H-1-NMR INVESTIGATION OF THE ELECTRONIC AND MOLECULAR-STRUCTURE OF THE 4-IRON CLUSTER FERREDOXIN FROM THE HYPERTHERMOPHILE PYROCOCCUS-FURIOSUS - IDENTIFICATION OF ASP-14 AS A CLUSTER LIGAND IN EACH OF THE 4 REDOX STATES

The molecular and electronic structure of the four-iron cluster of the ferredoxin (Fd) from the hyperthermophilic archaeon, Pyrococcus furio sus, Pf (which has only three Cys in the cluster binding consensus seq uence), has been investigated by H-1 NMR in order to determine the ide ntity of the non-cysteinyl cluster ligand in each of the four redox st ates [Gorst, C. M., Zhou, Z. H., Ma, K., Teng, Q., Howard, J. B., Adam s, M. W., & La Mar, G. N. (1995) Biochemistry 34, 8788-8795], and to c haracterize the electron spin ground state for the reduced cluster whi ch at 10 K exhibits an unusual predominant S = 3/2 ground state [Conov er, R. C., Kowal, A. T., Fu, W., Park, J.-B., Aono, S., Adams, M. W. W ., & Johnson, M. K. (1990) J. Biol. Chem. 265, 8533-8541]. It is demon strated that a combination of 1D and 2D NMR tailored to relaxed resona nces allows the location of four hyperfine shifted and paramagneticall y relaxed spin systems which dictates that all four cluster ligands ar e amino acid side chains, rather than a solvent water/hydroxide at the unique non-Cys ligation site. Three of the Ligands could be sequence- specifically assigned to the three Cys residues (positions 11, 17, and 56) in the consensus sequence for cluster binding, hence identifying the fourth ligand as Asp 14. It is concluded that the identification o f Asp ligation to a 4Fe cluster is readily achieved in the reduced, bu t not in the oxidized cluster of Fd. Analysis of the relaxation proper ties and pattern of the hyperfine shifts in PfFd reveals very strong s imilarities to other Fds with S = 1/2 ground states, leading to the co nclusion that the S = 3/2 ground state is not detected in solution at ambient temperatures, and this is independent of the redox state of th e two remaining Cys residues in the protein (positions 21 and 48). How ever, the electron self-exchange rate for 4Fe Pf Fd is significantly s lower than for other 4Fe Fd with complete Cys ligation. Changes in the pattern of hyperfine shifts between oxidized and reduced clusters for the four ligands in PfFd reveal that the most significant variation o ccurs for the Asp 14 orientation, suggesting that the altered Asp orie ntation may ''gate'' the electron transfer.