JBIC Journal of Biological Inorganic Chemistry

, Volume 24, Issue 1, pp 117–135 | Cite as

DEPC modification of the CuA protein from Thermus thermophilus

  • Taylor Devlin
  • Cristina R. Hofman
  • Zachary P. V. Acevedo
  • Kelsey R. Kohler
  • Lizhi Tao
  • R. David Britt
  • Kevin R. Hoke
  • Laura M. Hunsicker-WangEmail author
Original Paper


The CuA center is the initial electron acceptor in cytochrome c oxidase, and it consists of two copper ions bridged by two cysteines and ligated by two histidines, a methionine, and a carbonyl in the peptide backbone of a nearby glutamine. The two ligating histidines are of particular interest as they may influence the electronic and redox properties of the metal center. To test for the presence of reactive ligating histidines, a portion of cytochrome c oxidase from the bacteria Thermus thermophilus that contains the CuA site (the TtCuA protein) was treated with the chemical modifier diethyl pyrocarbonate (DEPC) and the reaction followed through UV–visible, circular dichroism, and electron paramagnetic resonance spectroscopies at pH 5.0–9.0. A mutant protein (H40A/H117A) with the non-ligating histidines removed was similarly tested. Introduction of an electron-withdrawing DEPC-modification onto the ligating histidine 157 of TtCuA increased the reduction potential by over 70 mV, as assessed by cyclic voltammetry. Results from both proteins indicate that DEPC reacts with one of the two ligating histidines, modification of a ligating histidine raises the reduction potential of the CuA site, and formation of the DEPC adduct is reversible at room temperature. The existence of the reactive ligating histidine suggests that this residue may play a role in modulating the electronic and redox properties of TtCuA through kinetically-controlled proton exchange with the solvent. Lack of reactivity by the metalloproteins Sco and azurin, both of which contain a mononuclear copper center, indicate that reactivity toward DEPC is not a characteristic of all ligating histidines.


CuA Cytochrome oxidase Diethyl pyrocarbonate UV–visible spectroscopy Circular dichroism Chemical modification Cyclic voltammetry Electrochemistry 



The research was supported by the Arnold and Mabel Beckman Foundation Beckman Scholars Program, and by the Semmes Distinguished Scholar in Science Award from Trinity University for TD. The FASTER grant SURF-National Science Foundation DUE S-STEM Award 1153796 supported CRH, and the T. Frank and Norine R. Murchison Faculty Development Fund helped support both ZA and CRH. The Berry College Faculty Development Program helped support KRH. The Trinity University Chemistry Department also helped support the work. We are grateful to the laboratory of Prof. Brian R. Crane for the plasmid used to express azurin, and to the late Jim Fee for the TtSco plasmid. We would also like to acknowledge the work of the Biochemistry Lab Students from Fall 2014 at Trinity University. The students performed the initial studies with fewer equivalents of DEPC reacting with TtCuA, which led to the study performed in Fig. 4, and was described in a book chapter on integrating research and teaching [69].

Supplementary material

775_2018_1632_MOESM1_ESM.pdf (3 mb)
Supplemental Information: Supporting information including additional UV and CD pH-dependent studies, equivalents studies, reversibility of the adduct at pH 6, control voltammograms and the TtSco unmodified CD spectrum over time, is available free of charge. Supplementary material final.pdf


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Copyright information

© Society for Biological Inorganic Chemistry (SBIC) 2018

Authors and Affiliations

  1. 1.Department of ChemistryTrinity UniversitySan AntonioUSA
  2. 2.Department of ChemistryUniversity of California at DavisDavisUSA
  3. 3.Department of Chemistry and BiochemistryBerry CollegeMount BerryUSA
  4. 4.Department of BiophysicsJohns Hopkins UniversityBaltimoreUSA

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