NOVEL HETERONUCLEAR METHODS OF ASSIGNMENT TRANSFER FROM A DIAMAGNETICTO A PARAMAGNETIC PROTEIN - APPLICATION TO RAT CYTOCHROME-B(5)

N-15 and H-1 resonance assignments for backbone and side-chain resonan ces of both equilibrium forms of rat ferricytochrome b5 have been obta ined, using a combination of novel heteronuclear assignment transfer m ethods from the known assignments of the diamagnetic protein [Guiles, R. D., Basus, V. J., Kuntz, I. D., & Waskell, L. A. (1992) Biochemistr y 31, 11365-11375] and computational methods which depend on an accura te determination of the orientation of the components of the susceptib ility tensor. The transfer of amide proton resonance assignments takes advantage of the apparent insensitivity of amide N-15 resonances to p seudocontact effects, evident in overlays of N-15-H-1 heteronuclear co rrelation spectra. Amide-proton resonance assignments tentatively tran sferred from the known diamagnetic assignments to the paramagnetic for m of the protein were confirmed using conventional assignment strategi es employing 600-MHz COSY, HOHAHA, and NOESY spectra of the oxidized p rotein. As was observed in rat ferrocytochrome b5, more than 40% of al l residues exhibited NMR detectable heterogeneity due to the two diffe rent orientations of the heme. Complete assignment of both forms enabl ed accurate determination of the orientation of the susceptibility ten sor for both conformations of the heme. The orientation of the z-compo nent of the susceptibility tensors for the two forms are indistinguish able, while the in-plane components appear to differ by about 6-degree s. Differences in the orientation of the in-plane susceptibility compo nents are undoubtedly due dominantly to the relative axial rotation of the heme of between 5-degrees and 10-degrees indicated by the NOESY c ontacts to the protein observed in the spectra of the ferrocytochrome [Guiles, R. D., Basus, V. J., Kuntz, I. D., & Waskell, L. A. (1992) Bi ochemistry 31,11365-11375; Pochapsky, T. C., Sligar, S. G., McLachlan, S. J., & LaMar, G. N. (1990) J. Am. Chem. Soc. 112,5258-5263].