for a potential archaeal A 1 A O ATP synthase with a V-type c subunit in a mesophilic organism revealed an A 1 A O ATP synthase cluster in the anaerobic, acetogenic bacterium Eubacterium limosum KIST612. The enzyme was purified to apparent homogeneity from cells grown on methanol to a specific activity of 1.2 UÁmg À1 with a yield of 12%. The enzyme contained subunits A, B, C, D, E, F, H, a, and c. Subunit c is predicted to be a typical V-type c subunit with only one ion (Na + )-binding site. Indeed, ATP hydrolysis was strictly Na + -dependent. N,N 0 -dicyclohexylcarbodiimide (DCCD) inhibited ATP hydrolysis, but inhibition was relieved by addition of Na + . Na + was shown directly to abolish binding of the fluorescence DCCD derivative, NCD-4, to subunit c, demonstrating a competition of Na + and DCCD/NCD-4 for a common binding site. After incorporation of the A 1 A O ATP synthase into liposomes, ATP-dependent primary transport of 22 Na + as well as DµNa + -driven ATP synthesis could be demonstrated. The Na + A 1 A O ATP synthase from E. limosum is the first ATP synthase with a V-type c subunit from a mesophilic organism. This will enable future bioenergetic analysis of these unique ATP synthases. AbbreviationsDµNa + , electrochemical sodium ion potential; DpNa, sodium ion potential; DΨ, membrane potential; DCCD, N,N 0 -dicyclohexylcarbodiimide; DDM, n-dodecyl-b-maltoside; ETH 2120, N,N,N,N 0 -tetra-cyclo-hexyl-1,2-phenylenedioxydiacetamide; MALDI, matrix-assisted laser desorption/ ionization; TCS, 3,3 0 ,4 0 ,5-tetrachlorosalicylanilide.

Biochemistry of methanol‐dependent acetogenesis in Eubacterium callanderi KIST612

“…In earlier studies, ATP synthase from methanol‐grown cells and Rnf from cells grown on glucose were shown to be Na + dependent in E . callanderi KIST612 (Jeong et al ., 2015; Litty and Müller, 2020). The Na + dependence of the Rnf could be verified with membranes isolated from cells grown on methanol (Fig.…”

Overcoming Energetic Barriers in Acetogenic C1 Conversion

“…The ion gradient established by the Rnf and Ech complex is then used by a F 1 F O -ATP-synthase to drive the synthesis of ATP. F 1 F O -ATP-synthases have been purified from only a few acetogens such as M. thermoacetica (Ivey and Ljungdahl, 1986), Moorella thermoauthotrophica (Das et al, 1997), A. woodii and Eubacterium limosum (Litty and Müller, 2020). F 1 F O -ATP-synthases are macromolecular machines that convert electrochemical energy via mechanical energy into chemical energy (ATP) (Müller and Grüber, 2003).…”

“…There are more residues involved in complexing Na + (Meier et al, 2005) but these three are the conserved ones that make the conserved Na +binding motif. C. ljungdahlii and C. autoethanogenum only have the active carboxylate but not the Na + -binding motif (Mayer et al, 1986), whereas A. woodii Matthies et al, 2014) as well as E. limosum (Litty and Müller, 2020) have the conserved Na + -binding motif. The activity of the purified enzymes is strictly Na + dependent and the enzymes translocate Na + after reconstitution into liposomes (Fritz and Müller, 2007;Litty and Müller, 2020).…”

Genome Sequence of Eubacterium limosum B2 and Evolution for Growth on a Mineral Medium with Methanol and CO2 as Sole Carbon Sources

“…Regarding the efficiency of the total protein extraction method for membrane-bound proteins, seven out of nine constitutive proteins of the ATP synthase complex were identified. The ATP synthase in E. callanderi KIST 612 was identified to be a unique combination of a Na + A 1 A 0 ATP synthase with a V-type c subunit organized in an operon of nine genes [41]. A similar operon was found in the genome of E. limosum B2, and seven out of nine constitutive proteins of the ATPase operon were identified by mass spectrometry (Figure 8B).…”