Characterization of a [2Fe-2S] protein encoded in the iron-hydrogenase operon of Thermotoga maritima

摘要

Thermotoga maritima grows optimally at 80 ℃ by fermenting carbohydrates to organic acids, CO_2 and H_2. The production of H_2 is catalyzed by a cytoplasmic, heterotrimeric (αβγ) Fe-hydrogenase. This is encoded by three genes, hydC(γ), HYDb(β) and hydA (α), organized within a single operon that contains five additional open reading frames (ORFs). The recombinant form of the first ORF of the operon, TM1420, was produced in Escherichia coli. It has a molecular mass of 8537±3 Da as determined by mass spectrometry, in agreement with the predicted amino acid sequence. Purified TM1420 is red in color, has a basic Pi (8.8), and contains 1.9 Fe atoms/mol that are present as a single [2Fe-2S] cluster, as determined by UV-Visible absorption and EPR spectroscopy. The protein contains five cysteine residues, but their arrangement is characteristic of a subunit or domain rather than of a ferredoxin-type protein. The reduction potential of the [2Fe-2S] cluster (-233 mV at pH 6.5 and 25 ℃) is pH independent but decreases linearly with temperature to -296 mV (-1.15 mV/℃) at 80 ℃. TM1420 is not reduced, in vitro, by the Fe-hydrogenase nor by a pyruvate ferredoxin oxidoreductase. The protein was unstable at 70 ℃ under anaerobic conditions with a half-life of ～30 min. The basic nature of TM1420, its instability at the growth temperature of T. maritima, and the unusual spacing of its cysteine residues suggest that this protein does not function as a ferredoxin-type electron carrier for the Fe-hydrogenase. Instead, TM1420 is more likely part of a thermostable multi-protein complex that is involved in metal cluster assembly of the hydrogenase holoenzyme.