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

Abstract

Thermotoga maritima grows optimally at 80 °C by fermenting carbohydrates to organic acids, CO₂, and H₂. The production of H₂ 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 °C) is pH independent but decreases linearly with temperature to −296 mV (−1.15 mV/°C) at 80 °C. TM1420 is not reduced, in vitro, by the Fe-hydrogenase nor by a pyruvate ferredoxin oxidoreductase. The protein was unstable at 70 °C 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.