Solution NMR characterization of the thermodynamics of the disulfide bond orientational isomerism and its effect of cluster electronic properties forthe hyperthermostable three-iron cluster ferredoxin from the archaeon Pyrococcus furiosus

The thermodynamics and dynamics of the Cys21-Cys48 disulfide "S" <----> "R" conformational isomerism in the three-iron, single cubane cluster ferredox in (Fd) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) have b een characterized by H-1 NMR spectroscopy in both water and water/ methanol mixed solvents. The mean interconversion rate at 25 degreesC is 3 x 10(3) s(-1) and DeltaG(298) = -0.2 kcal/mol [DeltaH = 4.0 kcal/mol; DeltaS = 14 c al/(mol.K)], with the S orientation as the more stable form at low temperat ure (<0 degreesC) but the R orientation predominating at > 100 degreesC, wh ere the organism thrives. The distinct pattern of ligated Cys beta -proton contact shifts for the resolved signals and their characteristic temperatur e behavior for the forms of the 3Fe Fd with alternate disulfide orientation s have been analyzed to determine the influences of disulfide orientation a nd methanol cosolvent on the topology of the inter-iron spin coupling in th e 3Fe cluster. The Cys21-Cys48 disulfide orientation influences primarily t he spin couplings involving the iron ligated to Cys17, whose carbonyl oxyge n is a hydrogen bond acceptor to the Cys21 peptide proton. Comparison of th e Cys beta -proton contact shift pattern for the alternate disulfide orient ations with the pattern exhibited upon cleaving the disulfide bridge confir ms 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, 8167-81781 proposal that the structure of the sam e Fd with the R disulfide orientation resembles that of the Fd upon cleavin g the disulfide bond.