Ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus is a monomeric protein (7.5 kDa) that contains a single [4Fe-4S]1+, 2+ cluster. The protein is unusual in that its cluster is coordinated by three Cys and one Asp residue, rather than by the typical four Cys residues. Site-directed mutagenesis has been used to obtain mutant forms in which the cluster-coordinating Asp was replaced by Cys (D14C) and also by Ser (D14S), together with a third mutant (A1K) which contained N-Met-Lys at the N-terminus instead of N-Ala. Analyses using UV-visible absorption, far-UV circular dichroism, and EPR spectroscopy showed that there were no gross structural differences between the native and the three mutant forms and that they each contained a [4Fe-4S] cluster. The reduction potentials, determined by direct electrochemistry (at 23 degrees C, pH 8.0), of the D14S, D14C, and A1K mutants were -490, -422, and -382 mV, respectively, which compare with values of -375 mV for native [4Fe-4S]-containing ferredoxin and -160 mV for the [3Fe-4S]-containing form. The native, D14C, and A1K proteins functioned as electron acceptors in vitroat 80 degrees C for pyruvate ferredoxin oxidoreductase (POR) and aldehyde ferredoxin oxidoreductase (AOR) from P. furiosus using pyruvate and crotonaldehyde as substrates, respectively. The calculated kcat/Km values were similar for the three proteins when ferredoxin reduction was measured either directly by visible absorption or indirectly by coupling ferredoxin reoxidation to the reduction of metronidazole. In contrast, using the D14S mutant and the 3Fe-form of the native ferredoxin as electron acceptors, the activity with AOR was virtually undetectable, and with POR the calculated kcat/Km values were at least 3-fold lower than those obtained with the native (4Fe-), D14C, and A1K proteins. The ability of this 4Fe-ferredoxin to accept electrons from two oxidoreductases of the same organism is therefore not absolutely dependent upon Asp14, as this residue can be effectively replaced by Cys. However, the efficiency of electron transfer is compromised if Asp14 is replaced by Ser, or if the 4Fe-cluster is converted to the 3Fe-form, but Asp14 does not appear to offer any kinetic advantage over the expected Cys.