An analysis of the magnetic field dependence of one-bond couplings has yielded the magneticsusceptibility anisotropies for
Clostridium pasteurianum rubredoxin (Rdx) in its oxidized Fe(III) and reducedFe(II) states. Experimental one-bond
1H
N-
15N and
1H
![](/images/gifchars/alpha.gif)
-
13C
![](/images/gifchars/alpha.gif)
couplings were measured at two field strengths(corresponding to 400 and 750 MHz
1H frequencies) and decomposed into their field-independent scalar (
1J)and field-dependent dipolar (
1D) components. The total numbers of measured dipolar couplings (
1H
N-
15Nplus
1H
![](/images/gifchars/alpha.gif)
-
13C
![](/images/gifchars/alpha.gif)
) were 50 for oxidized Rdx and 49 for reduced Rdx. The atom pairs giving rise to these signalsare located >11 Å from the iron; those closer to the iron are too broad to be resolved in two-dimensionalNMR spectra and may exhibit large Fermi contact shifts. A five-dimensional grid search and
Powell minimizationof the difference between each set of measured dipolar couplings and those calculated from an X-ray crystalstructure of Fe(III) Rdx yielded the magnitude and orientation of the magnetic susceptibility anisotropy ineach oxidation state. (The data for Fe(II) Rdx were analyzed in terms of the X-ray structure for Fe(III) Rdxbecause no X-ray coordinates were available for the reduced rubredoxin. The assumption underlying thisapproximation, that the conformations of the oxidized and reduced rubredoxin are very similar in proteinregions >11 Å from the iron, was validated by comparisons of experimental and calculated pseudocontactshifts.) The axial and rhombic magnetic susceptibility anisotropies were 5.3 × 10
-28 and 2.1 × 10
-28 cm
3/molecule, respectively, for oxidized Rdx, and 20.3 × 10
-28 and 9.7 × 10
-28 cm
3/molecule, respectively, forreduced Rdx. The derived susceptibility tensors were then used to calculate the pseudocontact contributions tothe backbone
1H
![](/images/gifchars/alpha.gif)
and
1H
N chemical shifts of Rdx in the two oxidation states. Oxidation-state-dependentpseudocontact shifts were found to account fully (within experimental error) for the experimental chemicalshift differences exhibited by these backbone signals. Thus, the results are consistent with the absence ofappreciable conformational differences between Fe(III) Rdx and Fe(II) Rdx in the protein regions representedby the NMR data (>11 Å from the iron).