Participation of the disulfide bridge in the redox cycle of the ferredoxin from the hyperthermophile Pyrococcus furiosus: super(1)H nuclear magnetic resonance time resolution of the four redox states at ambient temperature

• Published in: Biochemistry (Easton) 1995-01, Vol.34 (27), p.8788-8795
• Main Authors: Gorst, C M, Zhou, Zhi Hao, Ma, K, Teng, Q, Howard, J B, Adams, MWW, La-Mar, G N
• Format: Article
• Language: English
• Subjects: Pyrococcus furiosus
• ISSN: 0006-2960

The oxidized and reduced forms of the [4Fe-4S]-containing ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus, Pf, have been investigated by super(1)H nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy and thiol titrations. We have identified and isolated at ambient temperature four distinct redox states for the [4Fe-4S] form of the ferredoxin. These states differ in the redox 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. The protein, as isolated under anaerobic conditions, designated 4Fe Fd sub(B) super(red), contains the reduced cluster and two free thiols. The cluster, but not the thiols, is readily oxidized by brief exposure to O sub(2) to yield 4Fe Fd sub(B) super(ox). Prolonged O sub(2) treatment (>24 h at 30 degree C) is required to generate the protein with a disulfide (4Fe Fd sub(A) super(ox)) while this fully oxidized form is readily converted by brief reduction with sodium dithionite to the protein with a reduced cluster and a disulfide (4Fe Fd sub(A) super(ox)). Analyses of the magnitude and the number of hyperfine-shifted resonances in each of the four redox states are discussed.