Partial orientation of oxidized and reduced cytochrome b(5) at high magnetic fields: Magnetic susceptibility anisotropy contributions and consequences for protein solution structure determination

The backbone N-15-H-1 (1)J values have been measured for oxidized and reduc ed cytochrome b(5) at 500 and 800 MHz. Their field dependence, due to incre asing partial orientation of the molecule in solution at high magnetic fiel ds, provides structural constraints relative to the orientation of the NH b ond vector with respect to the principal directions of the molecular suscep tibility tensor. The constraints have been used in a distance geometry algo rithm together with the NOE constraints and with or without pseudocontact s hifts 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 N-15-H-1 (1)J values provide the principal direct ions and anisotropies of the molecular or overall magnetic susceptibility t ensor, chi(mol). The chi(mol) parameters obtained for the oxidized form dif fer from those for the diamagnetic reduced form essentially by the magnetic susceptibility of the paramagnetic ion, whose anisotropy is responsible fo r the occurrence of pseudocontact shifts. The chi(mol) tensor of the parama gnetic form displays a sizable rhombic anisotropy, thus permitting the full assessment of the orientation of individual amide vectors in the molecular axes frame.