Nuclear magnetic resonance studies of two-iron-two-sulfur ferredoxins. 5. Hyperfine-shifted peaks in proton and carbon-13 spectra

Self Cite

All the nitrogen signals from the amino acid side chains and 80 of the total of 98 backbone nitrogen signals of the oxidized form of the 2Fe.2S* ferredoxin from Anabaena sp. strain PCC 7120 were assigned by means of a series of heteronuclear two-dimensional experiments [Oh, B.-H. Mooberry, E. S., & Markley, J. L. (1990) Biochemistry (second paper of three in this issue )]. Two additional nitrogen signals were observed in the one-dimensional 15N NMR spectrum and classified as backbone amide resonances from residues whose proton resonances experience paramagnetic broadening. The one-dimensional 15N NMR spectrum shows nine resonances that are hyperfine shifted and broadened. From this inventory of diamagnetic nitrogen signals and the available X-ray coordinates of a related ferredoxin [Tsukihara, T., Fukuyama, K., Nakamura, M., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., Hase, T., & Matsubara, H. (1981) J. Biochem. 90, 1763-1773], the resolved hyperfine-shifted 15N peaks were attributed to backbone amide nitrogens of the nine amino acids that share electrons with the 2Fe.2S* center or to backbone amide nitrogens of two other amino acids that are close to the 2Fe.2S* center. The seven 15N signals that are missing and unaccounted for probably are buried under the envelope of amide signals. 1H NMR signals from all the amide protons directly bonded to the seven missing and nine hyperfine-shifted nitrogens were too broad to be resolved in conventional 2D NMR spectra.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Resultsmentioning

confidence: 92%

Multinuclear magnetic resonance studies of the 2Fe.cntdot.2S* ferredoxin from Anabaena species strain PCC 7120. 3. Detection and characterization of hyperfine-shifted nitrogen-15 and hydrogen-1 resonances of the oxidized form

Markley²

1990

Self Cite

“…One-dimensional NMR data from reduced Anabaena 7120 vegetative ferredoxin are shown in Figure 1. Peaks a-e, j, and k show weak Curie-type temperature dependence (the peaks shift toward their diamagnetic positions as the temperature is raised) whereas peaks f-i show anti-Curie temperature dependence (the peaks shift away from their diamagnetic positions as the temperature is raised) (Chan & Markley, 1983;Skjeldal et al, 1990). Signals with Curie scalar shifts are assigned to protons on residues that are ligated to Fe(III), and signals with anti-Curie scalar shifts are assigned to protons on residues that are ligated to Fe(II) (Dunham et al, 1971;Bertini et al, 1984).…”

Section: Resultsmentioning

confidence: 99%

Two-dimensional magnetization exchange spectroscopy of Anabaena 7120 ferredoxin. Nuclear Overhauser effect and electron self-exchange cross peaks from amino acid residues surrounding the iron-sulfur (2Fe-2S) cluster

Skjeldal¹,

Westler²,

Oh³

et al. 1991

Self Cite

Hyperfine 1H NMR signals of the 2Fe-2S* vegetative ferredoxin from Anabaena 7120 have been studied by two-dimensional (2D) magnetization exchange spectroscopy. The rapid longitudinal relaxation rates of these signals required the use of very short nuclear Overhauser effect (NOE) mixing times (0.5-20 ms). The resulting pattern of NOE cross-relaxation peaks when combined with previous 1D NOE results [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271] led to elucidation of the carbon-bound proton spin systems from each of the four cysteines ligated to the 2Fe-2S* cluster in the reduced ferredoxin. Additional NOE cross peaks were observed that provide information about other amino acid residues that interact with the iron-sulfur cluster. NOE cross peaks were assigned tentatively to Leu27, Arg42, and Ala43 on the basis of the X-ray coordinates of oxidized Anabaena 7120 ferredoxin [Rypniewski, W.R., Breiter, D.R., Benning, M.M., Wesenberg, G., Oh, B.-H., Markley, J.L., Rayment, I., & Holden, H. M. (1991) Biochemistry 30, 4126-4131]. Three chemical exchange cross peaks were detected in magnetization exchange spectra of half-reduced ferredoxin and assigned to the 1H alpha protons of Cys49 and Cys79 [both of whose sulfur atoms are ligated to Fe(III)] and Arg42 (whose amide nitrogen is hydrogen-bonded to one of the inorganic sulfurs of the 2Fe-2S* cluster). The chemical exchange cross peaks provide a means of extending assignments in the spectrum of reduced ferredoxin to assignments in the spectrum of the oxidized protein.(ABSTRACT TRUNCATED AT 250 WORDS)

“…For the nuclei surrounding the cluster, this paramagnetism gives rise to "hyperfine" shifted resonances and to line broadening due to shortened relaxation times. Many NMR studies have been performed on the chloroplasttype ferredoxins: the earlier studies concerned the observation of hyperfine-shifted 'H resonances (Poe et al, 1971;Salmeen & Palmer, 1972;Nagayama et al, 1983;Chan & Markley, 1983e;Bertini et al, 1993). Subsequently, the amino acid residues surrounding the [2Fe-2S] cluster have been studied in the reduced form of ferredoxin from the cyanobacteria Spirulina platensis and Anabaena and from the red alga Porphyra umbilicalis (Dugad & La Mar, 1990;Skjeldal et al, 1991).…”

mentioning

confidence: 99%

1H AND 15N NMR Sequential Assignment, Secondary Structure, and Tertiary Fold of [2Fe-2S] Ferredoxin from Synechocystis sp. PCC 6803

Lelong

Sétif²,

Bottin³

et al. 1995

The [2Fe-2S] ferredoxin extracted from Synechocystis sp. PCC 6803 was studied by 1H and 15N nuclear magnetic resonance. Sequence-specific 1H and 15N assignment of amino acid residues far from the paramagnetic cluster (distance higher than 8 A) was performed. Interresidue NOE constraints have allowed the identification of several secondary structure elements: one beta sheet composed of four beta strands, one alpha helix, and two alpha helix turns. The analysis of interresidue NOEs suggests the existence of a disulfide bridge between the cysteine residues 18 and 85. Such a disulfide bridge has never been observed in plant-type ferredoxins. Structure modeling using the X-PLOR program was performed with or without assuming the existence of a disulfide bridge. As a result, two structure families were obtained with rms deviations of 2.2 A. Due to the lack of NOE connectivities resulting from the paramagnetic effect from the [2Fe-2S] cluster, the structures were not well resolved in the region surrounding the [2Fe-2S] cluster, at both extremities of the alpha helix and the C and N terminus segments. In contrast, when taken separately, the beta sheet and the alpha helix were well defined. This work is the first report of a structure model of a plant-type [2Fe-2S] Fd in solution.