Determination of ligand conformation in reduced [2Fe-2S] ferredoxin from cysteine β-proton hyperfine couplings

The isotropic hyperfine couplings of cysteine beta-protons in iron-sulfur clusters of proteins provide information about the structure and conformation of the clusters if their magnetic resonance peaks can be resolved and assigned. The application of two-dimensional ESEEM (HYSCORE) spectroscopy to the reduced [2Fe-2S] cluster in ferredoxin from red marine algae Porfira umbilicalis is described. After deuterium substitution of the exchangeable protons, highly-resolved, orientationally-selected HYSCORE spectra show cross-peaks from strongly coupled, nonexchangeable protons. When cross-peaks from all the HYSCORE spectra are linearized and transformed to a common nuclear Zeeman frequency, they fall along five straight lines. Four of these sets of peaks are assigned to beta-protons of the cysteine ligands. The isotropic and anisotropic hyperfine couplings for these protons are extracted from the slopes and intercepts of these lines. Two rescaling procedures are examined for the conversion of the experimentally measured isotropic couplings from different irons in [2Fe-2S] and [4Fe-4S] clusters. The couplings from P. umbilicalis appear to fit the same empirical dependence on Fe-S-C-H dihedral angle as do the couplings from a [4Fe-4S] model cluster. A method to assign protons for proteins of unknown structure is proposed that yields the correct assignment as derived from the crystal structure of the highly homologous protein from Spirulina platensis. The conformations of the cysteines in the reduced protein, derived without any adjustable parameters from this procedure and the empirical functions, are consistent with those reported for the latest refinement of the crystal structure of the oxidized protein.