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 pi 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 b5 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Lee
K B
KB
Department of Chemistry, University of California, Davis 95616.
La Mar
G N
GN
Kehres
L A
LA
Fujinari
E M
EM
Smith
K M
KM
Pochapsky
T C
TC
Sligar
S G
SG
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002645:Chickens; D015786:Cytochromes b5 / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D006859:Hydrogen; D008099:Liver / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D011994:Recombinant Proteins; D006418:Heme; D006859:Hydrogen; D015786:Cytochromes b5