De novo design of a D2-symmetrical protein that reproduces the diheme four-helix bundle in cytochrome bc1.
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.
Ghirlanda
Giovanna
G
Johnson Research Foundation and Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA. gghirlanda@asu.edu
Osyczka
Artur
A
Liu
Weixia
W
Antolovich
Michael
M
Smith
Kevin M
KM
Dutton
P Leslie
PL
Wand
A Joshua
AJ
DeGrado
William F
WF
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
D003574:Cytochrome c Group; D006420:Hemeproteins; D010455:Peptides; D006427:Hemin