Journal
JOURNAL OF BACTERIOLOGY
Volume 194, Issue 14, Pages 3689-3699Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/JB.00385-12
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Funding
- Max Planck Society
- Fonds der Chemischen Industrie
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Moorella thermoacetica ferments glucose to three acetic acids. In the oxidative part of the fermentation, the hexose is converted to 2 acetic acids and 2 CO2 molecules with the formation of 2 NADH and 2 reduced ferredoxin (Fd(red)(2-)) molecules. In the reductive part, 2 CO2 molecules are reduced to acetic acid, consuming the 8 reducing equivalents generated in the oxidative part. An open question is how the two parts are electronically connected, since two of the four oxidoreductases involved in acetogenesis from CO2 are NADP specific rather than NAB specific. We report here that the 2 NADPH molecules required for CO2 reduction to acetic acid are generated by the reduction of 2 NADP(+) molecules with 1 NADH and 1 Fd(red)(2-) catalyzed by the electron-bifurcating NADH-dependent reduced ferredoxin:NADP(+) oxidoreductase (NfnAB). The cytoplasmic iron-sulfur flavoprotein was heterologously produced in Escherichia coli, purified, and characterized. The purified enzyme was composed of 30-kDa (NfnA) and 50-kDa (NfnB) subunits in a 1-to-1 stoichiometry. NfnA harbors a [2Fe2S] cluster and flavin adenine dinucleotide (FAD), and NfnB harbors two [4Fe4S] clusters and FAD. M. thermoacetica contains a second electron-bifurcating enzyme. Cell extracts catalyzed the coupled reduction of NAD(+) and Fd with 2 H-2 molecules. The specific activity of this cytoplasmic enzyme was 3-fold higher in H-2-CO2-grown cells than in glucose-grown cells. The function of this electron-bifurcating hydrogenase is not yet clear, since H-2-CO2-grown cells additionally contain high specific activities of an NADP(+)-dependent hydrogenase that catalyzes the reduction of NADP(+) with H-2. This activity is hardly detectable in glucose-grown cells.
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