Histidine Ligated Iron‐Sulfur Peptides

Abstract

Iron‐sulfur clusters are thought to be ancient cofactors that could have played a role in early protometabolic systems. Thus far, redox active, prebiotically plausible iron‐sulfur clusters have always contained cysteine ligands to the cluster. However, extant iron‐sulfur proteins can be found to exploit other modes of binding, including ligation by histidine residues, as seen with [2Fe‐2S] Rieske and MitoNEET proteins. Here, we investigated the ability of cysteine‐ and histidine‐containing peptides to coordinate a mononuclear Fe2+ center and a [2Fe‐2S] cluster and compare their properties with purified iron‐sulfur proteins. The iron‐sulfur peptides were characterized by UV‐vis, circular dichroism, and paramagnetic NMR spectroscopies and cyclic voltammetry. Small (≤6 amino acids) peptides can coordinate [2Fe‐2S] clusters through a combination of cysteine and histidine residues with similar reduction potentials as their corresponding proteins. Such complexes may have been important for early cell‐like systems.

Peptide Mimics of the Cysteine‐Rich Regions of HapX and SreA Bind a [2Fe‐2S] Cluster In Vitro

“…Peptide Synthesis: Peptides were synthesized by solid phase peptide synthesis, as previously described. [14] N,N-dimethyl formamide (DMF) was used as the solvent and Wang resin was used as the starting polymeric support. The building blocks employed were trityl-protected Fmoccysteine (Fmoc-Cys(Trt)-OH) and tert-butyl (OtBu) side chain-protected Fmoc-𝛼-amino acids.…”

“…Binding affinity was calculated as previously described. [14,36] Briefly, the peptide solutions were prepared under anoxic conditions by dissolving the peptides (1 mм) in 20 mм glycylglycine at pH 8.7. This pH was chosen to ensure that all the thiols in the peptide would be deprotonated and available for the binding of an iron-sulfur cluster.…”

“…The data were fit to the Hill equation to determine dissociation constants (Table 1; Figures S5-S9, Supporting Information), which were similar to values obtained previously with a 25 residue peptide that contained four cysteine residues within a region that possessed CXC and CX 2 C motifs separated by 16 residues. [14] To evaluate the ability of each peptide to stabilize the formation of an iron-sulfur cluster, 5 mм of each peptide was either incubated with 2.5 or 5 mм Fe 3+ and HS − . In each case, UVvis spectra showed characteristic bands at ≈415 and ≈450 nm (Figure 2; Figures S10 and S11, Supporting Information) indicative of a [2Fe-2S] cluster.…”

“…Such an approach builds on prior experience with small, cysteine-containing peptides (2-25 residues in length) that coordinate iron-sulfur clusters. [14,[19][20][21][22] Peptide analogs of iron-sulfur motifs found in proteins frequently retain the ability to coordinate their cognate iron-sulfur cluster. [23] Here, each synthesized peptide was identical in sequence to a region within HapX or SreA, with the exception of HapX CRR-C (Table 1).…”

“…CXC motifs have been demonstrated to bind Fe 2+ , Co 2+ , and Zn 2+ with high affinity and to stabilize the formation of [2Fe‐2S] clusters. [ 14 ] HapX CRR‐D contains two consecutive Cys separated by eight residues rather than a CX 2 C or CXC sequence. Consecutive metal‐binding Cys residues are found in metallothioneins.…”