Solution 1H NMR Determination of Secondary Structure for the Three-Iron Form of Ferredoxin from the Hyperthermophilic Archaeon Pyrococcus furiosus

Abstract

Two-dimensional 1H NMR data have been used to make sequence-specific assignments and define the secondary structure of the three-iron form of the oxidized ferredoxin, Fd, from the hyperthermophilic archaeon Pyrococcus furiosus, Pf. Signals for at least some protons were located for 65 of the 66 amino acids in the sequence, in spite of the paramagnetic (S = 1/2) ground state, but not all could be assigned. Unassigned and missing signals could be qualitatively correlated with the expected proximity of the protons to the paramagnetic cluster. The secondary structure was deduced from qualitative analysis of the 2D nuclear Overhauser effect, which identified two antiparallel beta-sheets, one triple-stranded including Ala1-Ser5, Val39-Glu41, and Thr62-Ala66, and one double-stranded consisting of Glu26-Asn28 and Lys32-Glu34, as well as an alpha-helix involving Glu43-Glu54. Three tight type I turns are located at residues Asp7-Thr10, Pro22-Phe25, and Asp29-Gly31. Comparison with the crystal structure of Desulfovibrio gigas, Dg, Fd (Kissinger et al., 1991) reveals a very similar folding topology, although several secondary structural elements are extended in Pf relative to Dg Fd. Thus the beta-sheet involving the two termini is expanded to include the two terminal residues and incorporates a third strand from the internal loop that is lengthened by several insertions in Pf relative to Dg Fd. The double-stranded beta-sheet in the interior of Pf Fd is lengthened slightly due to a much tighter type I turn between the two strands. The helix near the C-terminus is three residues longer in Pf than in Dg Fd, as well as being shifted toward the N-terminus. The disulfide link between the two nonligating Cys residues (Cys21 and Cys48) is conserved in Pf Fd, but the link near the C-terminus is in the middle of the long alpha-helix in Pf Fd, instead of at the N-terminus of the helix as in Dg Fd. The extensions of the beta-sheets and alpha-helix increase the number of main-chain hydrogen bonds in Pf Fd by approximately 8 relative to those in Dg Fd and likely contribute to its remarkable thermostability (it is unaffected by anaerobic incubation at 95 degrees C for 24 h).(ABSTRACT TRUNCATED AT 400 WORDS)

1H Nuclear‐Magnetic‐Resonance Investigation of Oxidized Fe4S4 Ferredoxin from Thermotoga maritima

“…Therefore, we recorded a large number of 2D spectra under a variety of temperature and solvent (H,O or D,O) conditions and found the slow exchange rate of a number of amide protons to be of crucial importance for the assignment process. Similarly to Teng et al (1994) in the case of f? furiosus Fd, we could not use the pH value as a parameter to influence HN chemical shifts because the 1D spectra of T maritima Fd at pH 6.0 and pH 8.0 were identical.…”

“…(D) Deduced secondary structural elements and slowly exchanging backbone amide protons (*) in P. furiosus Fd (Pf.) according to Teng et al (1994). a Chemical shift value was estimated from the 1D spectrum at 293 K depicted in Macedo et al (1993a).…”

“…They are based on weak HNHa cross peaks in all three cases, on strong Ha-Ha' cross peaks for the glycines and on sequential dCN NOES between G14 and V15, which is the only GV sequence motif in I: maritima Fd; G12 was assigned by exclusion as was its analogue G13 in Fe,S, Fd from l? furiosus (Teng et al, 1994). According to Backes et al (1991), the amide protons of these three residues are expected to form hydrogen bonds to cysteinyl or inorganic sulfur atoms of the Fe-S cluster which would preclude their detection as discussed above.…”

“…The most likely explanation for this finding is the presence of a paramagnetic Fe-S cluster which causes fast nuclear relaxation, and hence severe line broadening. Gaps or difficulties with partial spin systems in the sequence-specific assignment of Fe,S, or Fe,S, proteins are usually attributed to spatial proximity of amino acids to cubane cluster iron atoms (Gaillard et al, 1992(Gaillard et al, , 1993Nettesheim et al, 1992;Teng et al, 1994). Based on their results for the three-iron form of P. furiosus Fd, Teng et al (1994) suggest that protons in a distance of less than -0.5 nm from an iron atom are not detectable by standard 2D-NMR methods.…”

“…Moreover, we present sequence-specific assignments for 85 % of the amino acid residues from which we deduce information on the second- ary structure of I: maritima Fd. The results are compared to the crystal structure of Fe,S, ferredoxin (ferredoxin 11) from Desulfovibrio gigas (Kissinger et al, 1991) and to a recent NMR study on the three-iron form of the ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus (Teng et al, 1994). Both of these proteins show high sequence identities to I: maritima ferredoxin (Darimont and Sterner, 1994).…”