Characterization of a [2Fe-2S] protein encoded in the iron-hydrogenase operon of Thermotoga maritima.
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.
Pan
Guangliang
G
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA.
Menon
Angeli Lal
AL
Adams
Michael W W
MW
D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009876:Operon; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D020124:Thermotoga maritima / Q000378:metabolism*
D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; C118830:iron hydrogenase; D006864:Hydrogenase