Hyperfine-Shifted ¹³C and ¹⁵N NMR Signals from <i>Clostridium pasteurianum</i> Rubredoxin: Extensive Assignments and Quantum Chemical Verification

Stable isotope-labeling methods, coupled with novel techniques for detecting fast-relaxing NMR signals, now permit detailed investigations of paramagnetic centers of metalloproteins. We have utilized these advances to carry out comprehensive assignments of the hyperfine-shifted ¹³C and ¹⁵N signals of the rubredoxin from <i>Clostridium pasteurianum</i> (<i>Cp</i>Rd) in both its oxidized and reduced states. We used residue-specific labeling (by chemical synthesis) and residue-type-selective labeling (by biosynthesis) to assign signals detected by one-dimensional ¹⁵N NMR spectroscopy, to nitrogen atoms near the iron center. We refined and extended these ¹⁵N assignments to the adjacent carbonyl carbons by means of one-dimensional ¹³C[¹⁵N] decoupling difference experiments. We collected paramagnetic-optimized SuperWEFT ¹³C[¹³C] constant time COSY (SW-CT-COSY) data to complete the assignment of ¹³C signals of reduced <i>Cp</i>Rd. By following these ¹³C signals as the protein was gradually oxidized, we transferred these assignments to carbons in the oxidized state. We have compared these assignments with hyperfine chemical shifts calculated from available X-ray structures of <i>Cp</i>Rd in its oxidized and reduced forms. The results allow the evaluation of the X-ray structural models as representative of the solution structure of the protein, and they provide a framework for future investigation of the active site of this protein. The methods developed here should be applicable to other proteins that contain a paramagnetic center with high spin and slow electron exchange

Structural and Spectroscopic Characterization of Iron(II), Cobalt(II), and Nickel(II) <i>ortho</i>-Dihalophenolate Complexes: Insights into Metal–Halogen Secondary Bonding

Metal complexes incorporating the tris­(3,5-diphenylpyrazolyl)­borate ligand (Tp^Ph2) and <i>ortho</i>-dihalophenolates were synthesized and characterized in order to explore metal–halogen secondary bonding in biorelevant model complexes. The complexes Tp^Ph2ML were synthesized and structurally characterized, where M was Fe­(II), Co­(II), or Ni­(II) and L was either 2,6-dichloro- or 2,6-dibromophenolate. All six complexes exhibited metal–halogen secondary bonds in the solid state, with distances ranging from 2.56 Å for the Tp^Ph2Ni­(2,6-dichlorophenolate) complex to 2.88 Å for the Tp^Ph2Fe­(2,6-dibromophenolate) complex. Variable temperature NMR spectra of the Tp^Ph2Co­(2,6-dichlorophenolate) and Tp^Ph2Ni­(2,6-dichlorophenolate) complexes showed that rotation of the phenolate, which requires loss of the secondary bond, has an activation barrier of ∼30 and ∼37 kJ/mol, respectively. Density functional theory calculations support the presence of a barrier for disruption of the metal–halogen interaction during rotation of the phenolate. On the other hand, calculations using the spectroscopically calibrated angular overlap method suggest essentially no contribution of the halogen to the ligand-field splitting. Overall, these results provide the first quantitative measure of the strength of a metal–halogen secondary bond and demonstrate that it is a weak noncovalent interaction comparable in strength to a hydrogen bond. These results provide insight into the origin of the specificity of the enzyme 2,6-dichlorohydroquinone 1,2-dioxygenase (PcpA), which is specific for <i>ortho</i>-dihalohydroquinone substrates and phenol inhibitors