Methods to identify and characterize iron–sulfur oligopeptides in water

Abstract

Iron-sulfur clusters are ubiquitous cofactors that mediate central biological processes. However, despite their long history, these metallocofactors remain challenging to investigate when coordinated to small (≤ six amino acids) oligopeptides in aqueous solution. In addition to being often unstable in vitro, iron-sulfur clusters can be found in a wide variety of forms with varied characteristics, which makes it difficult to easily discern what is in solution. This difficulty is compounded by the dynamics of iron-sulfur peptides, which frequently coordinate multiple types of clusters simultaneously. To aid investigations of such complex samples, a summary of data from multiple techniques used to characterize both iron-sulfur proteins and peptides is provided. Although not all spectroscopic techniques are equally insightful, it is possible to use several, readily available methods to gain insight into the complex composition of aqueous solutions of iron-sulfur peptides.

Histidine Ligated Iron‐Sulfur Peptides

“…Near‐UV‐visible circular dichroism (CD) spectra showed a band at 470 nm that was similar to the 450 nm peak seen with ferredoxin, but with lower molar ellipticity (Figure S20A). Paramagnetic 1 H NMR spectra confirmed the presence of the [2Fe‐2S] 2+ cluster with a broad resonance at 30 ppm (Figure S9D) [28] . The NMR spectrum also revealed the continued presence of the mononuclear center that was not observable by UV‐vis absorption (Figure S9C).…”

“…To confirm the coordination of Fe 2+ , paramagnetic 1 H NMR spectra were acquired. The paramagnetism of the Fe 2+ center gives rise to hyperfine shifted resonances of Cys Hα in the 10–20 ppm region and Cys Hβ within the 100–300 ppm range [28] . The paramagnetic 1 H NMR spectra of the Cys‐containing peptides incubated with Fe 2+ showed the presence of such paramagnetically shifted resonances of Cys Hα and Cys Hβ (Figure S9A‐9B).…”

“…Such a result was not surprising, since peptides exist in an equilibrium between the coordination of a mononuclear center and a [2Fe-2S] cluster when in an environment containing iron and sulfide ions. [28] Conversely, UV-Vis absorption spectra of small (� 6 amino acids) peptides that possessed both Cys and His residues did not resemble the spectra of ferredoxin or the Rieske protein (Figure 4B-S18BC-S19B). Although there was absorption in the visible region, no clear peaks were discernable, and their CD spectra were featureless (Figure S14B).…”

“…The paramagnetism of the Fe 2 + center gives rise to hyperfine shifted resonances of Cys Hα in the 10-20 ppm region and Cys Hβ within the 100-300 ppm range. [28] The paramagnetic 1 H NMR spectra of the Cys-containing peptides incubated with Fe 2 + showed the presence of such paramagnetically shifted resonances of Cys Hα and Cys Hβ (Figure S9A-9B). Paramagnetically shifted resonances were not detected for peptides containing His (Figure S10A).…”

“…Paramagnetic 1 H NMR spectra confirmed the presence of the [2Fe-2S] 2 + cluster with a broad resonance at 30 ppm (Figure S9D). [28] The NMR spectrum also revealed the continued presence of the mononuclear center that was not observable by UV-vis absorption (Figure S9C). Such a result was not surprising, since peptides exist in an equilibrium between the coordination of a mononuclear center and a [2Fe-2S] cluster when in an environment containing iron and sulfide ions.…”