Redox-dependent magnetic alignment of Clostridium pasteurianum rubredoxin:Measurement of magnetic susceptibility anisotropy and prediction of pseudocontact shift contributions

An analysis of the magnetic field dependence of one-bond couplings has yiel ded the magnetic susceptibility anisotropies for Clostridium pasteurianum r ubredoxin (Rdx) in its oxidized Fe(III) and reduced Fe(II) states. Experime ntal one-bond (HN)-H-1-N-15 and (Halpha-13Calpha)-H-1 couplings were measur ed at two field strengths (corresponding to 400 and 750 MHz H-1 frequencies ) and decomposed into their field-independent scalar ((1)J) and field-depen dent dipolar (D-1) components. The total numbers of measured dipolar coupli ngs (H-1(N)-N-15 plus (1)Ha-C-13(alpha)) were 50 for oxidized Rdx and 49 fo r reduced Rdx. The atom pairs giving rise to these signals are located >11 Angstrom from the iron; those closer to the iron are too broad to be resolv ed in two-dimensional NMR spectra and may exhibit large Fermi contact shift s. A five-dimensional grid search and Powell minimization of the difference between each set of measured dipolar couplings and those calculated from a n X-ray crystal structure of Fe(III) Rdx yielded the magnitude and orientat ion of the magnetic susceptibility. anisotropy in each oxidation state. (Th e data for Fe(II) Rdx were analyzed in terms of the X-ray structure for Fe( III) Rdx because no X-ray coordinates were available for the reduced rubred oxin. The assumption underlying this approximation, that the conformations of the oxidized and reduced rubredoxin are very similar in protein regions >11 Angstrom from the iron, was validated by comparisons of experimental an d calculated pseudocontact shifts.) The axial and rhombic magnetic suscepti bility anisotropies were 5.3 x 10(-28) and 2.1 x 10(-28) cm(3)/molecule, re spectively, for oxidized Rdx, and 20.3 x 10(-28) and 9.7 x 10(-28) cm(3)/mo lecule, respectively, for reduced Rdx. The derived susceptibility tensors w ere then used to calculate the pseudocontact contributions to the backbone H-1(alpha) and H-1(N) chemical shifts of Rdx in the two oxidation states. O xidation-state-dependent pseudocontact shifts were found to account fully ( within experimental,error) for the experimental chemical shift differences exhibited by these backbone signals. Thus, the results are consistent with the absence of appreciable conformational differences between Fe(III) Rdx a nd Fe(ll) Rdx in the protein regions represented by the NMR data (>11 Angst rom from the iron).