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 (similar to 20 mg/liter of culture). One system was used when N-15-labeling the ferredoxin uniformly by growing E. coli with (NH4Cl)-N-15 as the nitrogen source; the other was used in conjunction with auxotrophic host strains to enrich the protein selectively by incorporating H-2-, C-13-, and N-15-labeled amino acids. The labeled ferredoxin samples were studied by H-1, H-2, C-13 and N-15 NMR spectroscopy. Results from H-1 and H-2 NMR studies of samples containing [H-2(alpha)]Cys, [H-2(beta 2,beta 3)]Cys, [C-13(beta)]-Cys, and [N-15]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 C-13 NMR peaks arising from the four cysteinyl beta-carbons and all four N-15 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 C-13(i)-N-15(i+1) coupling in a ferredoxin sample with incorporated [C-13']Ala and [N-15]Cys. The nitrogen signal of Cys(41) was assigned tentatively on the basis of its chemical shift and T-1 relaxation time. The cysteinyl beta-carbon resonances in the reduced state have been assigned to individual residues on the basis of correlations with their (previously assigned) beta-protons. The beta-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 beta-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 H-1, C-13, and N-15 signals of the cysteinyl ligands to the [2Fe-2S] cluster show that the slope of the temperature dependence (Delta delta/Delta T-1) can be different for different atom types within a given residue. For example, in the reduced ferredoxin, although Delta delta/Delta T-1 is positive for Cys(49) H-1(beta 2) and H-1(beta 3), it is negative for Cys(49) C-13(beta). Although Delta delta/Delta 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-N-15]Arg, [26% U-C-13]Arg, and [H-2(alpha,beta 2,beta 3)]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 (peak ''K'') in the spectrum of the reduced ferredoxin does not arise from H-1(alpha) of Arg(42) as previously postulated. (C) 1995 Academic Press, Inc.