Chemical modification of the rieske protein from thermus thermophilus using diethyl pyrocarbonate modifies ligating histidine 154 and reduces the [2Fe-2S] cluster

Mary E. Konkle, Kaitlin N. Elsenheimer, Kevin Hakala, Jennifer C. Robicheaux, Susan E Weintraub, Laura M. Hunsicker-Wang

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Rieske proteins are a class of electron transport proteins that are intricately involved in respiratory and photosynthetic processes. One unique property of Rieske proteins is that the reduction potential is pH-dependent. The ionizable groups responding to changes in pH have recently been shown to be the two histidine residues that ligate the [2Fe-2S] cluster. To probe the chemical reactivity toward and the accessibility of the ligating histidines to small molecules, akin to the substrate quinol and the inhibitor stigmatellin, the Thermus thermophilus Rieske protein was reacted with diethyl pyrocarbonate (DEPC) over a range of pH values. The modification was followed by UV-visible, circular dichroism, and EPR spectroscopies and the end product analyzed by mass spectrometry. The ligating His154, as well as the two nonligating histidines and surface-exposed lysines, were modified. Interestingly, modification of the protein by DEPC was also found to reduce the metal cluster. The ability to control the redox state was examined by the addition of oxidants and reductants and removal of the DEPC-histidine adduct by sodium hydroxide. Characterization of the DEPC-modified Rieske protein, which remains redox active, offers a probe to analyze the effects of small molecules that inhibit the function of the bc1 complex and that have also been shown to interact with the ligating histidines of the Rieske [2Fe-2S] cluster in crystal structures of the complex.

Original languageEnglish (US)
Pages (from-to)7272-7281
Number of pages10
JournalBiochemistry
Volume49
Issue number34
DOIs
StatePublished - Aug 31 2010

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Diethyl Pyrocarbonate
Thermus thermophilus
Chemical modification
Histidine
Proteins
Oxidation-Reduction
Hydroquinones
Sodium Hydroxide
Chemical reactivity
Molecules
Reducing Agents
Electron Transport
Circular Dichroism
Oxidants
Lysine
Mass spectrometry
Paramagnetic resonance
Mass Spectrometry
Spectrum Analysis
Carrier Proteins

ASJC Scopus subject areas

  • Biochemistry

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Chemical modification of the rieske protein from thermus thermophilus using diethyl pyrocarbonate modifies ligating histidine 154 and reduces the [2Fe-2S] cluster. / Konkle, Mary E.; Elsenheimer, Kaitlin N.; Hakala, Kevin; Robicheaux, Jennifer C.; Weintraub, Susan E; Hunsicker-Wang, Laura M.

In: Biochemistry, Vol. 49, No. 34, 31.08.2010, p. 7272-7281.

Research output: Contribution to journalArticle

Konkle, Mary E. ; Elsenheimer, Kaitlin N. ; Hakala, Kevin ; Robicheaux, Jennifer C. ; Weintraub, Susan E ; Hunsicker-Wang, Laura M. / Chemical modification of the rieske protein from thermus thermophilus using diethyl pyrocarbonate modifies ligating histidine 154 and reduces the [2Fe-2S] cluster. In: Biochemistry. 2010 ; Vol. 49, No. 34. pp. 7272-7281.
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abstract = "Rieske proteins are a class of electron transport proteins that are intricately involved in respiratory and photosynthetic processes. One unique property of Rieske proteins is that the reduction potential is pH-dependent. The ionizable groups responding to changes in pH have recently been shown to be the two histidine residues that ligate the [2Fe-2S] cluster. To probe the chemical reactivity toward and the accessibility of the ligating histidines to small molecules, akin to the substrate quinol and the inhibitor stigmatellin, the Thermus thermophilus Rieske protein was reacted with diethyl pyrocarbonate (DEPC) over a range of pH values. The modification was followed by UV-visible, circular dichroism, and EPR spectroscopies and the end product analyzed by mass spectrometry. The ligating His154, as well as the two nonligating histidines and surface-exposed lysines, were modified. Interestingly, modification of the protein by DEPC was also found to reduce the metal cluster. The ability to control the redox state was examined by the addition of oxidants and reductants and removal of the DEPC-histidine adduct by sodium hydroxide. Characterization of the DEPC-modified Rieske protein, which remains redox active, offers a probe to analyze the effects of small molecules that inhibit the function of the bc1 complex and that have also been shown to interact with the ligating histidines of the Rieske [2Fe-2S] cluster in crystal structures of the complex.",
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