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Design, synthesis, and characterization of a novel hemoprotein

Department of Chemistry, Rutgers, The State University of New Jersey, Newark, New Jersey 07102, USA

Reprint requests to: Dr. Ramy S. Farid, Department of Chemistry, Rutgers University, 73 Warren Street, Newark, New Jersey 07102-1811, USA; e-mail: rfarid{at}newark.rutgers.edu; fax: (973) 353-1264.

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 3 mM, K_D4 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.

Keywords: de novo proteins; heme; hemoprotein; protein design

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