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.
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Department of Chemistry, Rutgers, The State University of New Jersey, Newark, New Jersey 07102, USA.
Farid
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D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D006418:Heme / Q000378:metabolism; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010452:Peptide Biosynthesis; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D012984:Software; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000378:metabolism
D006420:Hemeproteins; C431347:hemoprotein 6H7H; D006418:Heme; D014364:Tryptophan