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.

Engineering the Assembly of Heme Cofactors in Man-Made Proteins

“…Like the maquette B, natural cytochromes b 562 and b 5 and oxygen transport proteins such as myoglobin are predominantly unstructured in the apo state, becoming substantially more structured when heme is bound. 26,42 In all cases, there is a small amount of structure in the apo state that restricts the conformational freedom of the unstructured regions. Apocytochrome b 562 retains two intact helices, while the helices that bind heme are random coil.…”

“…The stepwise redesign of maquettes from early multichain forms to a final single chain form that are sufficiently malleable in the apo state has increased the rate of cofactor self-assembly to a point comparable to natural proteins and limited by the physical chemical properties of the porphyrin itself. Like the maquette B, natural cytochromes b 562 and b 5 and oxygen transport proteins such as myoglobin are predominantly unstructured in the apo state, becoming substantially more structured when heme is bound. , In all cases, there is a small amount of structure in the apo state that restricts the conformational freedom of the unstructured regions. Apocytochrome b 562 retains two intact helices, while the helices that bind heme are random coil.…”