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

Abstract 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 15 N-labeling the ferredoxin uniformly by growing E. coli with 15 NH 4 Cl as the nitrogen source; the other was used in conjunction with auxotrophic host strains to enrich the protein selectively by incorporating 2 H-, 13 C-, and 15 N-labeled amino acids. The labeled ferredoxin samples were studied by 1 H, 2 H, 13 C and 15 N NMR spectroscopy. Results from 1 H and 2 H NMR studies of samples containing [ 2 H α ]Cys, [ 2 H β2,β3 ]Cys, [ 13 C β ]-Cys, and [ 15 N]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 13 C NMR peaks arising from the four cysteinyl β-carbons and all four 15 N NMR peaks from the four cysteinyl nitrogens were resolved in spectra of both the oxidized and reduced ferredoxins. The nitrogen resonance of Cys 46 , which is located in a unique (Ala-Cys) dipeptide, was assigned by detection of 13 C i - 15 N i +1 coupling in a ferredoxin sample with incorporated [ 13 C′]Ala and [ 15 N]Cys. The nitrogen signal of Cys 41 was assigned tentatively on the basis of its chemical shift and T 1 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 Cys 46 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 1 H, 13 C, and 15 N signals of the cysteinyl ligands to the [2Fe-2S] cluster show that the slope of the temperature dependence (Δδ/Δ T −1 ) can be different for different atom types within a given residue. For example, in the reduced ferredoxin, although Δδ/Δ T −1 is positive for Cys 49 1 H β2 and 1 H β3 , it is negative for Cys 49 13 C β . Although Δδ/Δ T −1 is negative for protons of cysteines ligated to Fe(II) and positive for protons of cysteines ligated to Fe(III), it is positive for all the cysteinyl nitrogens. Nearly complete assignments for the spin system of Arg 42 were derived from NMR studies of three selectively labeled samples: ferredoxin incorporating [U- 15 N]Arg, [26% U- 13 C]Arg, and [ 2 H α,β2,β3 ]Arg. The resonance arising from the backbone amide nitrogen exhibited an unusual chemical shift at 201.6 ppm in the oxidized state but was unresolved in the reduced state. The NMR results indicate that the hydrogen bond observed between the Arg 42 backbone nitrogen and a sulfide of the iron-sulfur cluster in the X-ray structure of the oxidized ferredoxin crystal (W. R. Rypniewski, W. R. Breiter, M. M. Benning, G. Wesenberg, B.-H. Oh, J. L. Markley, I. Rayment, and H. M. Holden (1991) Biochemistry 30, 4126-4131) is present in solution in both the oxidized and reduced forms of the protein. The results show that the noncysteinyl, hyperfine-shifted (peak "K") in the spectrum of the reduced ferredoxin does not arise from 1 H α of Arg 42 as previously postulated.