Supramolecular hemoprotein linear assembly by successive interprotein heme-heme pocket interactions

We demonstrate a new strategy for the construction of supramolecular hemoprotein assemblies. A synthetic heme was selectively introduced onto the surface Cys residue of the cytochrome b(562) single mutant (H63C) through a thioether bond. After removal of the native heme of the H63C mutant by acid denaturation followed by neutralization, the externally attached heme on the apoprotein surface was inserted into the vacant heme pocket of the other apoprotein. Therefore, the interprotein heme-heme pocket interaction produces a unique submicrometer-sized linear hemoprotein fiber, determined by size exclusion chromatography and atomic force microscopy. This methodology should be widely applicable to the creation of new nanobiomaterials based on a functional hemoprotein.