A novel and remarkably thermostable ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus.
The archaebacterium Pyrococcus furiosus is a strict anaerobe that grows optimally at 100 degrees C by a fermentative-type metabolism in which H2 and CO2 are the only detectable products. A ferredoxin, which functions as the electron donor to the hydrogenase of this organism was purified under anaerobic reducing conditions. It had a molecular weight of approximately 12,000 and contained 8 iron atoms and 8 cysteine residues/mol but lacked histidine or arginine residues. Reduction and oxidation of the ferredoxin each required 2 electrons/mol, which is consistent with the presence of two [4Fe-4S] clusters. The reduced protein gave rise to a broad rhombic electronic paramagnetic resonance spectrum, with gz = 2.10, gy = 1.86, gx = 1.80, and a midpoint potential of -345 mV (at pH 8). However, this spectrum represented a minor species, since it quantitated to only approximately 0.3 spins/mol. P. furiosus ferredoxin is therefore distinct from other ferredoxins in that the bulk of its iron is not present as iron-sulfur clusters with an S = 1/2 ground state. The apoferredoxin was reconstituted with iron and sulfide to give a protein that was indistinguishable from the native ferredoxin by its iron content and electron paramagnetic resonance properties, which showed that the novel iron-sulfur clusters were not artifacts of purification. The reduced ferredoxin also functioned as an electron donor for H2 evolution catalyzed by the hydrogenase of the mesophilic eubacterium Clostridium pasteurianum. P. furiosus ferredoxin was resistant to denaturation by sodium dodecyl sulfate (20%, wt/vol) and was remarkably thermostable. Its UV-visible absorption spectrum and electron carrier activity to P. furiosus hydrogenase were unaffected by a 12-h incubation of 95 degrees C.
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Department of Biochemistry, School of Chemical Sciences, University of Georgia, Athens 30602.
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D000596:Amino Acids / Q000032:analysis; D001105:Archaea / Q000502:physiology*; D018407:Bacterial Physiological Phenomena; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D006864:Hydrogenase / Q000378:metabolism; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D005288:Ferredoxins; D006864:Hydrogenase