NADP-Specific Electron-Bifurcating [FeFe]-Hydrogenase in a Functional Complex with Formate Dehydrogenase in Clostridium autoethanogenum Grown on CO

Abstract

c Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, one is a heteromeric ferredoxin-and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hydrogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was found in high concentrations (6% of the cytoplasmic proteins) in the acetogenic Clostridium autoethanogenum autotrophically grown on CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different subunits. As predicted from the sequence of the encoding clustered genes (fdhA/hytA-E) and from chemical analyses, the 78.8-kDa subunit (FdhA) is a selenocysteine-and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an ironsulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenase proper, and the 18.1-kDa (HytC), 28.6-kDa (HytD), 19.9-kDa (HytE1), and 20.1-kDa (HytE2) subunits are iron-sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP ؉ with H 2 or formate and the reversible formation of H 2 and CO 2 from formate. We propose the complex to have two functions in vivo, namely, to normally catalyze CO 2 reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H 2 formation from NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of C. autoethanogenum on CO (E 0= ‫؍‬ ؊520 mV). Five years ago it was discovered that in butyric acid-forming clostridia, the exergonic reduction of crotonyl coenzyme A (crotonyl-CoA; E 0 = ϭ Ϫ10 mV) with NADH (E 0 = ϭ Ϫ320 mV) is coupled with the endergonic reduction of ferredoxin (Fd) (E 0 = ϭ Ϫ400 mV) with NADH (reaction 1) catalyzed by the cytoplasmic butyryl-CoA dehydrogenase/electron transfer flavoprotein complex Bcd/EtfAB (1, 2).The available evidence indicates that electron bifurcation is flavin based: a protein-bound flavin is reduced by NADH to the hydroquinone, which is subsequently reoxidized by crotonylCoA to the semiquinone radical that has a redox potential sufficiently negative to reduce ferredoxin (3). The proposed mechanism is analogous to the mechanisms of ubiquinonebased electron bifurcation in the bc1 complex of the aerobic respiratory chain and plastoquinone-based electron bifurcation in the b6f-complex in oxygenic photosynthesis (4-6). The main differences between the electron bifurcation mechanisms are that flavin-based electron bifurcation is associated with cytoplasmic proteins and operates at more negative redox potentials (Ϫ300 mV Ϯ 200 mV), whereas ubiquinone/plastoquinone-based electron bifurcation is associated with membranes and operates at more positive redox potentials (ϩ100 mV Ϯ 200 mV) (3,(7)(8)(9).Since t...

Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica

“…Cell extracts of C. autoethanogenum do not catalyze the reduction of methylene-H 4 F with NADH or NADPH (18). Methylene-H 4 F reductase activity is found, as in cell extracts of M. thermoacetica, only with benzyl viologen.…”

“…thermoacetica contains three hydrogenases for H 2 activation: (i) a cytoplasmic electron-bifurcating [FeFe]-hydrogenase (HydABC), which uses NAD ϩ plus ferredoxin as electron acceptors (17); (ii) a cytoplasmic [FeFe]-hydrogenase (HytA to -F) (18), which uses NADP as an electron acceptor (19); and (iii) a membrane-associated, energy-converting [NiFe]-hydrogenase (EchABCE to -I) connected via an iron-sulfur protein to a formate dehydrogenase (FdhA2) (20) (Fig. 1).…”

Genomic and metagenomic surveys of hydrogenase distribution indicate H2 is a widely utilised energy source for microbial growth and survival

“…The group A3 [FeFe]-hydrogenases reversibly bifurcate electrons from H 2 to ferredoxin and NAD using trimeric or tetrameric complexes; in the reverse reaction, energy conserved during the oxidation of ferredoxin is used to drive the thermodynamically unfavourable production of H 2 from NADH (Schut and Adams, 2009;Schuchmann and Müller, 2012). A subtype of the group A4 [FeFe]-hydrogenases can also bifurcate electrons from H 2 to NADP and ferredoxin, and act physiologically in hexameric complexes with formate dehydrogenase (Wang et al, 2013). We analysed the genetic organisation of the 705 group A [FeFe]-hydrogenases represented in our database in order to identify putative electron-bifurcating complexes (Figure 2 and Supplementary Table S1).…”

Trash to treasure: production of biofuels and commodity chemicals via syngas fermenting microorganisms

“…This is driven by coupling the oxidation of reduced ferredoxin with the reduction of NAD + [27] (Figure 3). In the acetogen Acetobacterium woodii, a Na + gradient generated by the Rnf complex is converted to ATP by a Na [36]. An Nfn complex similar to that of Clostridium kluyveri [37] (Figure 3) was identified in M. thermoacetica as well [34 ].…”