Mössbauer spectroscopy and DFT calculations on all protonation states of the 2Fe-2S cluster of the Rieske protein

Apd1 and Aim32 Are Prototypes of Bishistidinyl-Coordinated Non-Rieske [2Fe–2S] Proteins

“…In the T. thermophilus Rieske protein, the changes of the Mossbauer parameters of the bishistidinyl-coordinated ferric ion are larger but show the same tendency (δ = 0.34, 0.29, and 0.29 mm/s, ΔE Q = 1.05, 0.78, and 0.71 mm/s). 44 These findings corroborate the assignment of subspectra 4S and 2S2N. The effects of ligand protonation on the two ferric ions support the choice of nesting of quadrupole doublets, i.e., which isomer shift is associated with which quadrupole splitting.…”

“…The isomer shift of subspectrum 4S was pH independent (0.23–0.24 mm/s) at 5 and 77 K (Table , Figures S20 and S21). For the T. thermophilus Rieske protein a similar trend for the all-sulfur coordinated ferric ion was observed upon deprotonation . A significant decrease of the isomer shift (0.36, 0.35, and 0.33 mm/s) and quadrupole splitting (1.11, 1.05, and 0.94 mm/s at 5 K) of the Apd1 subspectrum 2S2N occurred upon histidine deprotonation (Figure S22).…”

“…The effects of ligand protonation on the two ferric ions support the choice of nesting of quadrupole doublets, i.e., which isomer shift is associated with which quadrupole splitting. An assignment by the minimization of ferric ion isomer shifts for the Rieske protein 6,44 and for Apd1 holds for all protonation states. With the Mossbauer data on the protonation states of Apd1, its monohistidinyl-coordinated variants, and data from the literature (Table S5), we can compile a sufficiently densely populated diagram with isomer shifts along the x-axis and quadrupole splittings along the y-axis (Figure 5e).…”

“…For the T. thermophilus Rieske protein a similar trend for the all-sulfur coordinated ferric ion was observed upon deprotonation. 44 A significant decrease of the isomer shift (0.36, 0.35, and 0.33 mm/s) and quadrupole splitting (1.11, 1.05, and 0.94 mm/s at 5 K) of the Apd1 subspectrum 2S2N occurred upon histidine deprotonation (Figure S22). In the T. thermophilus Rieske protein, the changes of the Mossbauer parameters of the bishistidinyl-coordinated ferric ion are larger but show the same tendency (δ = 0.34, 0.29, and 0.29 mm/s, ΔE Q = 1.05, 0.78, and 0.71 mm/s).…”

“…The cloud for the all-sulfur-coordinated ferric ions in the lower left range of the diagram is separated from the mono-and bishistidinyl-coordinated ferric ions in the central part and top right part of the diagram, with the exception of the pH 10 data of the T. thermophilus Rieske protein. 45 For the T. thermophilus Rieske protein 44 and Apd1 (Figure 5a−c), protonation of histidine ligands shifts the position in the diagram progressively to the top right. More emphasis on defined protonation states in future Mossbauer studies, including monohistidinyl-coordinated clusters, will expand the application of this diagram for identification of the number of histidine ligands and their protonation state.…”