JBIC Journal of Biological Inorganic Chemistry

, Volume 19, Issue 7, pp 1121–1135 | Cite as

The reduction rates of DEPC-modified mutant Thermus thermophilus Rieske proteins differ when there is a negative charge proximal to the cluster

  • Nicholas E. Karagas
  • Christie N. Jones
  • Deborah J. Osborn
  • Anika L. Dzierlenga
  • Paul Oyala
  • Mary E. Konkle
  • Emily M. Whitney
  • R. David Britt
  • Laura M. Hunsicker-Wang
Original Paper

Abstract

Rieske and Rieske-type proteins are electron transport proteins involved in key biological processes such as respiration, photosynthesis, and detoxification. They have a [2Fe–2S] cluster ligated by two cysteines and two histidines. A series of mutations, L135E, L135R, L135A, and Y158F, of the Rieske protein from Thermus thermophilus has been produced which probe the effects of the neighboring residues, in the second sphere, on the dynamics of cluster reduction and the reactivity of the ligating histidines. These properties were probed using titrations and modifications with diethyl pyrocarbonate (DEPC) at various pH values monitored using UV–Visible and circular dichroism spectrophotometry. These results, along with results from EPR studies, provide information on ligating histidine modification and rate of reduction of each of the mutant proteins. L135R, L135A, and Y158F react with DEPC similarly to wild type, resulting in modified protein with a reduced [2Fe–2S] cluster in <90 min, whereas L135E requires >15 h under the same conditions. Thus, the negative charge slows down the rate of reduction and provides an explanation as to why negatively charged residues are rarely, if ever, found in the equivalent position of other Rieske and Rieske-type proteins.

Keywords

Electron transfer Iron–sulfur cluster Rieske proteins DEPC Circular dichroism 

Abbreviations

bc1

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

CAPS

3-(Cyclohexylamino)-1-propanesulfonic acid

CD

Circular dichroism

CW-EPR

Continuous-wave electron paramagnetic resonance

DEPC

Diethyl pyrocarbonate

DMSO

Dimethyl sulfoxide

HEPES

4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid

IPTG

Isopropyl β-d-1-thiogalactopyranoside

LMCT

Ligand-to-metal charge transfer

MES

2-(N-Morpholino)ethanesulfonic acid hydrate

MOPS

3-(N-Morpholino)propanesulfonic acid

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TAPS

N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid

Tris

2-Amino-2-(hydroxymethyl)-1,3-propanediol

truncTtRp

The truncated version of the Rieske protein from Thermus thermophilus

TtRp

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

Notes

Acknowledgments

This work was supported by funds from the National Science Foundation (CHE-1058273) and the Welch Foundation (W-0031). Funding for EMW was provided in part by a grant from the Howard Hughes Medical Institute. We would like to acknowledge the work of Ravi Pokhrel who made the L135A mutant and Sarah Muellner who made the Y158F mutant. We would also like to acknowledge Abhishek Chhetri who discovered the need to change the buffer conditions for the pH-dependent UV–Visible titrations. We would also like to thank Drs. Bert Chandler and Nancy Mills for helpful discussions.

Supplementary material

775_2014_1167_MOESM1_ESM.pdf (2 mb)
Supplementary material 1 (PDF 2074 kb)

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Copyright information

© SBIC 2014

Authors and Affiliations

  • Nicholas E. Karagas
    • 1
    • 3
  • Christie N. Jones
    • 1
  • Deborah J. Osborn
    • 1
  • Anika L. Dzierlenga
    • 1
    • 4
  • Paul Oyala
    • 2
  • Mary E. Konkle
    • 1
    • 5
  • Emily M. Whitney
    • 1
    • 6
  • R. David Britt
    • 2
  • Laura M. Hunsicker-Wang
    • 1
  1. 1.Department of ChemistryTrinity UniversitySan AntonioUSA
  2. 2.Department of ChemistryUniversity of California at DavisDavisUSA
  3. 3.University of Texas at Houston Medical SchoolHoustonUSA
  4. 4.Department of Pharmacology and ToxicologyUniversity of ArizonaTucsonUSA
  5. 5.Department of ChemistryEastern Illinois UniversityCharlestonUSA
  6. 6.University of Georgia College of PharmacyAthensUSA

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