Binding of α-synuclein with Fe(III) and with Fe(II) and biological implications of the resultant complexes

doi:10.1016/j.jinorgbio.2009.11.005

Journal of Inorganic Biochemistry

Volume 104, Issue 4, April 2010, Pages 365-370

https://doi.org/10.1016/j.jinorgbio.2009.11.005 Get rights and content

Abstract

Parkinson’s disease (PD) is hallmarked by the abnormal intracellular inclusions (Lewy bodies or LBs) in dopaminergic cells. Amyloidogenic protein α-synuclein (α-syn) and iron (including both Fe(III) and Fe(II)) are both found to be present in LBs. The interaction between iron and α-syn might have important biological relevance to PD etiology. Previously, a moderate binding affinity between α-syn and Fe(II) (5.8 × 10³ M⁻¹) has been measured, but studies on the binding between α-syn and Fe(III) have not been reported. In this work, electrospray mass spectrometry (ES-MS), cyclic voltammetry (CV), and fluorescence spectroscopy were used to study the binding between α-syn and Fe(II) and the redox property of the resultant α-syn–Fe(II) complex. The complex is of a 1:1 stoichiometry and can be readily oxidized electrochemically and chemically (by O₂) to the putative α-syn–Fe(III) complex, with H₂O₂ as a co-product. The reduction potential was estimated to be 0.025 V vs. Ag/AgCl, which represents a shift by −0.550 V vs. the standard reduction potential of the free Fe(III)/Fe(II) couple. Such a shift allows a binding constant between α-syn and Fe(III), 1.2 × 10¹³ M⁻¹, to be deduced. Despite the relatively high binding affinity, α-syn–Fe(III) generated from the oxidation of α-syn–Fe(II) still dissociates due to the stronger tendency of Fe(III) to hydrolyze to Fe(OH)₃ and/or ferrihydrite gel. The roles of α-syn and its interaction with Fe(III) and/or Fe(II) are discussed in the context of oxidative stress, metal-catalyzed α-syn aggregation, and iron transfer processes.

Introduction

Parkinson’s disease (PD) is the second most common neurodegenerative disease (next only to Alzheimer’s disease or AD) and affecting more than 1% of the people over the age of 65 in the United States [1]. Pathologically, PD is characterized by a progressive loss of the dopaminergic cells in the substantia nigra, a small brain region producing dopamine [2]. The surviving dopaminergic cells of PD patients contain cytosolic filamentous inclusions known as the Lewy bodies (LBs) [3], within which α-synuclein (α-syn) is a major component [3], [4]. α-Syn comprises 140 amino acids and its exact function is still unknown [5]. Mounting evidence has shown that α-syn can misfold and subsequently aggregate and the resultant aggregates are neurotoxic [6]. It is also reported that abnormal iron homeostasis appears to exacerbate with PD progression. For example, unusually high concentrations of iron and a greater Fe(III)/Fe(II) ratio have been found in the substantia nigra of PD patients [7]. In addition, postmortem studies have revealed Fe(III) and Fe(II) presence in the LBs [8], [9]. The role in PD pathology could be at least twofold. On one hand, in vitro studies have shown that iron can accelerate the α-syn aggregation and microscopic techniques (i.e., the atomic force microscopy and scanning electron microscopy) have shown that α-syn also forms fibrous aggregates in the absence and presence of iron [6], [10], [11], [12]. On the other hand, iron is a redox active metal and its interaction with α-syn could induce or be involved in oxidative stress. Lending support to the second aspect is the redox metal-induced oxidative stress associated with other neurodegenerative diseases such as AD [13]. Metal ions (e.g., Cu(II), Fe(II), and Fe(III)) can complex with amyloid-β (Aβ) peptides and the resultant complexes react with other cellular redox molecules to generate reactive oxygen species (ROS) [13], [14]. The ROS generated can subsequently cause damages to a variety of molecules and organelles (DNA, protein, lipid, and mitochondrial), leading eventually to neuronal cell death.

A better understanding of the roles of iron in PD pathology can be gained from studies of the interaction between iron and α-syn. Although binding between Fe(II) and α-syn has been studied and a binding constant of 5.8 × 10³ M⁻¹ was reported [15], any description of the functions of iron is incomplete without the knowledge about the binding between α-syn and Fe(III). Specifically, important questions such as the effect of iron (including Fe(II) and Fe(III)) on α-syn aggregation, possible redox reactions of the α-syn–iron complexes, and the role of α-syn in iron transfer, cannot be elucidated. The lack of the knowledge about the binding between α-syn and Fe(III) largely stems from the fact that in vitro studies are hampered by the extensive hydrolysis of Fe(III) at physiological pH (i.e., pH 7.4). Particularly intriguing is whether Fe(III) complexation by α-syn would inhibit the Fe(III) hydrolysis. Of equal interest is whether α-syn–Fe(II) and the putative α-syn–Fe(III) complex can react with cellular redox species. As aforementioned, such reactions could potentially produce ROS that will impose oxidative stress to neuronal cells [13], [14].

We wish to report our mass spectrometric, fluorescence, and electrochemical studies of the interaction between iron and α-syn. Cyclic voltammetry (CV) is a simple and accurate technique to measure the redox potential of many biologically important species. We have successfully utilized CV to determine the binding affinity of Aβ to Fe(III) or Fe(II) by comparing the experimentally measured redox potentials of the complexes to the standard reduction potential of the free Fe(III)/Fe(II) couple [14]. Extension of this approach to the studies of the putative α-syn–Fe(III) complex not only yields the binding constant, but also enables us to gain insight into the possible roles of α-syn in iron transport and ROS generation. The possible effects of the oxidation of α-syn–Fe(II) on the α-syn aggregation process are also discussed.

Section snippets

Materials

Sodium dihydrogen phosphate, sodium hydroxide, sodium sulfate, ammonium sulfate, trifluoroacetic acid, ferrous ammonium sulfate (Fe(NH₄)₂(SO₄)₂), Trizma base (Tris), isopropyl β-d-thiogalactopyranoside (IPTG), and acetonitrile were purchased from Thermo Fisher Scientific, Inc. (Pittsburgh, PA). All aqueous solutions were prepared using Millipore water (18 MΩ cm⁻¹). Escherichia coli BL21 (DE3) and lysozyme were purchased from Invitrogen Corp (Carlsbad, CA) and EMD, Inc. (Gibbstown, NJ),

Identification of α-syn–Fe(II) by ES-MS

We first attempted to identify the α-syn–Fe(II) complex by ES-MS. Fig. 1 depicts an ES-MS spectrum of a mixture of Fe(NH₄)₂(SO₄)₂ and α-syn. The multiply charged (protonated to 10–18) α-syn peaks are easily discernable. Next to each α-syn peak is one whose m/z value corresponds to the α-syn–Fe(II) complex (identified by an asterisk in Fig. 1). We should note that each of the complex peaks contains one Fe(II) and two Na⁺ ions (given rise by Na⁺ in the phosphate buffer used for the α-syn

Discussion

Our ES-MS results confirmed the formation of the α-syn–Fe(II) complex (Fig. 1) and the dissociation of Fe(III) from the complex after being oxidized by O₂. The CV (Fig. 2A) and fluorescence data also showed the loss of Fe(II) when the solution was exposed to air. Fluorescence studies verified that, similar to the complexes formed between Aβ and redox metal ions, the reaction between α-syn–Fe(II) and O₂ can generate H₂O₂ (Fig. 4). This is reasonable because the potential of the

Acknowledgements

We thank Renee Williams and Prof. Yinsheng Wang (University of California-Riverside) for their help on the ES-MS measurements and Lin Liu for help on the voltammetric experiment. Partial support of this work by a NINDS Grant (No. SC1NS070155-01), the NIH-RIMI Program at California State University, Los Angeles (P20-MD001824-01 to F.Z.), and the Natural Science Foundation of China (Nos. 20676153 and 20876179 to Y.L.) is gratefully acknowledged. Y.P. also thanks the China Scholarship Council for

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¹: These authors contributed equally.

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Binding of α-synuclein with Fe(III) and with Fe(II) and biological implications of the resultant complexes

Abstract

Introduction

Section snippets

Materials

Identification of α-syn–Fe(II) by ES-MS

Discussion

Acknowledgements

Neuron

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Free Radical Biol. Med.

J. Biol. Chem.

Free Radical Biol. Med.

J. Inorg. Biochem.

J. Biol. Chem.

Annu. Rev. Neurosci.

Proc. Natl. Acad. Sci. USA

Nature

Biochemistry

Acc. Chem. Res.

J. Neurochem.

Acta Neuropathol.

Br. J. Pharmacol.

Protein Sci.

Biochemistry

Biochemistry