The gene encoding ferredoxin was cloned from hyperthermophilic archaeon Pyrococcus sp. KOD1, and the nucleotide sequence was determined. The ferredoxin gene encodes a polypeptide of 62 amino acids that incorporates a single [4Fe-4S] cluster with a motif (CXXDXXCXXXCP) in the N-terminal region, where one cysteine residue is replaced by an aspartate residue. Sequence comparison of KOD1 ferredoxin indicated high sequence homology to the ferredoxins from other hyperthermophilic archaea Pyrococcus furiosus (75%), Thermococcus litoralis (67%) and hyperthermophilic bacterium Thermotoga maritima (58%). Based on the sequence information, the phylogenetic tree for the ferredoxins containing single [4Fe-4S] cluster was constructed, which indicates a close evolutionary relationship among hyperthermophiles. Natural ferredoxin was purified from KOD1 cells to homogeneity, and its structure was compared with that of recombinant KOD1 ferredoxin expressed in E. coli. Amino terminal sequences for both natural and recombinant ferredoxins were determined, showing that both ferredoxins lacked the initial methionine. Circular dichroism (CD) spectra of these proteins were distinctly different from each other. Upon heat treatment (90 degrees C, 3 h), the structure of recombinant ferredoxin was mostly converted to that of natural ferredoxin, suggesting that exposure to high temperature environment plays an important role for proper folding of natural KOD1 ferredoxin.