Reactive sites and course of reduction in the Rieske protein

  • Si Ying Li
  • Paul H. Oyala
  • R. David Britt
  • Susan T. Weintraub
  • Laura M. Hunsicker-Wang
Original Paper


Rieske proteins play an essential role in electron transfer in the bc1 complex. Rieske proteins contain a [2Fe–2S] cluster with one iron ligated by two histidines and the other iron ligated by two cysteines. All Rieske proteins have pH-dependent reduction potentials with the histidines ligating the cluster deprotonating in response to increases in pH. The addition of diethylpyrocarbonate (DEPC) modifies deprotonated histidines. The previous studies on the isolated Thermus thermophilus Rieske protein have used large excesses of DEPC, and this study examines what amino acids become modified under different molar equivalents of DEPC to protein. Increasing amounts of DEPC result in more modification, and higher pH values result in faster reaction. Upon modification, the protein also becomes reduced and ~6 equivalents of DEPC are needed for 50% of the reduction to occur. Which amino acids are modified first also points to the most reactive species on the protein. Mass spectrometry analysis shows that lysine 68 is the most reactive amino acid, followed by the ligating histidine 154 and two other surfaces lysines, 76 and 43. The modification of the ligating histidine at low numbers of DEPC equivalents and correlation with a similar number of equivalents needed to reduce the protein shows that this histidine can interact with neighboring groups, and these results can be extended to the protein within the bc1 complex, where interaction with neighboring residues or molecules may allow reduction to occur. These results may shed light on how Rieske transfers electrons and protons in the bc1 complex.


Iron–sulfur cluster Rieske proteins DEPC Circular dichroism Mass spectrometry 



The cytochrome bc1 complex, complex III of the electron transport chain


Circular dichroism


Continuous-wave electron paramagnetic resonance




Ethylenediamine tetraacetic acid


HPLC-electrospray ionization-tandem mass spectrometry


Isopropyl β-d-1-thiogalactopyranoside


Ligand to metal charge transfer




Sodium dodecyl sulfate polyacrylamide gel electrophoresis


The truncated version of the Rieske protein from Thermus thermophilus


The Thermus thermophilus Rieske protein that has the additional 17 amino acids



This work was supported by funds from the National Science Foundation (CHE-1058273) and the Welch Foundation (W-0031). LHW would also like to acknowledge the work of the Biochemistry Lab (CHEM 3131) in Fall 2013. Their preliminary work on differing amounts of DEPC added to truncTtRp led to this study. We would also like to thank Dr. Mary Konkle for helpful comments on the manuscript.

Supplementary material

775_2017_1445_MOESM1_ESM.pdf (1.9 mb)
Supplementary material 1 (PDF 1984 kb)


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Copyright information

© SBIC 2017

Authors and Affiliations

  • Si Ying Li
    • 1
  • Paul H. Oyala
    • 2
  • R. David Britt
    • 2
  • Susan T. Weintraub
    • 3
  • Laura M. Hunsicker-Wang
    • 1
  1. 1.Department of ChemistryTrinity UniversitySan AntonioUSA
  2. 2.Department of ChemistryUniversity of California at DavisDavisUSA
  3. 3.Department of BiochemistryUniversity of Texas Health Science Center at San AntonioSan AntonioUSA

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