De novo design of a D2-symmetrical protein that reproduces the diheme four-helix bundle in cytochrome bc1. Academic Article uri icon


  • An idealized, water-soluble D(2)-symmetric diheme protein is constructed based on a mathematical parametrization of the backbone coordinates of the transmembrane diheme four-helix bundle in cytochrome bc(1). Each heme is coordinated by two His residues from diagonally apposed helices. In the model, the imidazole rings of the His ligands are held in a somewhat unusual perpendicular orientation as found in cytochrome bc(1), which is maintained by a second-shell hydrogen bond to a Thr side chain on a neighboring helix. The resulting peptide is unfolded in the apo state but assembles cooperatively upon binding to heme into a well-folded tetramer. Each tetramer binds two hemes with high affinity at low micromolar concentrations. The equilibrium reduction midpoint potential varies between -76 mV and -124 mV vs SHE in the reducing and oxidizing direction, respectively. The EPR spectrum of the ferric complex indicates the presence of a low-spin species, with a g(max) value of 3.35 comparable to those obtained for hemes b of cytochrome bc(1) (3.79 and 3.44). This provides strong support for the designed perpendicular orientation of the imidazole ligands. Moreover, NMR spectra show that the protein exists in solution in a unique conformation and is amenable to structural studies. This protein may provide a useful scaffold for determining how second-shell ligands affect the redox potential of the heme cofactor.

published proceedings

  • J Am Chem Soc

author list (cited authors)

  • Ghirlanda, G., Osyczka, A., Liu, W., Antolovich, M., Smith, K. M., Dutton, P. L., Wand, A. J., & DeGrado, W. F.

citation count

  • 67

complete list of authors

  • Ghirlanda, Giovanna||Osyczka, Artur||Liu, Weixia||Antolovich, Michael||Smith, Kevin M||Dutton, P Leslie||Wand, A Joshua||DeGrado, William F

publication date

  • July 2004