De novo design of a D2-symmetrical protein that reproduces the diheme four-helix bundle in cytochrome bc1

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

Research output: Contribution to journalArticlepeer-review

67 Scopus citations


An idealized, water-soluble D2-symmetric diheme protein is constructed based on a mathematical parametrization of the backbone coordinates of the transmembrane diheme four-helix bundle in cytochrome bc1. 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 bc1, 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 bc1 (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.

Original languageEnglish (US)
Pages (from-to)8141-8147
Number of pages7
JournalJournal of the American Chemical Society
Issue number26
StatePublished - Jul 7 2004

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry


Dive into the research topics of 'De novo design of a D2-symmetrical protein that reproduces the diheme four-helix bundle in cytochrome bc1'. Together they form a unique fingerprint.

Cite this