SITE-DIRECTED MUTATIONS OF THE 4FE-FERREDOXIN FROM THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-FURIOSUS - ROLE OF THE CLUSTER-COORDINATING ASPARTATE IN PHYSIOLOGICAL ELECTRON-TRANSFER REACTIONS

Ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus is a monomeric protein (7.5 KDa) that contains a single [4Fe-4S](+,2+) cl uster. The protein is unusual in that its cluster is coordinated by th ree Cys and one Asp residue, rather than by the typical four Cys resid ues. Site-directed mutagenesis has been used to obtain mutant forms in which the cluster-coordinating Asp was replaced by Cys (D14C) and als o 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 tha t there were no gross structural differences between the native and th e 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, -42 2, and -382 mV, respectively, which compare with values of -375 mV for native [4Fe-4S]-containing ferredoxin and -160 mV for the [3Fe-4S]-co ntaining form. The native, D14C, and A1K proteins functioned as electr on accepters in vitro at 80 degrees C for pyruvate ferredoxin oxidored uctase (POR) and aldehyde ferredoxin oxidoreductase (AOR) from P. furi osus using pyruvate and crotonaldehyde as substrates, respectively. Th e calculated k(cat)/K-M values were similar fur the three proteins whe n ferredoxin reduction was measured either directly by visible absorpt ion 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 accepters, the activity with AOR was virtually undetectable, and with POR the calculated k(cat)/K-M val ues were at least 3-fold lower than those obtained with the native (4F e-), D14C, and A1K proteins. The ability of this 4Fe-ferredoxin to acc ept electrons from two oxidoreductases of the same organism is therefo re not absolutely dependent upon Asp14, as this residue can be effecti vely replaced by Cys. However, the efficiency of electron transfer is compromised if Asp14 is replaced by Ser, or if the 4Fe-cluster is conv erted to the 3Fe-form, but Asp14, does not appear to offer any kinetic advantage over the expected Cys.