A De Novo Designed 2[4Fe-4S] Ferredoxin Mimic Mediates Electron Transfer

Abstract

[Fe-S] clusters, nature's modular electron transfer units, are often arranged in chains that support long-range electron transfer. Despite considerable interest, the design of biomimetic artificial systems emulating multicluster-binding proteins, with the final goal of integrating them in man-made oxidoreductases, remains elusive. Here, we report a novel bis-[4Fe-4S] cluster binding protein, DSD-Fdm, in which the two clusters are positioned within a distance of 12 Å, compatible with the electronic coupling necessary for efficient electron transfer. The design exploits the structural repeat of coiled coils as well as the symmetry of the starting scaffold, a homodimeric helical protein (DSD). In total, eight hydrophobic residues in the core of DSD were replaced by eight cysteine residues that serve as ligands to the [4Fe-4S] clusters. Incorporation of two [4Fe-4S] clusters proceeds with high yield. The two [4Fe-4S] clusters are located in the hydrophobic core of the helical bundle as characterized by various biophysical techniques. The secondary structure of the apo and holo proteins is conserved; further, the incorporation of clusters results in stabilization of the protein with respect to chemical denaturation. Most importantly, this de novo designed protein can mimic the function of natural ferredoxins: we show here that reduced DSD-Fdm transfers electrons to cytochrome c, thus generating the reduced cyt c stoichiometrically.

pH dependent binding in de novo hetero bimetallic coiled coils

“…4), but which is consistent with Hg(II) binding as HgCys 2 to Cys located in "a" sites at pH 7.0, 17 as would be the case here. However, as the pH is raised to 7.5 and ultimately 8.6, we observe the appearance of the characteristic signature for trigonal HgS 3 . 17 This pH dependence is consistent with the previously reported apparent pK a of 7.6, associated with coordination of trigonal HgS 3 to an "a" site.…”

“…In order to establish if the lack of Tb(III) luminescence signal at basic pH, is in fact a result of enhanced quenching due to deprotonation of water bound to the TbAsp 3 site, an analogous luminescence pH titration was performed with Tb(MB1-2) 3 , in which the Tb(III) is coordinatively saturated and is not bound directly to any water molecules. A similar profile is observed, though with a much larger pH range for optimal Tb(III) binding (ca.…”

“…These include sites inspired by those found in nature, often in an effort to gain insight into the native site, and ultimately to recreate its function. Notable examples include mononuclear sites mimicking the active site of carbonic anhydrase; 1 dinuclear sites, including dioxygen-activating di-iron sites; 2 multinuclear clusters, such as the cubane-like [4Fe-4S] cluster; 3 and binding of haem cofactors. 4 But similarly, various mononuclear sites have been introduced, which though not found in nature, have appealing chemical properties for exploitation in protein design.…”

“…Truly hetero bimetallic coiled coils, with different metal ions binding to two distinct sites, should be easier to design, as they exploit real intrinsic differences in metal ion coordination chemistry, such as hard-soft-acid-base (HSAB) theory, to achieve selectivity and control. The first example of these features a Ru(bipyridyl) 3 site at one end of a coiled coil, which helps assemble and preorganise the second CuHis 3 binding site located at the opposite end. 15 The advantage of including two distinct mononuclear metal ion sites with different properties within a single assembly is clear, as it allows multi functionality and complexity, relating to interdependence, to be introduced.…”

Maquette Strategy for Creation of Light- and Redox-Active Proteins

“…Early demonstrations showed iron sulfur clusters spontaneously assemble in maquettes 2 singly or in pairs, with or without heme(s); later singlechain variants were designed [Gibney et al, 1996;Mulholland et al, 1998;Musgrave et al, 2002;Nanda et al, 2005;Grzyb et al, 2010;Nanda et al, 2016]. Maquette frames of the kind in Figure 7 have also proved to offer a creative environment for metals alone and in clusters ( Figure 5C) [Dieckmann et al, 1997;Farrer et al, 2000;Calhoun et al, 2005;Geremia et al, 2005;Touw et al, 2007;Calhoun et al, 2008;Tegoni et al, 2012;Zastrow et al, 2012;Roy et al, 2014].…”