Abstract
The methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme in acetogenic CO2 fixation. The MetVF-type enzyme has been purified from four different species and the physiological electron donor was hypothesized to be reduced ferredoxin. We have purified the MTHFR from Clostridium ljungdahlii to apparent homogeneity. It is a dimer consisting of two of MetVF heterodimers, has 14.9 ± 0.2 mol iron per mol enzyme, 16.2 ± 1.0 mol acid-labile sulfur per mol enzyme, and contains 1.87 mol FMN per mol dimeric heterodimer. NADH and NADPH were not used as electron donor, but reduced ferredoxin was. Based on the published electron carrier specificities for Clostridium formicoaceticum, Thermoanaerobacter kivui, Eubacterium callanderi, and Clostridium aceticum, we provide evidence using metabolic models that reduced ferredoxin cannot be the physiological electron donor in vivo, since growth by acetogenesis from H2 + CO2 has a negative ATP yield. We discuss the possible basis for the discrepancy between in vitro and in vivo functions and present a model how the MetVF-type MTHFR can be incorporated into the metabolism, leading to a positive ATP yield. This model is also applicable to acetogenesis from other substrates and proves to be feasible also to the Ech-containing acetogen T. kivui as well as to methanol metabolism in E. callanderi.
Highlights
Acetogenic bacteria are polyphyletic and characterized by a special pathway for acetate formation from CO2, the WoodLjungdahl pathway (WLP). This two-branched linear pathway is considered an ancient pathway of C O2 fixation since it is the only of the known pathways for C O2 fixation that is coupled to net ATP synthesis, allowing the bacteria to grow lithotrophically on H2 + CO2 or CO (Martin and Russell 2007; Martin 2012)
For the purification of the methylene-tetrahydrofolate reductase (MTHFR), C. ljungdahlii was grown in 20 l medium with fructose (28 mM) as carbon and electron source
The type II-MTHFR uses F dred as reductant in vitro, this is to be excluded in vivo since the ATP yield of acetogenesis from H2 + CO2 becomes negative
Summary
Acetogenic bacteria are polyphyletic and characterized by a special pathway for acetate formation from CO2, the WoodLjungdahl pathway (WLP) This two-branched linear pathway is considered an ancient pathway of C O2 fixation since it is the only of the known pathways for C O2 fixation that is coupled to net ATP synthesis, allowing the bacteria to grow lithotrophically on H2 + CO2 or CO (Martin and Russell 2007; Martin 2012). Christian Öppinger and Florian Kremp contributed to this study How this pathway is coupled to the synthesis of net ATP has been an enigma for decades (Müller 2003), but in the last couple of years, it was demonstrated unequivocally that acetogens have a respiratory chain consisting of an ATP synthase and a respiratory redox enzyme (Schuchmann and Müller 2014).
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