Molecular biology and biochemical characterization of the CO dehydrogenase-linked ferredoxin from Methanosarcina thermophila strain TM-1
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The gene encoding the CO dehydrogenase-linked ferredoxin (fdxA) in Ms. thermophila had the coding capacity for a 6,230-Da protein which contained eight cysteines with spacings typical of 2[4Fe-4S] ferredoxins. A second open reading frame (ORF1) was also identified which had the potential to encode a 2[4Fe-4S] bacterial-like ferredoxin (5,850 Da). The deduced proteins from fdxA and ORF1 were 62% identical. fdxA and ORFI were present as single copies in the genome and each was transcribed on a monocistronic mRNA. Both fdxA and ORF1 were transcribed in cells grown on methanol and trimethylamine, but only the fdxA -specific transcript was detected in acetate-grown cells. The apparent transcriptional start sites offdxA and ORFI were downstream of sequences which had high identity with the consensus methanogen promoter.
The heterodisulfide of two cofactors unique to the methanogenic microorganisms, HS-HTP and HS-CoM, was enzymatically reduced in cell extracts of Ms. thermophila using electrons from the oxidation of either H2 or CO. The homodisulfides of either HS-HTP or HS-CoM were not reduced under the same conditions. The results indicated that methane is formed by reductive demethylation of CH3-S-CoM using HS-HTP as a reductant in Ms. thermophila. Coupling of CO oxidation with reduction of the heterodisulfide suggested that the CO dehydrogenase-linked ferredoxin may be involved, although the details of electron flow are not known.
- Doctoral Dissertations