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 journalArticle

54 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)8141-8147
Number of pages7
JournalJournal of the American Chemical Society
Volume126
Issue number26
DOIs
Publication statusPublished - Jul 7 2004

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Electron Transport Complex III
Heme
Proteins
Ligands
Cytochrome b Group
Deuterium Oxide
Scaffolds
Oxidation-Reduction
Paramagnetic resonance
Conformations
Hydrogen
Hydrogen bonds
Nuclear magnetic resonance
Scaffolds (biology)
Peptides
Water
imidazole

ASJC Scopus subject areas

  • Chemistry(all)

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De novo design of a D2-symmetrical protein that reproduces the diheme four-helix bundle in cytochrome bc1. / Ghirlanda, Giovanna; Osyczka, Artur; Liu, Weixia; Antolovich, Michael; Smith, Kevin M.; Dutton, P. Leslie; Wand, A. Joshua; DeGrado, William F.

In: Journal of the American Chemical Society, Vol. 126, No. 26, 07.07.2004, p. 8141-8147.

Research output: Contribution to journalArticle

Ghirlanda, G, Osyczka, A, Liu, W, Antolovich, M, Smith, KM, Dutton, PL, Wand, AJ & DeGrado, WF 2004, 'De novo design of a D2-symmetrical protein that reproduces the diheme four-helix bundle in cytochrome bc1', Journal of the American Chemical Society, vol. 126, no. 26, pp. 8141-8147. https://doi.org/10.1021/ja039935g
Ghirlanda, Giovanna ; Osyczka, Artur ; Liu, Weixia ; Antolovich, Michael ; Smith, Kevin M. ; Dutton, P. Leslie ; Wand, A. Joshua ; DeGrado, William F. / De novo design of a D2-symmetrical protein that reproduces the diheme four-helix bundle in cytochrome bc1. In: Journal of the American Chemical Society. 2004 ; Vol. 126, No. 26. pp. 8141-8147.
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