Solution NMR Study of the Electronic and Molecular Structures of Hyperthermostable Ferredoxins

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9600759 LaMar The molecular and electronic structural and dynamic properties of ferredoxins (Fds), from a series of hyperthermophilic archaea, Pyroccus furiosus, Pf, Thermococcus litoralis, Tl, and the as yet unnamed ES-4 and JDF-3, will be elucidated by NMR. These small electron transfer Fd possess paramagnetic clusters at ambient temperature in both oxidation states. The appropriate tailoring of 1D and 2D NMR methods, as augmented by 3D NMR on select N-15 labeled Fd, to detect and assign all paramagnetically influenced resonances is expected to provide: the sequence-specific identification of the valence states for he individual iron in the reduced cluster; elucidation of the influence of ligand mutation and/or exogenous ligands in modulating the overall cluster redox potential as well as the individual iron oxidation states; the spectral signatures of non cysteine ligands for identifying protein ligands in other iron-sulfur cluster enzymes; a protocol for utilizing various paramagnetic spectral data for use as structural constraints to substitute for lost conventional NMR constraints near the cluster; and, lastly, but most importantly, robust molecular models for these Fds. The pf, ES-4 and JDF-3 Fds also possess a pair of remote Cys which serve as 2e- 2H+ redox sites at essentially the same potential as the cluster. The complete time resolution of the resulting four Fd redox states at ambient temperature is expected to provide unique insight into the influence of each step in modulating the protein architecture both near and remote from the cluster, as well as the structural basis of the large reorganizational energy for cluster electron transfer in these Fd. The resulting molecular structures are expected to shed light on what structural properties impart the remarkable thermostability (negligible denaturation at 95(C over 24h) to these proteins in spite of significant sequence homology to much less stable Fds from mesophiles. The identification of "stabilizing structural motifs" i n the present Fds may permit future introduction by mutagenesis of the "motifs" to increase thermostability of industrial biocatalysts. The characterization of these ferredoxins will also contribute to the understanding of these novel organisms (archaea) that are proposed to be the most ancient and slowly evolving form of life and possibly the closest living descendant to a universal life form. %%% The molecular structures of a series of small ((60 amino acid residues) electron transfer proteins called ferredoxins will be determined by solution proton nuclear magnetic resonance and the resulting structures used to interpret properties relevant to their role in electron transfer. The ferredoxins are selected from a number of archaebacteria (i.e. Pyrococcus furiosus, Thermococcus litoralis) that live in deep ocean volcano vents at temperatures of boiling water. The goal is to define the interactions among the various pairs of residues in these ferredoxins and to relate differences in these interactions in the high temperature-state archaebacteria ferredoxins with similar proteins of more normal eubacteria which are stable at 37(C, but rapidly denature at temperatures much higher than 50(C. The understanding of the types of interactions that contribute to the high temperature stability may provide clues as how to increase by site-directed mutagenesis the high temperature stability of enzymes used in the biotechnology industry. The proposed studies will also contribute to the understanding of these relatively recently discovered archaebacteria which are proposed to be the closest living descendants of the earliest forms of life on this planet. ***