Design, synthesis, and characterization of a novel hemoprotein

Here we describe a synthetic protein (6H7H) designed to bind four heme grou ps via bis-histidine axial ligation. The hemes are designed to bind perpend icular to another in an orientation that mimics the relative geometry of th e two heme a groups in the active site of cytochrome c oxidase. Our newly d eveloped protein-design program, called CORE, was implemented in the design of this novel hemoprotein. Heme titration studies resolved four distinct K -D values (K-D1 = 80 nM, K-D2 = 18 nM, K-D3 greater than or equal to 3 mM, K-D4 less than or equal to 570 nM, with K-D3 x K-D4 = 1700); positive coope rativity in binding between the first and second heme, as well as substanti al positive cooperativity between the third and forth heme, was observed. C hemical and thermal denaturation studies reveal a stable protein with nativ e-like properties. Visible circular dichroism spectroscopy of holo-6H7H ind icates excitonic coupling between heme groups. Further electrochemical and spectroscopic characterization of the hole-protein support a structure that is consistent with the predefined target structure.