An NMR‐Derived Model for the Solution Structure of Oxidized Thermotoga maritima 1[Fe₄‐S₄] Ferredoxin

Abstract

The solution structure of the 60-residue 1 [Fe,-S,] ferredoxin from the hyperthermophilic bacterium Thermotoga maritima was determined based on 683 distance and 35 dihedral angle restraints that were obtained from NMR data. In addition, data known from crystallographic studies of ferredoxins was used for modeling of the iron-sulfur cluster and its environment. The protein shows a globular fold very similar to the fold of the related 1 [Fe,-S,] ferredoxins from Desulfovibrio gigas and Desuljovibrio africanus, and elements of regular secondary structure similar to those in other ferredoxins were found in the 2: maritima protein. In particular, the ?: rnaritimu protein displayed a @-sheet structure made up of strands located at the very NH, and COOH termini of the protein, and an internal rx-helix. The internal @-sheet observed in the D. gigas and D. africunus ferredoxins could not be confirmed in 7: muritima ferredoxin and is thus suggested to be only weakly present or even absent in this protein. This result suggests that thermostability in ferredoxins is not necessarily correlated with the content of stable elements of regular secondary structure.Keywords; Ferredoxin; NMR ; protein structure ; iron-sulfur proteins ; Thermotoga maritima. Thermotoga maritima is a hyperthermophilic bacterium (Stetter et al., 1990) with a maximal growth temperature of 90°C (Huber et al., 1986). Together with Aquifex pyrophilus (Huber et al., 1992), 7: maritima is the only known exception to the rule that hyperthermophilic microorganisms can be classified as Archaea. In addition, as 7: maritima and A. pyrophilus represent the most original and most slowly evolving branches of bacteria, these species may have retained more characteristic properties of the presumed last common ancestor of life than most other known organisms.Ferredoxins are small iron-sulfur electron transport proteins that are involved in crucial metabolic processes such as photosynthesis, oxidative phosphorylation, or nitrogen fixation (for reviews see Beinert, 1990;Matsubara and Saeki, 1992). The function of these proteins is accomplished by Fe,S, clusters (mainly in plants and animals) or Fe,S, (Fe,S,) clusters (mainly in prokaryotes).Ferredoxin from 7: muritima (Fd,,) functions as an electron carrier for pyruvate oxidoreductase (Blarney et al., 1994). Recently, the gene encoding Fd,,,, was cloned and expressed in Escherichia coli (Darimont and Sterner, 1994). These studies Bayreuth, D-95440 Bayreuth, Germany revealed that Fd.,, contains a single Fe,S, cluster and a disulfide bond. Fd.,.,, is extremely thermostable and shows high sequence similarities to ferredoxins of hyperthermophilic Archaea, with identities of 73% and 45% between FdT,, and the proteins from Thermococcus litoralis (Fd,,) and Pyr;ococcus furiosus (Fd,,;Busse et al., 1992), respectively ( Table 1). The sequence identity to ferredoxins from mesophilic bacteria such as Desulfovibrio gigas (Fd,,;Bruschi, 1979;Travis et al., 1971) and Desulfovibrio africanus (Fd,,;Bruschi and Hatschikian, 1982) is 3...

Sequence Determination of Reduction Potentials by Cysteinyl Hydrogen Bonds and Peptide Dipoles in [4Fe-4S] Ferredoxins

“…In the Ϫ60°orientation, the CysS␥ hydrogen bonds to the redox site S␥ 4 . In AvI Fd, the CysS␥⅐⅐⅐S␥ 4 and CysH␥⅐⅐⅐S␥ 4 distances are 3.64 and 2.43 Å, respectively, while the (Backes et al, 1991), Cv (Moulis et al, 1996), Ca (Dauter et al, 1997;Duée et al, 1994), Cp (Bertini et al, 1995), AvI (Stout et al, 1998;Stout, 1993;Schipke et al, 1999), AvIЈ (Soman et al, 1991), Bs and BsЈ (Aono et al, 1998), Sa (Fujii et al, 1996), DaI (Séry et al, 1994), Bt (Fukuyama et al, 1989), Tm (Sticht et al, 1996), DgII (Kissinger et al, 1991 CysS␥⅐⅐⅐S* 2 and CysH␥⅐⅐⅐S* 2 distances are 3.36 and 3.58 Å, respectively, and the CysS␥⅐⅐⅐S* 3 and CysH␥⅐⅐⅐S* 3 distances are 3.89 and 3.60 Å, respectively. Because the S␥OH␥ bond length is 1.325 Å, a linear hydrogen bond is formed to S␥ 4 whereas only perpendicular hydrogen bonds can be made to S* 2 and S* 3 .…”

Small structural changes account for the high thermostability of 1[4Fe–4S] ferredoxin from the hyperthermophilic bacterium Thermotoga maritima

“…Of the ferredoxins containing 1[4Fe-4S] or 1[3Fe-4S] clusters (monocluster-type ferredoxins) the X-ray structures of 1[3Fe-4S] Fd Dg [4], 1[4Fe-4S] Fd Daf [5] and 1[4Fe-4S] Fd Bt [6] are known. The secondary structures of the 1[4Fe-4S] Fd Tl [7] and 1[3Fe-4S] Fd Pf [8] have also been determined by 1 H NMR and recently a NMR-derived model of 1[4Fe-4S] Fd Tm has been reported [9].…”

A Divide-and-Conquer Implementation of the Discrete Variational DFT Method for Large Molecular and Solid Systems

“…The atomic structure used in this work was obtained from the Brookhaven Protein Database under the entry 1ROF (Structure 1) and is based on solution-NMR data [45]. This protein is a small iron-sulfur electron carrier of the bacterial species Thermotoga maritima.…”

“…By changing its oxidation state, this cluster is generally believed to be the primary carrier site for electrons. The structure used is that of the fully oxidized form of Ferredoxin with formal Fe 4 S 4 cluster charge of +3 [45].…”

“…The effect of increasing fragment size on computed properties is studied for a solid silicate structure (Annite). Finally a trial run is presented to compute the electronic structure of the 845 atom protein Thermotoga maritima 1 [Fe 4 S 4 ] Ferredoxin [45].…”