Abstract

The hyperthermophilic archaeon Pyrococcus woesei, is a strict anaerobe heterotroph that grows up to 110 degrees C. Its ferredoxin has now been purified to homogeneity under anaerobic conditions. P. woesei ferredoxin has a Mr value of 7,000 by gel electrophoresis. The Mr value was determined to be 14 000 and 6 500 by gel filtration in 0.2 M NaCl and 0.5 M NaCl, respectively, suggesting that the purified protein exists as a dimer at lower ionic strength. Air-oxidized P! woesei ferredoxin had an UV-visible absorption spectra typical of 4Fe-type ferredoxins with an A(390)/A(280) ratio of 0.63. Its reduction potential was -360mV at room temperature as determined by direct electrochemistry. P. woesei ferredoxin showed to be extremely thermostable, being unaffected by a 12h incubation at 95 degrees C. Interestingly, its N-terminus sequence, which represents approximately 1/3 of the total sequence, was determined to be more closely related to the sequences of extremely thermophilic bacterial ferredoxins (greater than or equal to 76%) than it was to the sequence of a hyperthermophilic ferredoxin (56%) isolated from an archaeon from the same genus, P. furiosus. (C) 2000 Academic Press.