De Novo Design of a D2-Symmetrical Protein that Reproduces the Diheme Four-Helix Bundle in Cytochrome bc1

J. Am. Chem. Soc., 126 (26), 8141 -8147, 2004. 10.1021/ja039935g S0002-7863(03)09935-9
Web Release Date: June 15, 2004

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, and William F. DeGrado*

Contribution from The Johnson Research Foundation and Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia Pennsylvania 19104; the Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287; the Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and the Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803

gghirlanda@asu.edu; wdegrado@mail.med.upenn.edu

Received December 3, 2003

Abstract:

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.

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