PARTICIPATION OF THE DISULFIDE BRIDGE IN THE REDOX CYCLE OF THE FERREDOXIN FROM THE HYPERTHERMOPHILE PYROCOCCUS-FURIOSUS - H-1 NUCLEAR-MAGNETIC-RESONANCE TIME RESOLUTION OF THE 4 REDOX STATES AT AMBIENT-TEMPERATURE

The oxidized and reduced forms of the (4Fe-4S]-containing ferredoxin f rom the hyperthermophilic archaeon Pyrococcus furiosus, Pf, have been investigated by H-1 nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy and thiol titrations. We have iden tified and isolated at ambient temperature four distinct redox states for the [4Fe-4S] form of the ferredoxin. These states differ in the re dox state of the cluster, which is coordinated by Cys 11, Asp 14, Cys 17, and Cys 56, and of a disulfide bridge between Cys 21 and Cys 48, T he protein, as isolated under anaerobic conditions, designated 4Fe Fd( B)(red), contains the reduced cluster and two free thiols. The cluster , but not the thiols, is readily oxidized by brief exposure to O-2 to yield 4Fe Fd(B)(ox). Prolonged O-2 treatment (> 24 h at 30 degrees C) is required to generate the protein with a disulfide (4Fe Fd(A)(ox)) w hile this fully oxidized form is readily converted by brief reduction with sodium dithionite to the protein with a reduced cluster and a dis ulfide (4Fe Fd(A)(red)), Analyses of the magnitude and the number of h yperfine-shifted resonances in each of the four redox states are discu ssed.