Design, synthesis, and characterization of a novel hemoprotein

Here we describe a synthetic protein (6H7H) designed to bind four heme groups via bis-histidine axial ligation. The hemes are designed to bind perpendicular to another in an orientation that mimics the relative geometry of the two heme a groups in the active site of cytochrome c oxidase. Our newly developed 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) > or = 3 mM, K(D4) < or = 570 nM, with K(D3) x K(D4) = 1700); positive cooperativity in binding between the first and second heme, as well as substantial positive cooperativity between the third and forth heme, was observed. Chemical and thermal denaturation studies reveal a stable protein with native-like properties. Visible circular dichroism spectroscopy of holo-6H7H indicates excitonic coupling between heme groups. Further electrochemical and spectroscopic characterization of the holo-protein support a structure that is consistent with the predefined target structure.