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Flavin nucleotides act as electron shuttles mediating reduction of the [2Fe-2S] clusters in mitochondrial outer membrane protein mitoNEET.

ABSTRACT: MitoNEET, a primary target of type II diabetes drug pioglitazone, has an essential role in regulating energy metabolism, iron homeostasis, and production of reactive oxygen species in mitochondria. Structurally, mitoNEET is anchored to the mitochondrial outer membrane via its N-terminal transmembrane ?-helix. The C-terminal cytosolic domain of mitoNEET hosts a redox active [2Fe-2S] cluster via three cysteine and one histidine residues. Here we report that the reduced flavin nucleotides can rapidly reduce the mitoNEET [2Fe-2S] clusters under anaerobic or aerobic conditions. In the presence of NADH and flavin reductase, 1 molecule of flavin nucleotide is sufficient to reduce about 100 molecules of the mitoNEET [2Fe-2S] clusters in 4min under aerobic conditions. The electron paramagnetic resonance (EPR) measurements show that flavin mononucleotide (FMN), but not flavin adenine dinucleotide (FAD), has a specific interaction with mitoNEET. Molecular docking models further reveal that flavin mononucleotide binds mitoNEET at the region between the N-terminal transmembrane ?-helix and the [2Fe-2S] cluster binding domain. The closest distance between the [2Fe-2S] cluster and the bound flavin mononucleotide in mitoNEET is about 10Å, which could facilitate rapid electron transfer from the reduced flavin nucleotide to the [2Fe-2S] cluster in mitoNEET. The results suggest that flavin nucleotides may act as electron shuttles to reduce the mitoNEET [2Fe-2S] clusters and regulate mitochondrial functions in human cells.

SUBMITTER: Landry AP

PROVIDER: S-EPMC5209285 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Flavin nucleotides act as electron shuttles mediating reduction of the [2Fe-2S] clusters in mitochondrial outer membrane protein mitoNEET.

Landry Aaron P AP Wang Yiming Y Cheng Zishuo Z Crochet Robert B RB Lee Yong-Hwan YH Ding Huangen H

Free radical biology & medicine 20161203

MitoNEET, a primary target of type II diabetes drug pioglitazone, has an essential role in regulating energy metabolism, iron homeostasis, and production of reactive oxygen species in mitochondria. Structurally, mitoNEET is anchored to the mitochondrial outer membrane via its N-terminal transmembrane α-helix. The C-terminal cytosolic domain of mitoNEET hosts a redox active [2Fe-2S] cluster via three cysteine and one histidine residues. Here we report that the reduced flavin nucleotides can rapid ...[more]

PMID: 27923678

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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

Flavin nucleotides act as electron shuttles mediating reduction of the [2Fe-2S] clusters in mitochondrial outer membrane protein mitoNEET.

Publications

Flavin nucleotides act as electron shuttles mediating reduction of the [2Fe-2S] clusters in mitochondrial outer membrane protein mitoNEET.

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