Novel heteronuclear methods of assignment transfer from a diamagnetic to a paramagnetic protein: application to rat cytochrome b5

Abstract

15N and 1H resonance assignments for backbone and side-chain resonances of both equilibrium forms of rat ferricytochrome b5 have been obtained, using a combination of novel heteronuclear assignment transfer methods from the known assignments of the diamagnetic protein [Guiles, R. D., Basus, V. J., Kuntz, I. D., & Waskell, L. A. (1992) Biochemistry 31, 11365-11375] and computational methods which depend on an accurate determination of the orientation of the components of the susceptibility tensor. The transfer of amide proton resonance assignments takes advantage of the apparent insensitivity of amide 15N resonances to pseudocontact effects, evident in overlays of 15N-1H heteronuclear correlation spectra. Amide-proton resonance assignments tentatively transferred from the known diamagnetic assignments to the paramagnetic form of the protein were confirmed using conventional assignment strategies employing 600-MHz COSY, HOHAHA, and NOESY spectra of the oxidized protein. As was observed in rat ferrocytochrome b5, more than 40% of all residues exhibited NMR detectable heterogeneity due to the two different orientations of the heme. Complete assignment of both forms enabled accurate determination of the orientation of the susceptibility tensor for both conformations of the heme. The orientation of the z-component of the susceptibility tensors for the two forms are indistinguishable, while the in-plane components appear to differ by about 6 degrees. Differences in the orientation of the in-plane susceptibility components are undoubtedly due dominantly to the relative axial rotation of the heme of between 5 degrees and 10 degrees indicated by the NOESY contacts to the protein observed in the spectra of the ferrocytochrome [Guiles, R. D., Basus, V. J., Kuntz, I. D., & Waskell, L. A. (1992) Biochemistry 31, 11365-11375; Pochapsky, T. C., Sligar, S. G., McLachlan, S. J., & LaMar, G. N. (1990) J. Am. Chem. Soc. 112, 5258-5263].

Analysis of the paramagnetic shifts of haem carbon resonances in bovine ferricytochrome b 5

“…The change in the shift of Gly 42 is difficult to interpret in the presence of a modified dipolar field, but the NOE data have been used to propose that the haem in the B form is rotated by a few degrees about the haem normal with respect to the A form, in addition to being flipped over about the 5CH-15CH axis. The proposed sense of rotation shifts the 5CH of the B form towards the 20CH position found in the A form (Pochapsky et al 1990), and Guiles et al (1993) found a rotation of 5-10 ° between the magnetic susceptibility tensors in the A and B forms of rat cytochrome b 5 which they took to be a confirmation of the earlier conclusion. It is important to note that quite different reference frames apply: the NOE data relate the position of one methyl group in the protein to the position of the haem whereas the magnetic axes are determined with respect to the protein structure and do not provide any definitive information about the orientation of the haem itself.…”

Redox-Related Chemical Shift Perturbations on Backbone Nuclei of High-Potential Iron−Sulfur Proteins

“…9 These redox shifts, not yet fully understood, might have implications in the electron-transfer process. Similar effects have been reported for Fe n+ (Cys4) rubredoxins, 10 as well as for ferredoxins 11 and putidaredoxins, 12,13 which contain an [Fe 2 S 2 ] n+ cluster, whereas, to our knowledge, no such effects have been reported so far for any protein of the [Fe 4 S 4 ] n+ family.…”

Dimethyl propionate ester heme-containing cytochrome b 5: structure and stability

“…The only residue which has not been identified is Pro90. 15 N HSQC spectra are very similar to those of the native protein [37,38]. 15 N shift differences larger than 1 ppm between backbone amide protons in the native and in the modified proteins are detected for residues 1, 3, 31, 44, 56, 57, 58, 61, 63, 64, 65, 66, 68, 83, 85, and 93.…”