title: De novo design of a D2-symmetrical protein that reproduces the diheme four-helix bundle in cytochrome bc1.
abstract: 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.
authors.affiliation: Johnson Research Foundation and Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA. gghirlanda@asu.edu
authors.forename: Giovanna
authors.initials: G
authors.lastname: Ghirlanda
authors.forename: Artur
authors.initials: A
authors.lastname: Osyczka
authors.forename: Weixia
authors.initials: W
authors.lastname: Liu
authors.forename: Michael
authors.initials: M
authors.lastname: Antolovich
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Smith
authors.forename: P Leslie
authors.initials: PL
authors.lastname: Dutton
authors.forename: A Joshua
authors.initials: AJ
authors.lastname: Wand
authors.forename: William F
authors.initials: WF
authors.lastname: DeGrado
mesh_terms: D001665:Binding Sites; D003574:Cytochrome c Group / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D006420:Hemeproteins / Q000737:chemistry; D006427:Hemin / Q000737:chemistry*; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D003574:Cytochrome c Group; D006420:Hemeproteins; D010455:Peptides; D006427:Hemin