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NADPH inhibits [2Fe-2S] cluster protein transfer from diabetes drug target MitoNEET to an apo-acceptor protein.

ABSTRACT: MitoNEET (mNT) is the founding member of the recently discovered CDGSH family of [2Fe-2S] proteins capable of [2Fe-2S] cluster transfer to apo-acceptor proteins. It is a target of the thiazolidinedione (TZD) class of anti-diabetes drugs whose binding modulate both electron transfer and cluster transfer properties. The [2Fe-2S] cluster in mNT is destabilized upon binding of NADPH, which leads to loss of the [2Fe-2S] cluster to the solution environment. Because mNT is capable of transferring [2Fe-2S] clusters to apo-acceptor proteins, we sought to determine whether NADPH binding also affects cluster transfer. We show that NADPH inhibits transfer of the [2Fe-2S] cluster to an apo-acceptor protein with an inhibition constant (K(i)) of 200 ?m, which reflects that of NADPH concentrations expected under physiological conditions. In addition, we determined that the strictly conserved cluster interacting residue Asp-84 in the CDGSH domain is necessary for the NADPH-dependent inhibition of [2Fe-2S] cluster transfer. The most critical cellular function of NADPH is in the maintenance of a pool of reducing equivalents, which is essential to counteract oxidative damage. Taken together, our findings suggest that NADPH can regulate both mNT [2Fe-2S] cluster levels in the cell as well as the ability of the protein to transfer [2Fe-2S] clusters to cytosolic or mitochondrial acceptors.

SUBMITTER: Zuris JA

PROVIDER: S-EPMC3320914 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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NADPH inhibits [2Fe-2S] cluster protein transfer from diabetes drug target MitoNEET to an apo-acceptor protein.

Zuris John A JA Ali Syed S SS Yeh Howard H Nguyen Tung A TA Nechushtai Rachel R Paddock Mark L ML Jennings Patricia A PA

The Journal of biological chemistry 20120217 15

MitoNEET (mNT) is the founding member of the recently discovered CDGSH family of [2Fe-2S] proteins capable of [2Fe-2S] cluster transfer to apo-acceptor proteins. It is a target of the thiazolidinedione (TZD) class of anti-diabetes drugs whose binding modulate both electron transfer and cluster transfer properties. The [2Fe-2S] cluster in mNT is destabilized upon binding of NADPH, which leads to loss of the [2Fe-2S] cluster to the solution environment. Because mNT is capable of transferring [2Fe- ...[more]

PMID: 22351774

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Project description:Human mitoNEET is a homodimeric iron-sulfur protein located in the outer mitochondrial membrane with unknown function, but which is known to interact with thiazolidinedione diabetes drugs. Each monomer houses a [2Fe-2S] cluster with an unusual (Cys)(3)(His)(1) ligation. The His ligand is important for enabling cluster release and for tuning the redox potential. We use multifrequency (X-, Ka-, and Q-band) and multitechnique (continuous-wave, electron spin-echo envelope modulation (ESEEM), pulsed electron-nuclear double resonance (ENDOR), and hyperfine sublevel correlation (HYSCORE)) electron paramagnetic resonance spectroscopy to investigate the cluster in its paramagnetic reduced [Fe(2+)Fe(3+)] (S = 1/2) state. It has a rhombic g tensor (2.007, 1.937, 1.897) with an average g value of 1.947 that falls between those of Rieske-type and ferredoxin-type [2Fe-2S] clusters. Simulation and least-squares fitting of orientation-selective Ka- and Q-band ENDOR, 1D ESEEM, and HYSCORE spectra of (14)N and (15)N-labeled mitoNEET yield the principal values and orientations of both the hyperfine tensor ((14)N, A(iso) = -6.25 MHz, T = -0.94 MHz) and the quadrupolar tensor (e(2)Qq/h = -2.47 MHz, eta = 0.38) of the ligating histidine nitrogen N(delta). From these, we can infer the absolute g tensor orientation with respect to the cluster: The g(2) axis is close to perpendicular to the [2Fe-2S] plane, and g(1) and g(3) are in-plane, but skewed from the Fe-Fe and S-S axes. In X-band ENDOR and ESEEM spectra, a weakly coupled nitrogen is visible, most likely the N(epsilon) of the histidine in the protonated state. We find that the cluster is in a valence-localized state, where Fe(2+) is His-bound. The field-sweep spectra show evidence of intercluster dipolar coupling that can be simulated using an uncoupled spin model for each cluster (S(Fe(2+)) = 2, S(Fe(3+)) = 5/2). The parameters determined in this work can function as reporters on how the cluster structure is altered upon pH changes and drug binding.

Dataset Information

NADPH inhibits [2Fe-2S] cluster protein transfer from diabetes drug target MitoNEET to an apo-acceptor protein.

Publications

NADPH inhibits [2Fe-2S] cluster protein transfer from diabetes drug target MitoNEET to an apo-acceptor protein.

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