Effects of pH on the Rieske Protein from Thermus thermophilus: A Spectroscopic and Structural Analysis^,

Abstract

The Rieske protein from Thermus thermophilus (TtRp) and a truncated version of the protein (truncTtRp), produced to achieve a low-pH crystallization condition, have been characterized using UV-visible and circular dichroism spectroscopies. TtRp and truncTtRp undergo a change in the UV-visible spectra with increasing pH. The LMCT band at 458 nm shifts to 436 nm and increases in intensity. The increase at 436 nm versus pH can be fit using the sum of two Henderson-Hasselbalch equations, yielding two pK(a) values for the oxidized protein. For TtRp, pK(ox1) = 7.48 +/- 0.12 and pK(ox2) = 10.07 +/- 0.17. For truncTtRp, pK(ox1) = 7.87 +/- 0.17 and pK(ox2) = 9.84 +/- 0.42. The shift to shorter wavelength and the increase in intensity for the LMCT band with increasing pH are consistent with deprotonation of the histidine ligands. A pH titration of truncTtRp monitored by circular dichroism also showed pH-dependent changes at 315 and 340 nm. At 340 nm, the fit gives pK(ox1) = 7.14 +/- 0.26 and pK(ox2) = 9.32 +/- 0.36. The change at 315 nm is best fit for a single deprotonation event, giving pK(ox1) = 7.82 +/- 0.10. The lower wavelength region of the CD spectra was unaffected by pH, indicating that the overall fold of the protein remains unchanged, which is consistent with crystallographic results of truncTtRp. The structure of truncTtRp crystallized at pH 6.2 is very similar to TtRp at pH 8.5 and contains only subtle changes localized at the [2Fe-2S] cluster. These titration and structural results further elucidate the histidine ligand characteristics and are consistent with important roles for these amino acids.

Chemical Modification of the Rieske Protein from Thermus thermophilus Using Diethyl Pyrocarbonate Modifies Ligating Histidine 154 and Reduces the [2Fe-2S] Cluster

“…The ligating histidine that was modified by DEPC, His154, is in bold. and 1.80 (37). The EPR spectrum of DEPC-modified H120Q/ H162Q was consistent with that of a reduced Rieske protein (Figure 6d).…”

“…The purity of each protein was assessed by SDS-PAGE. The concentration of each protein was determined by the difference in AU 572 of the isolated oxidized form and the reduced form obtained by the addition of an excess of sodium dithionite (37). The expression of H120Q/ H162Q (4.3 mg/L of culture) was lower than that of truncTtRp (20 mg/L of culture).…”

Metalloproteins Containing Cytochrome, Iron–Sulfur, or Copper Redox Centers

“…803 Shifts in the UV–vis absorption peaks and CD features upon pH titration are consistent with the two protonation states of the oxidized form. 804 Several studies have shown that multiple inhibitors can bind to the sites close to the cluster and affect the reduction potential of the site. 787,805,806 …”

Probing the Hydrogen Bonding Structure in the Rieske Protein

“…However, some local modifications were observed around the cluster. [26] The crucial role of the hydrogen bonds in the immediate cluster environment on the redox potential and on the proton affinity of the two histidines was discussed on the basis of the first X-ray structures [17,27] and it was shown that all sulfur atoms of the cluster participate to the formation of a complex hydrogen-bonded network. The interplay of this network was explored on the basis of site directed mutagenesis studies.…”