Effects of pH on the rieske protein from thermus thermophilus: A spectroscopic and structural analysis

Mary E. Konkle, Sarah K. Muellner, Anika L. Schwander, Michelle M. Dicus, Ravi Pokhrel, R. David Britt, Alexander B. Taylor, Laura M. Hunsicker-Wang

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

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 pKa values for the oxidized protein. For TtRp, pKox1 = 7.48 ± 0.12 and pK ox2 = 10.07 ± 0.17. For truncTtRp, pKox1 = 7.87 ± 0.17 and pKox2 = 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 pKox1 = 7.14 ± 0.26 and pKox2 = 9.32 ± 0.36. The change at 315 nmis best fit for a single deprotonation event, giving pKox1 = 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 pH6.2 is very similar to TtRpat pH8.5 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.

Original languageEnglish (US)
Pages (from-to)9848-9857
Number of pages10
JournalBiochemistry
Volume48
Issue number41
DOIs
StatePublished - Oct 20 2009

ASJC Scopus subject areas

  • Biochemistry

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