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Engineering the redox potential over a wide range within a new class of FeS proteins.

Zuris JA, Halim DA, Conlan AR, Abresch EC, Nechushtai R, Paddock ML, Jennings PA - J. Am. Chem. Soc. (2010)

Bottom Line: Both the fold and the coordination of the [2Fe-2S] centers suggest that it could have novel properties compared to other known [2Fe-2S] proteins.We tested the robustness of mitoNEET to mutation and the range over which the redox potential (E(M)) could be tuned.We found that the protein could tolerate an array of mutations that modified the E(M) of the [2Fe-2S] center over a range of ∼700 mV, which is the largest E(M) range engineered in an FeS protein and, importantly, spans the cellular redox range (+200 to -300 mV).

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093, USA.

ABSTRACT
MitoNEET is a newly discovered mitochondrial protein and a target of the TZD class of antidiabetes drugs. MitoNEET is homodimeric with each protomer binding a [2Fe-2S] center through a rare 3-Cys and 1-His coordination geometry. Both the fold and the coordination of the [2Fe-2S] centers suggest that it could have novel properties compared to other known [2Fe-2S] proteins. We tested the robustness of mitoNEET to mutation and the range over which the redox potential (E(M)) could be tuned. We found that the protein could tolerate an array of mutations that modified the E(M) of the [2Fe-2S] center over a range of ∼700 mV, which is the largest E(M) range engineered in an FeS protein and, importantly, spans the cellular redox range (+200 to -300 mV). These properties make mitoNEET potentially useful for both physiological studies and industrial applications as a stable, water-soluble, redox agent.

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EM values of WT and mutant mitoNEET have been engineered over a range of ∼700 mV and can be tuned to obtain nearly any value within the EM range shown. Measurements adjusted to SHE values, with errors (±10 mV) indicated by cross bars. *EM extremes (see Figure 3B). The EM values of several cellular environments are shown on the left bar. Abbreviations: Resp. (Respiratory tract fluid, +200 mV), β-Cell (Cytoplasm of beta cells, producers of insulin in the pancreas, +55 mV), Cyt (Cytoplasm, −205 mV), ER (Endoplasmic Reticulum, −217 mV), Mito (Mitochondria, −260 mV).(4)
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fig2: EM values of WT and mutant mitoNEET have been engineered over a range of ∼700 mV and can be tuned to obtain nearly any value within the EM range shown. Measurements adjusted to SHE values, with errors (±10 mV) indicated by cross bars. *EM extremes (see Figure 3B). The EM values of several cellular environments are shown on the left bar. Abbreviations: Resp. (Respiratory tract fluid, +200 mV), β-Cell (Cytoplasm of beta cells, producers of insulin in the pancreas, +55 mV), Cyt (Cytoplasm, −205 mV), ER (Endoplasmic Reticulum, −217 mV), Mito (Mitochondria, −260 mV).(4)

Mentions: MitoNEET is a newly discovered mitochondrial target of the TZD class of antidiabetes drugs, such as pioglitazone (Actos) and rosiglitazone (Avandia).(5) Human mitoNEET defines a unique class of [2Fe-2S] proteins. The crystal structure shows that mitoNEET is a homodimer, with each protomer binding a [2Fe-2S] center through a rare 3-Cys and 1-His coordination geometry (Figure 1).(6) Both the fold and the coordination of the [2Fe-2S] centers suggest that mitoNEET could have novel properties compared to other known [2Fe-2S] proteins. For example, the unusual coordination of the [2Fe-2S] center contributes to the atypical proton-coupled EM,7 of +25 mV at pH 7.0.(7) We used potentiometric redox titrations(8) and protein-film voltammetry(9) to assess changes in the EM,7 resulting from mutagenesis of first and second shell residues near the [2Fe-2S] center. Single and double mutations were designed to test both the robustness of the [2Fe-2S] center and the EM range that could be achieved. We found that the EM could be tuned over a wide range (Figure 2, Table S1).


Engineering the redox potential over a wide range within a new class of FeS proteins.

Zuris JA, Halim DA, Conlan AR, Abresch EC, Nechushtai R, Paddock ML, Jennings PA - J. Am. Chem. Soc. (2010)

EM values of WT and mutant mitoNEET have been engineered over a range of ∼700 mV and can be tuned to obtain nearly any value within the EM range shown. Measurements adjusted to SHE values, with errors (±10 mV) indicated by cross bars. *EM extremes (see Figure 3B). The EM values of several cellular environments are shown on the left bar. Abbreviations: Resp. (Respiratory tract fluid, +200 mV), β-Cell (Cytoplasm of beta cells, producers of insulin in the pancreas, +55 mV), Cyt (Cytoplasm, −205 mV), ER (Endoplasmic Reticulum, −217 mV), Mito (Mitochondria, −260 mV).(4)
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fig2: EM values of WT and mutant mitoNEET have been engineered over a range of ∼700 mV and can be tuned to obtain nearly any value within the EM range shown. Measurements adjusted to SHE values, with errors (±10 mV) indicated by cross bars. *EM extremes (see Figure 3B). The EM values of several cellular environments are shown on the left bar. Abbreviations: Resp. (Respiratory tract fluid, +200 mV), β-Cell (Cytoplasm of beta cells, producers of insulin in the pancreas, +55 mV), Cyt (Cytoplasm, −205 mV), ER (Endoplasmic Reticulum, −217 mV), Mito (Mitochondria, −260 mV).(4)
Mentions: MitoNEET is a newly discovered mitochondrial target of the TZD class of antidiabetes drugs, such as pioglitazone (Actos) and rosiglitazone (Avandia).(5) Human mitoNEET defines a unique class of [2Fe-2S] proteins. The crystal structure shows that mitoNEET is a homodimer, with each protomer binding a [2Fe-2S] center through a rare 3-Cys and 1-His coordination geometry (Figure 1).(6) Both the fold and the coordination of the [2Fe-2S] centers suggest that mitoNEET could have novel properties compared to other known [2Fe-2S] proteins. For example, the unusual coordination of the [2Fe-2S] center contributes to the atypical proton-coupled EM,7 of +25 mV at pH 7.0.(7) We used potentiometric redox titrations(8) and protein-film voltammetry(9) to assess changes in the EM,7 resulting from mutagenesis of first and second shell residues near the [2Fe-2S] center. Single and double mutations were designed to test both the robustness of the [2Fe-2S] center and the EM range that could be achieved. We found that the EM could be tuned over a wide range (Figure 2, Table S1).

Bottom Line: Both the fold and the coordination of the [2Fe-2S] centers suggest that it could have novel properties compared to other known [2Fe-2S] proteins.We tested the robustness of mitoNEET to mutation and the range over which the redox potential (E(M)) could be tuned.We found that the protein could tolerate an array of mutations that modified the E(M) of the [2Fe-2S] center over a range of ∼700 mV, which is the largest E(M) range engineered in an FeS protein and, importantly, spans the cellular redox range (+200 to -300 mV).

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093, USA.

ABSTRACT
MitoNEET is a newly discovered mitochondrial protein and a target of the TZD class of antidiabetes drugs. MitoNEET is homodimeric with each protomer binding a [2Fe-2S] center through a rare 3-Cys and 1-His coordination geometry. Both the fold and the coordination of the [2Fe-2S] centers suggest that it could have novel properties compared to other known [2Fe-2S] proteins. We tested the robustness of mitoNEET to mutation and the range over which the redox potential (E(M)) could be tuned. We found that the protein could tolerate an array of mutations that modified the E(M) of the [2Fe-2S] center over a range of ∼700 mV, which is the largest E(M) range engineered in an FeS protein and, importantly, spans the cellular redox range (+200 to -300 mV). These properties make mitoNEET potentially useful for both physiological studies and industrial applications as a stable, water-soluble, redox agent.

Show MeSH