The dicluster-type ferredoxin is a key electron carrier in the cytoplasm of the aerobic and thermoacidophilic archaeon, Sulfolobus sp. strain 7, and contains 1 aspartate and 7 cysteine residues as possible ligands to two FeS clusters. The optical, electron paramagnetic resonance (EPR), and cyclic voltammetric studies suggest the presence of one each of [3Fe-4S]1+,0 (-280 mV) and [4Fe-4S]2+,1+ (-530 mV) clusters in the purified Sulfolobus ferredoxin, and the lower potential [4Fe-4S] center was scarcely reducible by excess dithionite even at pH 9. While the Sulfolobus ferredoxin has been known to function as an electron acceptor of 2-oxoacid:ferredoxin oxidoreductase (Kerscher, L., Nowitzki, S., and Oesterhelt, D. (1982) Eur. J. Biochem. 128, 223-230), it is not known whether one or both of two clusters is reduced during the steady-state turnover of the enzyme. Here we show by combinations of the optical and EPR spectroscopies that only the higher potential [3Fe-4S] cluster is reduced at the physiological pH during the steady-state turnover of the purified 2-oxoacid:ferredoxin oxidoreductase at 50 degrees C. The functional significance and evolutionary implications of the [3Fe-4S] center in dicluster-type ferredoxins are discussed.