Proton NMR investigation of the oxidized three-iron clusters in the ferredoxins from the hyperthermophilic archae Pyrococcus furiosus and Thermococcus litoralis

Abstract

The 3Fe forms of ferredoxins (Fd) from the hyperthermophilic archaebacteria Pyrococcus furiosus (Pf) and Thermococcus litoralis (Tl) have been investigated by 1H NMR. A combination of one-dimensional nuclear Overhauser and two-dimensional NOESY and bond correlation spectroscopy provides the assignment of the aromatic residues, one conserved valine, and the location of the signals for each of the three cysteines coordinated to the clusters. Dipolar contacts between the Trp 2 and Tyr 46 in Pf Fd and from an invariant phenylalanine to an invariant valine and a cluster cysteine in both Fd confirm a folding pattern for these proteins that is very similar to that of the crystallographically characterized ferredoxin from the mesophile Desulfovibro gigas. The sequence-specific assignment of the buried cysteine near the invariant phenylalanine has been made. The temperature dependence of the contact-shifted cysteinyl residues reveals a distinct 2:1 asymmetry in the magnetic coupling among the three high-spin ferric ions, in that one cysteine exhibits Curie behavior, while the other two cysteines display anti-Curie behavior. These magnetic properties are rationalized qualitatively on the basis of a magnetic coupling scheme where two iron couple to yield an intermediate spin of 2 which couples to the remaining S = 5/2 iron to yield the total cluster spin 1/2. This magnetic asymmetry appears to be a characteristic feature of oxidized 3 Fe clusters. Pf Fd also undergoes a dynamic equilibrium between two alternate forms that differ slightly in the environment of two of the coordinated cysteines. Analysis of the pattern of the contact shifts for the three cysteines in the two ferredoxins suggests that the cysteine coordinated to the unique iron does not have the same sequence origin.

Desulfovibrio gigas ferredoxin II: redox structural modulation of the [3Fe–4S] cluster

“…4). The experimental chemical shifts available for the b-CH 2 protons of DgFdII, Thermococus litoralis 3Fe Fd [73,74] and A. vinelandii 7Fe Fd [75] were used (after subtracting 2.8 ppm, the intrinsic diamagnetic chemical shift for a b-CH 2 proton, and assuming that the contact term is dominant) [76] together with the h values from the X-ray crystal structures of Dg [44] and A. vinelandii [77,78] Fds. T. litoralis 3Fe Fd was used in this fitting considering the similarity between the NMR data from this protein and those from DgFdII.…”

Characterization of aromatic aminotransferases from the hyperthermophilic archaeon Thermococcus litoralis

“…This organism also possesses significant concentrations of a new enzyme termed indolepyruvate ferredoxin oxidoreductase. This catalyzes the CoASH-dependent oxidative decarboxylation of indolepyruvate, phenylpyruvate and p-hydroxypyruvate to the corresponding aryl acyl-CoA using 7: litoralis ferredoxin [38] as the electron acceptor (Mai, X. and Adams, M. W. W., unpublished data: see 14, 81). Reduced ferredoxin is thought to be oxidized by 7: litoralis hydrogenase leading to H, production (see [39]) but the fate of the aryl acyl-CoA derivatives is not known.…”

The pH dependent spectral properties of Clostridium pasteurianum 2[Fe4S4] ferredoxin

“…The protein has been the subject of several spectroscopic investigations aimed at the determination of its solution structure [4,5] and the identification of the factors modulating the redox potential of the two clusters [&9]. Interestingly, the two weakly paramagnetic Fe&, clusters produce a characteristic 'H NMR spectrum with eight well-resolved hyperfine signals in the downfield region [lo].…”

“…Interestingly, the two weakly paramagnetic Fe&, clusters produce a characteristic 'H NMR spectrum with eight well-resolved hyperfine signals in the downfield region [lo]. Very recently, thanks to the application of paramagnetic 2D NMR techniques, the eight hyperfine signals have been assigned specifically to the /3-CH, protons of the cysteines coordinated to either cluster [5,6,. These assignments give us the chance to monitor in detail the perturbations that the protein experiences under different solution conditions.…”