Solution NMR Characterization of the Thermodynamics of the Disulfide Bond Orientational Isomerism and Its Effect of Cluster Electronic Properties for the Hyperthermostable Three-Iron Cluster Ferredoxin from the Archaeon Pyrococcus furiosus

• Published in: Biochemistry (Easton) 2001-10, Vol.40 (42), p.12575-12583
• Main Authors: Webba da Silva, Mateus, Sham, Simon, Gorst, Carol M, Calzolai, Luigi, Brereton, Philip S, Adams, Michael W. W, La Mar, Gerd N
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bridged-Ring Compounds - chemistry; Cysteine - chemistry; Disulfides - chemistry; Dithionite - chemistry; Electrons; Ferredoxins - chemistry; Hydrolysis; Iron - chemistry; Methanol - chemistry; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Protein Structure, Secondary; Pyrococcus furiosus - chemistry; Solutions; Stereoisomerism; Temperature; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0106179

The thermodynamics and dynamics of the Cys21−Cys48 disulfide “S” ⇔ “R” conformational isomerism in the three-iron, single cubane cluster ferredoxin (Fd) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) have been characterized by 1H NMR spectroscopy in both water and water/methanol mixed solvents. The mean interconversion rate at 25 °C is 3 × 103 s- 1 and ΔG 298 = −0.2 kcal/mol [ΔH = 4.0 kcal/mol; ΔS = 14 cal/(mol·K)], with the S orientation as the more stable form at low temperature (100 °C, where the organism thrives. The distinct pattern of ligated Cys β-proton contact shifts for the resolved signals and their characteristic temperature behavior for the forms of the 3Fe Fd with alternate disulfide orientations have been analyzed to determine the influences of disulfide orientation and methanol cosolvent on the topology of the inter-iron spin coupling in the 3Fe cluster. The Cys21−Cys48 disulfide orientation influences primarily the spin couplings involving the iron ligated to Cys17, whose carbonyl oxygen is a hydrogen bond acceptor to the Cys21 peptide proton. Comparison of the Cys β-proton contact shift pattern for the alternate disulfide orientations with the pattern exhibited upon cleaving the disulfide bridge confirms an earlier [Wang, P.-L., Calzolai, L., Bren, K. L., Teng, Q., Jenney, F. E., Jr., Brereton, P. S., Howard, J. B., Adams, M. W. W., and La Mar, G. N. (1999) Biochemistry 38, 8167−8178] proposal that the structure of the same Fd with the R disulfide orientation resembles that of the Fd upon cleaving the disulfide bond.