Effects of pH on the Rieske protein from Thermus thermophilus: a spectroscopic and structural analysis.
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.
Konkle
Mary E
ME
Department of Chemistry, Trinity University, One Trinity Place, San Antonio, Texas 78212, USA.
Muellner
Sarah K
SK
Schwander
Anika L
AL
Dicus
Michelle M
MM
Pokhrel
Ravi
R
Britt
R David
RD
Taylor
Alexander B
AB
Hunsicker-Wang
Laura M
LM
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D013053:Spectrophotometry; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; C037989:Rieske iron-sulfur protein; D014450:Electron Transport Complex III