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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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Protein backbone of the cytoplasmic exposed domain of the outer-mitochondrial membrane protein mitoNEET (PDB code 2QH7) showing the redox active [2Fe-2S] centers.(6a) MitoNEET is a homodimer, with one protomer shown in magenta and the other in green. Each protomer contains a [2Fe-2S] center (shown as spheres) coordinated by 3-Cys and 1-His, with the hallmark single-coordinating His87 indicated. The distance between the [2Fe-2S] centers is ∼16 Å from center-to-center. His87 coordinates to the outer, more solvent exposed, Fe of the [2Fe-2S] center, where the electron is predominantly localized upon reduction.
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fig1: Protein backbone of the cytoplasmic exposed domain of the outer-mitochondrial membrane protein mitoNEET (PDB code 2QH7) showing the redox active [2Fe-2S] centers.(6a) MitoNEET is a homodimer, with one protomer shown in magenta and the other in green. Each protomer contains a [2Fe-2S] center (shown as spheres) coordinated by 3-Cys and 1-His, with the hallmark single-coordinating His87 indicated. The distance between the [2Fe-2S] centers is ∼16 Å from center-to-center. His87 coordinates to the outer, more solvent exposed, Fe of the [2Fe-2S] center, where the electron is predominantly localized upon reduction.

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)

Protein backbone of the cytoplasmic exposed domain of the outer-mitochondrial membrane protein mitoNEET (PDB code 2QH7) showing the redox active [2Fe-2S] centers.(6a) MitoNEET is a homodimer, with one protomer shown in magenta and the other in green. Each protomer contains a [2Fe-2S] center (shown as spheres) coordinated by 3-Cys and 1-His, with the hallmark single-coordinating His87 indicated. The distance between the [2Fe-2S] centers is ∼16 Å from center-to-center. His87 coordinates to the outer, more solvent exposed, Fe of the [2Fe-2S] center, where the electron is predominantly localized upon reduction.
© Copyright Policy - open-access
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fig1: Protein backbone of the cytoplasmic exposed domain of the outer-mitochondrial membrane protein mitoNEET (PDB code 2QH7) showing the redox active [2Fe-2S] centers.(6a) MitoNEET is a homodimer, with one protomer shown in magenta and the other in green. Each protomer contains a [2Fe-2S] center (shown as spheres) coordinated by 3-Cys and 1-His, with the hallmark single-coordinating His87 indicated. The distance between the [2Fe-2S] centers is ∼16 Å from center-to-center. His87 coordinates to the outer, more solvent exposed, Fe of the [2Fe-2S] center, where the electron is predominantly localized upon reduction.
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