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

Abstract

Thermotoga maritima grows optimally at 80 degrees C by fermenting carbohydrates to organic acids, CO(2), and H(2). The production of H(2) is catalyzed by a cytoplasmic, heterotrimeric (alphabetagamma) Fe-hydrogenase. This is encoded by three genes, hydC (gamma), hydB (beta) and hydA (alpha), 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 p I (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 degrees C) is pH independent but decreases linearly with temperature to -296 mV (-1.15 mV/ degrees C) at 80 degrees C. TM1420 is not reduced, in vitro, by the Fe-hydrogenase nor by a pyruvate ferredoxin oxidoreductase. The protein was unstable at 70 degrees C under anaerobic conditions with a half-life of approximately 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.

Thioredoxin‐Like [2Fe–2S] Ferredoxin

“…properties have been thoroughly characterized. 42 While its quaternary structure is not firmly established, the data suggest a monomeric structure, 42 unlike the canonic Trx-like-Fd. It is believed to be part of a multiprotein complex possibly involved in redox processes or metal center assembly.…”

“…maritima, 42 and P. marinus. 7 The purification of the highly thermostable A. aeolicus protein includes a heat treatment (10 min at 70°C) of the soluble extract, which disposes of most of the E. coli proteins, thus allowing an easier and more efficient purification (up to 10 mg of pure protein/L of culture).…”

Discovery of Two Novel Radical S-Adenosylmethionine Proteins Required for the Assembly of an Active [Fe] Hydrogenase

“…It should also be noted that the HydF domain of the HydEF protein contains a putative GTPase domain, and the HypB protein, which also has GTPase activity, facilitates nickel incorporation into the active site of [NiFe] hydrogenases. Interestingly, neither the HydEF nor the HydG proteins are highly homologous to the TM1420 protein characterized from T. maritima (23). The latter is only 8.5 kDa long and does not contain a characteristic Radical SAM motif.…”

“…In this organism, an open reading frame encoding an [FeS] protein, TM1420, was identified in the hydrogenase operon. Characterization of TM1420 purified from Escherichia coli led to the suggestion that it may function in the assembly of the T. maritima [Fe] hydrogenase (23); however, its mode of action was not characterized, and TM1420 may be specific for the heterotrimeric T. maritima [Fe] hydrogenase.…”

A multisubunit membrane-bound [NiFe] hydrogenase and an NADH-dependent Fe-only hydrogenase in the fermenting bacterium Thermoanaerobacter tengcongensis

“…Three of the five accessory genes associated with the hydrogenase genes in Tt. maritima encode iron-sulfur proteins but their function is not yet known (Pan et al, 2003). In the Ta.…”