Electron transfer activity of the nanodisc-bound mitochondrial outer membrane protein mitoNEET

Abstract

MitoNEET is the first iron-sulfur protein found in mitochondrial outer membrane. Abnormal expression of mitoNEET in cells has been linked to several types of cancer, type II diabetes, and neurodegenerative diseases. Structurally, mitoNEET is anchored to mitochondrial outer membrane via its N-terminal single transmembrane alpha helix. The C-terminal cytosolic domain of mitoNEET binds a [2Fe-2S] cluster via three cysteine and one histidine residues. It has been shown that mitoNEET has a crucial role in energy metabolism, iron homeostasis, and free radical production in cells. However, the exact function of mitoNEET remains elusive. Previously, we reported that the C-terminal soluble domain of mitoNEET has a specific binding site for flavin mononucleotide (FMN) and can transfer electrons from FMNH₂ to oxygen or ubiquinone-2 via its [2Fe-2S] cluster. Here we have constructed a hybrid protein using the N-terminal transmembrane domain of Escherichia coli YneM and the C-terminal soluble domain of human mitoNEET and assembled the hybrid protein YneM-mitoNEET into phospholipid nanodiscs. The results show that the [2Fe-S] clusters in the nanodisc-bound YneM-mitoNEET can be rapidly reduced by FMNH₂ which is reduced by flavin reductase using NADH as the electron donor. Addition of lumichrome, a FMN analog, effectively inhibits the FMNH₂-mediated reduction of the [2Fe-2S] clusters in the nanodisc-bound YneM-mitoNEET. The reduced [2Fe-2S] clusters in the nanodisc-bound YneM-mitoNEET are quickly oxidized by oxygen under aerobic conditions or by ubiquinone-10 in the nanodiscs under anaerobic conditions. Because NADH oxidation is required for cellular glycolytic activity, we propose that the mitochondrial outer membrane protein mitoNEET may promote glycolysis by transferring electrons from FMNH₂ to oxygen or ubiquinone-10 in mitochondria.

Keywords: Electron transfer activity; FMN; Mitochondria; NADH; Nanodisc; mitoNEET.

Figure 1.. Preparation of the nanodisc-bound YneM-mitoNEET.

A), a model for the hybrid YneM-mitoNEET protein. B), SDS-PAGE gel of purified hybrid YneM-mitoNEET. Lane 1, molecular weight markers (PAGE-Master, GenScript co.); lane 2, soluble domain of mitoNEET; lane 3, hybrid YneM-mitoNEET. C), UV-Visible absorption spectrum of purified YneM-mitoNEET (containing 8 μM [2Fe-2S] clusters) dissolved in TSG10 buffer (containing Tris (pH 8.0, 50 mM), NaCl (100 mM), glycerol (10% v/v), and Triton-x-100 (0.5% v/v)). D), an elution profile of the nanodisc containing YneM-mitoNEET. E), the transmission electron microscopy (TEM) image of prepared nanodiscs. The scale of 200 nm is indicated in the panel.

Figure 2.. Reduction of the [2Fe-2S] clusters in the nanodisc-bound YneM-mitoNEET by FMNH₂.

A), UV-visible absorption spectra of the [2Fe-2S] clusters in the nanodisc-bound YneM-mitoNEET before and after reduction with FMNH₂ under anaerobic condition. The nanodisc-bound YneM-mitoNEET (containing 6 μM [2Fe-2S] clusters) was incubated with FMN (50 nM) and Fre (0.2 μM) under anaerobic conditions. NADH (20 μM) was added to the solution anaerobically to initiate the reaction. UV-visible absorption spectra were taken before (spectrum 1) and 5 minutes after addition of NADH (spectrum 2). B), EPR spectra of the nanodisc-bound YneM-mitoNEET. Spectrum 1, C-terminal soluble domain of mitoNEET (containing 6 μM [2Fe-2S] clusters). Spectrum 2, C-terminal soluble domain of mitoNEET (containing 6 μM [2Fe-2S] clusters) was reduced with sodium dithionite (4 mM). Spectrum 3, the nanodisc-bound YneM-mitoNEET (containing 6 μM [2Fe-2S] clusters). Spectrum 4, the nanodisc-bound YneM-mitoNEET (containing 6 μM [2Fe-2S] clusters) was reduced with NADH, flavin reductase and FMN. Spectrum 5, the nanodisc-bound YneM-mitoNEET (containing 6 μM [2Fe-2S] clusters) was reduced with sodium dithionite (4 mM). The data were representative of three independent experiments.

Figure 3.. Oxidation of the reduced [2Fe-2S] clusters in the nanodisc-bound YneM-mitoNEET by oxygen.

A), the nanodisc-bound YneM-mitoNEET (containing 6 μM [2Fe-2S] clusters) was incubated with FMN (0.2 μM), and Fre (0.2 μM) in a buffer containing Tris (20 mM, pH 7.9) and NaCl (20 mM) under aerobic condition. NADH (10 μM) was added to the solution under aerobic conditions. The redox state of the [2Fe-2S] clusters in the nanodisc-bound YneM-mitoNEET was monitored at 458 nm. B), same as A), except the concentration of NADH in the reaction solution was monitored at 340 nm. C), the nanodisc-bound YneM-mitoNEET (containing 6 μM [2Fe-2S] clusters) was incubated with FMN (0.2 μM), and Fre (0.2 μM) in a buffer containing Tris (20 mM, pH 7.9) and NaCl (20 mM) under aerobic condition. NADH (100 μM) was added to the reaction solution under aerobic conditions. The redox state of the YneM-mitoNEET [2Fe-2S] clusters was monitored at 458 nm. D), same as C), except the concentration of NADH in the reaction solution was monitored at 340 nm. The data were representative of three independent experiments.

Figure 4.. FMNH₂ fails to reduce the [2Fe-2S] clusters in the nanodisc-bound YneM-mitoNEET mutant H87C.

A) the nanodisc-bound YneM-mitoNEET (containing 6 μM [2Fe-2S] clusters) was incubated with FMN (0.2 μM), and Fre (0.2 μM) in a buffer containing Tris (20 mM, pH 7.9) and NaCl (20 mM) under aerobic condition. NADH (100 μM) was added to the solution to initiate the reaction. The redox state of the [2Fe-2S] clusters in the nanodisc-bound YneM-mitoNEET was monitored at 458 nm. B), same as A), except the YneM-mitoNEET mutant H87C was used in the nanodiscs. The data were representative of three independent experiments.

Figure 5.. The FMNH analog lumichrome inhibits the FMNH₂-mediated reduction of the [2Fe-2S] clusters in the nanodisc-bound YneM-mitoNEET.

A) the nanodisc-bound YneM-mitoNEET (containing 6 μM [2Fe-2S] clusters) was incubated with FMN (0.2 μM), and Fre (0.2 μM) in a buffer containing Tris (20 mM, pH 7.9) and NaCl (20 mM) under aerobic condition. NADH (100 μM) was added to the reaction solution to initiate the reaction. The redox state of the [2Fe-2S] clusters in the nanodisc-bound YneM-mitoNEET was monitored at 458 nm. B), same as A), except the nanodiscs were pre-incubated with lumichrome (500 nM) under aerobic conditions. The data were representative of three independent experiments.

Figure 6.. Oxidation of the reduced [2Fe-2S] clusters in the nanodisc-bound YneM-mitoNEET by ubiquinone-10 under anaerobic conditions.

A), the nanodisc-bound YneM-mitoNEET (containing 6 μM [2Fe-2S] clusters, with no ubiquinone-10) was incubated with FMN (50 nM) and Fre (0.2 μM) in a buffer containing Tris (20 mM, pH 7.9) and NaCl (20 mM) under anaerobic conditions. The reaction was initiated by adding NADH (20 μM) anaerobically. The redox state of the [2Fe-2S] clusters in the nanodisc-bound YneM-mitoNEET was monitored at 458 nm. B), same as A), except ubiquinone-10 was pre-incorporated into the nanodiscs at a ratio of ubiquinone-10:phospholipid of 1:13. The data were representative of three independent experiments.

Figure 7.. A schematic electron transfer pathway for the membrane-bound mitoNEET.

A), a diagram of the proposed electron transfer activity of the YneM-mitoNEET in nanodiscs. NADH is oxidized by flavin reductase, which reduces FMN to FMNH₂. FMNH₂ then binds to the YneM-mitoNEET and reduces the [2Fe-2S] clusters in the YneM-mitoNEET. The reduced [2Fe-2S] clusters in the YneM-mitoNEET are oxidized by oxygen or ubiquinone-10 in the nanodiscs. B), a proposed model for the electron transfer activity of mitoNEET in mitochondrial outer membrane. In cytosol, glycolysis reduces NAD⁺ to NADH. MitoNEET, together with flavin reductase, transfers electrons from NADH to oxygen or ubiquinone-10 in mitochondrial outer membrane and promotes glycolysis activity in cytosol.