Interaction of Quinone-Related Electron Acceptors with

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Interaction of quinone-related electron acceptors with hydropersulfide NaS: Evidence for one-electron reduction reaction 2

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Yumi Abiko, Yumi Nakai, Nho Cong Luong, Christopher L Bianco, Jon M. Fukuto, and Yoshito Kumagai Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00158 • Publication Date (Web): 05 Feb 2019 Downloaded from http://pubs.acs.org on February 6, 2019

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Chemical Research in Toxicology

Interaction of quinone-related electron acceptors with hydropersulfide Na2S2: Evidence for one-electron reduction reaction Yumi Abiko†, Yumi Nakai‡, Nho C. Luong║,┴, Christopher L. Bianco#, Jon M. Fukuto§ and Yoshito Kumagai†,* †

Environmental Biology Section, Faculty of Medicine, University of Tsukuba, 1-1-1

Tennodai, Tsukuba, Ibaraki 305-8575, Japan ‡ ║

JEOL Resonance Inc., Tokyo 196-8558, Japan Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1

Tennodai, Tsukuba, Ibaraki 305-8575, Japan. ┴

Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue University, 06

Ngo Quyen, Hue, Vietnam #

Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, United

States §

Sonoma State University, Rohnert Park, CA 94928, United States.

Keywords: Electron acceptor, perthiyl radical, persulfide, polysulfide, redox cycle, thiyl radical, 9,10-phenanthraquinone

* Corresponding author. Yoshito Kumagai, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Tel: +81-29-853-3297; Fax: +81-29-853-3259; E-mail: [email protected]

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Table of Contents Graphic

Electron acceptors

H-SS–

e.g., 9,10-PQ

–e– H-SS

O2– –e– +e–

+e– Radical species

O2


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ABSTRACT: We previously reported that 9,10-phenanthraquinone (9,10-PQ), an atmospheric electron acceptor, undergoes redox cycling with dithiols as electron donors, resulting in the formation of semiquinone radicals and monothiyl radicals; however, monothiols have little reactivity. Because persulfide and polysulfide species are highly reducing, we speculate that 9,10-PQ might undergo one-electron reduction with these reactive sulfides. In the present study, we explored the redox cycling capability of a variety of quinone-related electron acceptors, including 9,10-PQ, during interactions with the hydropersulfide Na2S2 and its related polysulfides. No reaction occurred when 9,10-PQ was incubated with Na2S; however, when 5 µM 9,10-PQ was incubated with either 250 µM Na2S2 or Na2S4, we detected extensive consumption of dissolved oxygen (84 µM). Under these conditions, both the semiquinone radicals of 9,10-PQ and their thiyl radical species were also detected using ESR, suggesting that a redox cycle reaction occurred utilizing one-electron reduction processes. Notably, the perthiyl radicals remained stable even under aerobic conditions. A similar phenomenon has also been observed with other electron acceptors, such as pyrroloquinoline quinone, vitamin K3 and coenzyme Q10. Our experiments with N-methoxycarbonyl penicillamine persulfide (MCPSSH), a precursor for endogenous cysteine persulfide, suggested the possibility of a redox coupling reaction with 9,10-PQ inside cells. Our study indicates that hydropersulfide and its related polysulfides are efficient electron donors that interact with quinones. Redox coupling reactions between quinoid electron acceptors and such highly reactive thiols might occur in biological systems.



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INTRODUCTION Redox systems are crucial for maintaining cellular homeostasis. Many enzymes use NADPH

or

1,5-dihydro-flavin-adenine

dinucleotide

(FADH2)

as

reducing

cofactors/cosubstrates to transfer electrons to electron acceptors. The transfer of electrons within the electron transport chain generates ATP. The presence of exogenous electron acceptors or electron donors from environmental sources can change the redox state of a cell through reactions with endogenous electron donor/acceptor species. For example, diesel exhaust particles, which are common air pollutants, contain chemicals such as redox active 9,10-phenanthraquinone (9,10-PQ)

1,2

and are thus considered to

contribute to pulmonary diseases caused by oxidative stress 3. It is known that 9,10-PQ can undergo one-electron reduction by cytochrome P450 reductase in the presence of NADPH forming an intermediate semiquinone radical of 9,10-PQ (9,10-PQ·–),4 which can react with O2 to generate superoxide and reforming the quinone. Hence, 9,10-PQ is capable of catalyzing the reduction of O2 to O2·– by biological reductants (termed “redox cycling”) with potential toxicological consequences. Furthermore, 9,10-PQ interacts with neuronal nitric oxide synthase (which has a P450 reductase domain) and inhibits its catalytic activity by shunting electrons from NADPH 5. In our previous study, we

showed

that

dithiol

compounds,

such

as

dithiothreitol,

2,3-dimercapto-1-propanesulfonic acid, 2,3-dimercapto-1-propanol and dihydrolipoic acid, but not monothiols such as GSH, cysteine, and N-acetylcysteine, also interacted with the quinone to yield 9,10-PQ·– and the corresponding thiyl radical 6,7. Recent studies have identified other thiol-derived/related species that may also have the ability to participate in the redox chemistry described above. There are hydropersulfide and hydropolysulfide species such as Cys hydropersulfide (Cys-SSH, ca. 1 µM), GSH hydropersulfide (GSSH, ca. 60 µM), and their polysulfides (Cys-SSnH, GSSnH, n > 1) in mouse liver 8. Unlike monosulfide species, hydropersulfide species predominantly exist as anionic species because of their lower pKa values. Also, persulfide species appear to have increased nucleophilic and reducing capabilities compared to the corresponding thiols

9,10

. One-electron oxidation of a hydropersulfide

to the relatively stable perthiyl radical (RSS·) is facile as Bianco et al. has shown that N-methoxycarbonyl penicillamine persulfide (MCPSSH), a model alkyl hydropersulfide, is oxidized by weak one-electron oxidants such as TEMPOL, TEMPO, K3Fe(CN)6, and MbFeIII to form the corresponding perthiyl radical (MCPSS·), which can ultimately 4 ACS Paragon Plus Environment

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dimerize to the tetrasulfide species11. These results suggest that hydropersulfide is an effective one-electron donor, resulting in MCPSS formation. •

Importantly, the purely

inorganic per- and poly-sulfide species should exhibit similar chemistry. That is the redox chemistry of Cys-SS- and HSS- are expected to be analogous. Based on the above observations, it is hypothesized that 9,10-PQ can undergo redox cycling, involving the reaction of hydropersulfide with 9,10-PQ, to form 9,10-PQ·– that can further react with O2 to make O2·–. To address this, we measured O2 consumption and detected semiquinone radicals and thiyl radicals during reaction of quinones and persulfides in this study. MATERIALS AND METHODS Materials. Coenzyme Q10 (CoQ10, 99% purity determined by HPLC), GSH (99.7% purity determined by HPLC), and 9,10-PQ (99.9% purity determined by HPLC) were from Sigma-Aldrich (San Diego, CA, USA). Sodium disulfide (Na2S2), sodium trisulfide (Na2S3), and sodium tetrasulfide (Na2S4) were from Dojindo Laboratories (Kumamoto, Japan). Acetone (environmental analysis grade), DTT, sodium sulfide (Na2S), and pyrroloquinoline quinone (PQQ, 100% purity determined by HPLC) were from Wako Pure Chemical Industries (Osaka, Japan). Vitamin K3 (VK3) was from Kohjin Life Sciences (Tokyo, Japan). S-Methoxycarbonyl penicillamine disulfide (MCPD) was prepared as described previously11,12. All other reagents were of the highest grade available. Oxygen consumption. When sulfur compounds (final concentration 500 µM for GSH, DTT, Na2S, N-acetyl-D-penicillamine, and MCPD or 250 µM for Na2S2, Na2S3, and Na2S4) were added to an aqueous solution of electron acceptors containing 2.5% dimethyl sulfoxide (DMSO), the reactions started and O2 consumption was monitored with a dissolved O2 (DO) meter (B-505, Iijima Electronics Corp., Aichi, Japan) at room temperature. Each experiment was performed at least three times. Detection of radical species. ESR analysis was performed at 25°C with a JES-X320 spectrometer (JEOL Ltd., Tokyo, Japan). To start reactions between electron acceptors and sulfur compounds, sulfur compounds (final concentration of 10 mM) were added to solutions containing electron acceptors (final concentration, 0.2 mM), 50% acetone and 5 ACS Paragon Plus Environment


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10% DMSO. For the reaction of 0.2 mM 9,10-PQ with 2 mM MCPD or N-acetyl-D-penicillamine, we used 50% acetone–10% DMSO–20 mM HEPES (pH 7.5) as a reaction buffer. We used a lower concentration of MCPD than that used for the other per/polysulfides because at high concentrations MCPD rapidly degrades to yield its dimer. The mixtures were incubated at 25°C for 30 s and then analyzed by ESR spectroscopy. The instrument settings were as follows: modulation frequency, 100 kHz; modulation width 0.6 mT; sweep time, 1 min; time constant, 0.1 s; microwave power, 8 mW; microwave frequency, 9418 MHz. MnO was measured as a standard with constant amount, 650. Each experiment was performed at least three times and representative data are shown. Detection of one-electron oxidation-reduction potential. Cyclic voltammetry was performed at 25°C under an argon gas flow using an ALC electron chemical analyzer and three electrodes cell (BAS Ltd., Tokyo, Japan). The reference electrode and counter electrode were Ag/AgCl and Pt wire, respectively. A glassy carbon (GC) electrode was used as the working electrode. Na2S2 (1 mM) in 0.1 N NaOH-100 mM NaNO3 were analyzed at a scan rate of 50 mV/s from –1 to 1 V. Cell culture. A431 cells were obtained from RIKEN Cell Bank, Tsukuba, Japan. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-alanyl-L-glutamine (Gibco, USA) in an incubator supplemented with 5% CO2 at 37ºC.

For each experiment, the cells were seeded at 8.3 × 104 cells/cm2 and were pre-incubated in serum-free medium for 24 h before treatment with compounds. Detection of persulfide species in A431cells. A431 cells were exposed to 9,10-PQ for 30 min, and then the cells were washed with ice-cold PBS three times after removal of the

medium.

The

collected

cells

were

homogenized

into

1

mM

β-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM, Molecular Bioscience, Boulder, CO, USA) in methanol and sonicated on ice, following incubation at 37°C for 30 min to yield HPE-AM adducts. The homogenate was centrifuged at 14,000 × g at 4°C for 10 min, and the resultant supernatant was diluted 4 times with 0.1% formic acid containing known amounts of isotope-labeled internal standards as prepared previously described13. 6 ACS Paragon Plus Environment

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The remained pellet was incubated with 2% SDS at 95°C for 20 min, and protein concentrations were determined by using the BCA Protein Assay (Nacalai Tesque, Kyoto, Japan). The aliquot of sample containing HPE-AM adducts was subjected to LC-ESI-MS/MS (Bruker, CA, USA) using a YMC-Triart phenyl column (50 × 2.0 mm inner diameter, 3 µm particle size, 12 nm pore size) that was maintained at 40°C. Mobile phase A [water containing 0.1 % (v/v) formic acid] and B [methanol containing 0.1 % (v/v) formic acid] were linearly mixed by a gradient program at a flow rate of 0.2 mL/min. The mobile phase composition was set as follows: 3% B for 3 min; linear increase over 15 min to 95% B; maintain at 95% B for 1 min before returning linearly to 3% B over 21 min. The eluted compounds were then transferred to the electrospray source of the triple quadrupole mass EVOQ Qube spectrometer (Bruker), whose control and analyses were performed using Bruker MS Workstation software ver. 8.1.2. The ESI was used with a spray voltage of 4 kV and collision energy was set as previously described13. The temperature of the ESI cone, and heated probe, at which MS spectra were obtained, was 350°C, and 250°C, respectively. The nebulizer, cone, and probe gas flows were set to 50, 25, and 50 psi, respectively. Statistical analysis. Each experiment was performed at least three times. ANOVA with correction for multiple comparisons in post-hoc analysis was performed using Graphpad Prism (Graphpad Software, San Diego, CA, USA). A p-value of

Interaction of Quinone-Related Electron Acceptors with

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