Partial Orientation of Oxidized and Reduced Cytochromeb5at High Magnetic Fields: Magnetic Susceptibility Anisotropy Contributions and Consequences for Protein Solution Structure Determination

Abstract

The backbone 15N−1H 1J values have been measured for oxidized and reduced cytochrome b5 at 500 and 800 MHz. Their field dependence, due to increasing partial orientation of the molecule in solution at high magnetic fields, provides structural constraints relative to the orientation of the NH bond vector with respect to the principal directions of the molecular susceptibility tensor. The constraints have been used in a distance geometry algorithm together with the NOE constraints and with or without pseudocontact shifts constraints. The three sets of constraints are found to be consistent with one another, and their relative contribution to the definition of the structure depends on the number of constraints and their assigned weight. The orientation-dependent 15N−1H 1J values provide the principal directions and anisotropies of the molecular or overall magnetic susceptibility tensor, χmol. The χmol parameters obtained for the oxidized form differ from those for the diamagnetic reduced form essentially by the magnetic susceptibility of the paramagnetic ion, whose anisotropy is responsible for the occurrence of pseudocontact shifts. The χmol tensor of the paramagnetic form displays a sizable rhombic anisotropy, thus permitting the full assessment of the orientation of individual amide vectors in the molecular axes frame.