Protein Expression, Selective Isotopic Labeling, and Analysis of Hyperfine-Shifted NMR Signals of Anabaena 7120 Vegetative [2Fe-2S]Ferredoxin

Two alternative T7 RNA promoter/polymerase systems have been employed for the heterologous expression of a plant-type [2Fe-2S]ferredoxin, Anabaena 7120 vegetative ferredoxin, in Escherichia coli at high levels (∼20 mg/liter of culture). One system was used when 15N-labeling the ferredoxin uniformly by growing E. coli with 15NH4Cl as the nitrogen source; the other was used in conjunction with auxotrophic host strains to enrich the protein selectively by incorporating 2H-, 13C-, and 15N-labeled amino acids. The labeled ferredoxin samples were studied by 1H, 2H, 13C and 15N NMR spectroscopy. Results from 1H and 2H NMR studies of samples containing [2Hα]Cys, [2Hβ2,β3]Cys, [13Cβ]-Cys, and [15N]Cys have confirmed previous cysteinyl proton resonance assignments (L. Skjeldal, W. M. Westler, B.-H. Oh, A. M. Krezel, H. M., Holden, B. L. Jacobson, I. Rayment, and J. L. Markley (1991) Biochemistry 30, 7363-7368). All four 13C NMR peaks arising from the four cysteinyl β-carbons and all four 15N NMR peaks from the four cysteinyl nitrogens were resolved in spectra of both the oxidized and reduced ferredoxins. The nitrogen resonance of Cys46, which is located in a unique (Ala-Cys) dipeptide, was assigned by detection of 13Ci-15Ni+1 coupling in a ferredoxin sample with incorporated [13C′]Ala and [15N]Cys. The nitrogen signal of Cys41 was assigned tentatively on the basis of its chemical shift and T1 relaxation time. The cysteinyl β-carbon resonances in the reduced state have been assigned to individual residues on the basis of correlations with their (previously assigned) β-protons. The β-carbon resonance from Cys46 in the oxidized state has been assigned by its correlation with the corresponding resonance in the reduced state; this was accomplished by following the progressive air oxidation of a protein sample reduced by dithionite in the presence of methyl viologen. The spin-lattice relaxation times of the β-carbons of the two cysteines coordinated to Fe(III) were similar in the oxidized and reduced states. This suggests that the antiferromagnetic coupling present in the reduced cluster has little influence on the electronic relaxation time of the Fe(III). Studies of the temperature dependence of the 1H, 13C, and 15N signals of the cysteinyl ligands to the [2Fe-2S] cluster show that the slope of the temperature dependence (Δδ/ΔT−1) can be different for different atom types within a given residue. For example, in the reduced ferredoxin, although Δδ/ΔT−1 is positive for Cy