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De novo design of an artificial bis[4Fe-4S] binding protein.

Authors
Type
Published Article
Journal
Biochemistry
1520-4995
Publisher
American Chemical Society
Publication Date
Volume
52
Issue
43
Pages
7586–7594
Identifiers
DOI: 10.1021/bi401199s
PMID: 24090184
Source
Medline
License
Unknown

Abstract

In nature, protein subunits containing multiple iron-sulfur clusters often mediate the delivery of reducing equivalents from metabolic pathways to the active site of redox proteins. The de novo design of redox active proteins should include the engineering of a conduit for the delivery of electrons to and from the active site, in which multiple redox active centers are arranged in a controlled manner. Here, we describe a designed three-helix protein, DSD-bis[4Fe-4S], that coordinates two iron-sulfur clusters within its hydrophobic core. The design exploits the pseudo two-fold symmetry of the protein scaffold, DSD, which is a homodimeric three-helix bundle. Starting from the sequence of the parent peptide, we mutated eight leucine residues per dimer in the hydrophobic core to cysteine to provide the first coordination sphere for cubane-type iron-sulfur clusters. Incorporation of two clusters per dimer is readily achieved by in situ reconstitution and imparts increased stability to thermal denaturation compared to that of the apo form of the peptide as assessed by circular dichroism-monitored thermal denaturation. The presence of [4Fe-4S] clusters in intact proteins is confirmed by UV-vis spectroscopy, gel filtration, analytical ultracentrifugation, and electron paramagnetic resonance spectroscopy. Pulsed electron-electron double-resonance experiments have detected a magnetic dipole interaction between the two clusters ~0.7 MHz, which is consistent with the expected intercluster distance of 29-34 Å. Taken together, our data demonstrate the successful design of an artificial multi-iron-sulfur cluster protein with evidence of cluster-cluster interaction. The design principles implemented here can be extended to the design of multicluster molecular wires.

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