Identification of a Novel Fumarate Reductase Potentially Involved in Electron Bifurcation

Abstract

In the succinic acid-producing bacterium Pseudoclostridium thermosuccinogenes the fumarate reductase (FRD) genes reside in an operon together with those encoding an electron bifurcating complex (FlxABCD-HdrABC) that shuttles electrons from NADH to ferredoxin and a disulfide bond. Based on phylogeny and genomic co-occurrence we propose two hypothetical mechanisms via which the FRD is involved in electron bifurcation: (I) A disulfide bond from a hitherto unknown cofactor is reduced by the electron-bifurcating FlxABCD-HdrABC complex, using NADH to generate two thiol groups, while facilitating the unfavourable reduction of ferredoxin by NADH. The disulfide bond is subsequently regenerated via the reduction of fumarate by the FRD using the previously formed thiol groups. (II) The FRD forms an integral part of the FlxABCD-HdrABC complex, and NADH is used to reduce ferredoxin and fumarate directly, without an intermediate disulfide-forming cofactor. Either way enables the conservation of additional energy by a soluble FRD, analogous to fumarate respiration.

Effects of CO2 limitation on the metabolism of Pseudoclostridium thermosuccinogenes

“…During 1% CO 2 , 0.42 mol extra ferredoxin per mole glucose was reduced by PFOR, resulting from the decreased pyruvate formate lyase (PFL) flux. Assuming that fumarate reductase is indeed linked to ferredoxin reduction (through NADH dehydrogenase/heterodisulfide reductase complex, as proposed previously [14,15]), it follows that overall, 0.26 mol extra ferredoxin is reduced during CO 2 limitation. Simultaneously, 0.25 mol fewer NADH and 0.18 mol extra NADPH is oxidized (the latter through the NADPH-dependent alcohol dehydrogenase of P. thermosuccinogenes [16]), assuming the transhydrogenase activity of the malate shunt has not changed [17].…”

“…This is surprising, as in most natural succinate producers H 2 addition enhances succinate production [27][28][29]. Nevertheless, it fits the notion that P. thermosuccinogenes has a unique fumarate reductase, coupling succinate formation to ferredoxin reduction [15], since inhibition of hydrogenases by high H 2 partial pressure would impair ferredoxin reducing reactions [30,31]. In the same study, confusingly, decreased culture redox potential (CRP; assumed to increase NADH availability) by addition of Na 2 S resulted in the complete opposite effect as H 2 addition.…”

“…Conversely, the succinic acid operon, harbouring the bifurcating NADH dehydrogenase/ heterodisulfide reductase complex (Flx-Hdr), is upregulated, albeit also not statistically significant for all subunits. RNF transfers electrons from ferredoxin to NAD + , whereas Flx-Hdr transfers electrons from NADH to ferredoxin (and thiols) [14,15]. The transcriptional effects could therefore indicate that the excess of NADH is more pressing than the excess of ferredoxin, as they would result in reduced transfer of electrons from ferredoxin to NADH.…”

Metagenomic insights into the environmental adaptation and metabolism of Candidatus Haloplasmatales, one archaeal order thriving in saline lakes

“…Indeed, it is worth to further research whether the regeneration of heterodisulfide is coupled with fumarate reduction or other compounds in the last common ancestor. Considering the lack of biosynthesis CoM and CoB (Table S12), heterodisulfide may be an alternative thio/disulfide molecule pair or a coenzyme like the FlxABCD‐HdrABC in Pseudoclostridium thermosuccinogenes (Van Kranenburg and Koendjbiharie, 2019). In addition, the direct electron transfer from hydrogen to fumarate cannot be excluded.…”