title: Structural propensities in the heme binding region of apocytochrome b5. II. Heme conjugates.
abstract: In the absence of heme cofactor, the water-soluble domain of rat microsomal cytochrome b5 (cyt b5) contains a long flexible region within its 42-residue heme-binding loop. Heme capture induces this region to fold into a well-defined structure containing helices H3-H5, each separated by a turn, with His39 and His63 serving as axial ligands to the heme iron. We have shown that the H4 region of the apoprotein has the greatest tendency for disorder within the isolated binding loop. Here, the effect of the His63-iron bond and proximity of heme plane on the population of helical conformation in H4 and H5 was investigated by synthesis and characterization of a peptide-sandwiched mesoheme construct in which two H4-H5 peptides were covalently attached to a single cofactor. Spectroscopic data indicated that a holoprotein-like bis-histidine coordination state was achieved over a pH range from 7 to 9. Trifluoroethanol titrations of the construct and the analogous free peptide under these pH conditions revealed that heme proximity and iron ligation were insufficient to promote helix formation in H4 and H5. These observations were used to assess the role of disordered regions in heme capture and the loop-scaffold interface in holoprotein folding and stability.
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Ronald B
authors.initials: RB
authors.lastname: Davis
authors.forename: Juliette T J
authors.initials: JT
authors.lastname: Lecomte
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D015786:Cytochromes b5 / Q000737:chemistry*; D006418:Heme / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D051381:Rats; D013056:Spectrophotometry, Ultraviolet; D017186:Titrimetry; D014270:Trifluoroethanol
chemical_list: D010455:Peptides; D006418:Heme; D014270:Trifluoroethanol; D015786:Cytochromes b5