1H NMR Study of the Influence of Hydrophobic Contacts on Protein-Prosthetic Group Recognition in Bovine and Rat Ferricytochrome b5
The proton nuclear magnetic resonance spectra of the soluble fragment of native bovine and genetically engineered wild-type rat ferricytochrome b5 reconstituted with a wide variety of hemes chemically modified at 2-and/or 4-positions have been recorded and analyzed. While all but one nonsymmetric heme yielded comparable amounts of the two heme orientations immediately after reconstitution, the relative proportion of the two orientations at equilibrium varied widely. The unpaired spin density distribution in the heme π system leads to substituent hyperfine shift patterns in these paramagnetic complexes that are completely diagnostic of the heme orientation in the protein matrix. An empirical assignment strategy is outlined and applied which allows unequivocal assignment of the absolute orientation of a derivatized heme within the protein matrix. Using a series of hemes lacking 2-fold symmetry solely due to a single substitution, the preferences for localized site occupation of vinyls, methyls, and hydrogens are developed. The large differences in relative stability of the two orientations of native protohemin in the two cytochromes bs is shown to result from the additivity of localized effects for the bovine protein and the near cancellation of competing effects in the rat protein. The major determinant of the heme orientation is judged to be a repulsive interaction between a vinyl and a hydrophobic cluster of amino acids including positions 23 and 25. The differences in this heme orientational preference among bovine, rat, and chicken ferricytochromes b5 could be correlated with the relative steric bulk of the residues at positions 23 and 25. Detailed thermodynamic analysis of the orientational preferences of native protoheme reveals that, while the same orientation as found in X-ray crystal structures of bovine cytochrome b5 predominate at 25 °C in both proteins, the preference in the bovine protein is primarily for enthalpic reasons while in the rat protein the preference is due to entropic factors. © 1990, American Chemical Society. All rights reserved.
Publication Source (Journal or Book title)
Lee, K., La Mar, G., Kehres, L., Fujinari, E., Smith, K., Pochapsky, T., & Sligar, S. (1990). 1H NMR Study of the Influence of Hydrophobic Contacts on Protein-Prosthetic Group Recognition in Bovine and Rat Ferricytochrome b5. Biochemistry, 29 (41), 9623-9631. https://doi.org/10.1021/bi00493a017