Electrostatic effects on water-soluble fullerene derivatives interaction with cytochrome c and cytochrome c oxidase

Abstract

Water-soluble fullerene derivatives are good candidates for various biological applications such as anticancer or antimicrobial therapy, cytoprotection, enzyme inhibition, and many others. Their toxicity, both in tissue culture and in vivo, is a critical characteristic for the development and restriction of these applications. The effects of six water-soluble cationic and anionic polysubstituted fullerene derivatives on cytochrome c oxidase activity in rat brain mitochondria and the possibility of cytochrome c binding were studied. We found that the ability of these fullerene derivatives to bind with cytochrome c oxidase and charged molecules like eosin Y strongly depends on their electrostatic charge. As was shown, the cationic fullerene derivative inhibits cytochrome c oxidase that has the overall negative electrostatic potential completely, unlike anionic derivatives. Thus, it confirms the essential role of electrostatic interactions in the interaction of fullerene derivatives with the active site of enzymes. The results explore how cationic fullerene derivatives play a role in mitochondrial dysfunction, oxidative stress, and cytotoxicity.

Introduction

Fullerenes are interesting molecules that have attracted considerable attention of researchers due to their unique molecular architecture. Pristine fullerene is extremely hydrophobic, and this is critical for biological applications. Various methods of synthesis of water-soluble fullerene derivatives have been developed, including the attachment of several cationic or anionic functional groups to the carbon cage [1–5].

Thus, water-soluble fullerene derivatives are amphiphilic compounds with a hydrophobic carbon cage and hydrophilic functional groups [6]. These compounds can penetrate cell membranes, interact with hydrophobic sites of proteins, and selectively bind to active centers of enzymes due to electrostatic interactions and hydrogen bonds.

Since water-soluble fullerene derivatives have been designed, they were shown to possess various biological activities, including membranotropic and antioxidant properties, neuroprotective, antibacterial, cytotoxic, antitumor, and antiproliferative activities [7–11]. Some derivatives are reported to possess significant antiviral activity due to the inhibition of HIV protease, HIV reverse transcriptase, NS3/4A protease, and NS5B polymerase of the hepatitis virus [12–14]. Some cationic fullerene derivatives were found to be able to modulate acetylcholinesterase activity [15].

We have previously shown that polysubstituted cationic and anionic fullerene derivatives (PFDs), similar to those studied in this work, can penetrate liposomal membranes [16], possess antioxidant and free-radical scavenging properties [17], and inhibit the catalytic activity of monoamine oxidase A and B [18], sorbitol dehydrogenase, aldose reductase [19], sarcoplasmic reticulum Ca²⁺-ATPase [20], exhibit neurotropic activity, acting as positive modulators of Purkinje cell AMPA receptors in rats [21].

The mitochondrial respiratory chain is a system of four membrane-bound enzymes, which catalyzes the oxidation of nicotinamide adenine dinucleotide (NADH), using the terminal electron acceptor oxygen in the inner mitochondrial membrane during the process of oxidative phosphorylation [22]. Finally, complex III of the respiratory chain delivers its electrons to cytochrome c, which transfers them to complex IV (cytochrome c oxidase) with the generation of a proton gradient across the inner membrane of a mitochondrion. Cytochrome c contains a heme surrounded by a cluster of positively charged lysine residues that carry the electron to cytochrome c oxidase [23–25]. Electrostatic interactions are formed between positively charged Lys residues on the cytochrome c and the negatively charged amino acid residues in the vicinity of the Cu²⁺-containing active center of cytochrome c oxidase [26]. Blocking of such electrostatic interactions leads to inhibition of the function of the mitochondrial respiratory chain. Moreover, it is known that cytochrome c can induce apoptosis in various cell types by the activation cascade of caspases once it is released into the cytosol [27].

Various biological activities of fullerene derivatives with anionic or cationic functional groups may be due, among other things, to their ability to penetrate the cell and mitochondrial membrane and selectively bind to cytochrome c or cytochrome c oxidase via a combination of hydrophobic and electrostatic interactions. Such events might lead to the inhibition of electron transport and thereby disrupt the normal functioning of the cell. Therefore, the study of the biological effects of water-soluble fullerene derivatives with cationic and anionic functional groups due to their interaction with cytochrome c and cytochrome c oxidase is of great interest.

In the present work, we studied the effects of six water-soluble cationic and anionic polysubstituted fullerene derivatives on cytochrome c oxidase activity in rat brain mitochondria and the possibility of cytochrome c binding to negatively charged molecules.

Materials and methods

In the present work, we have studied six different water-soluble fullerene derivatives (Fig. 1). The synthesis and spectral data of investigated polysubstituted fullerene derivatives were reported previously (compounds 1: [5], 2: [21], 3: [28], 4: [29], 5: [30], 6: [31]). Information on the aggregational behavior of synthesized fullerene derivatives in aqueous solutions is also available [32–37]. It demonstrates that investigated amphiphilic compounds form supramolecular structures (micelles, vesicles, etc.) in aqueous media with the sizes varying from several nanometers to hundreds of micrometers. Compounds 1, 2, and 4 are [60]fullerene derivatives that have five negatively charged carboxyl groups. Compound 3 is a polycarboxylic derivative of [70]fullerene. Compound 5 is a mixture of sodium fullerenolates. Compound 6 is a salt of aminofullerene, and it has five cationic functional groups. All these fullerene derivatives show high solubility in water (> 50 mg/mL).

Fig. 1.

Molecular structures of the investigated water-soluble C₆₀ and C₇₀ derivatives

Registration of diffusive interactions of cytochrome c and PFDs with eosin Y in an aqueous solution

The quenching of eosin Y phosphorescence by cytochrome c heme was used to analyze the influence of electrostatics on diffusive interactions between cytochrome c and anionic molecules. The kinetics of eosin Y (Sigma-Aldrich) phosphorescence decay were recorded using the Agilent Cary Eclipse fluorescence spectrometer. The eosin Y concentration was 2 μM, the cytochrome c concentration increased sequentially in the range from 0 to 2 μM, and the PFD concentration increased from 0 to 3.7 μM. The ionic strength of the solution was changed by adding a concentrated NaCl solution into the sample in the range from 0 to 0.82 M. Before measurements, oxygen was removed from the sample volume by adding 1 μg of glucose oxidase and 1 mg of glucose into the solution, as described previously [38].

Tissue preparation

The work was carried out following the EU Directive 2010/63/EU. Wistar male rats (about six months old) were sacrificed by decapitation. Each brain was rapidly excised, frozen in liquid nitrogen, and stored at –80 °C until use. The brains were thawed and homogenized using a Wisd WiseTis HG-15D homogenizer for 2 min in a buffer (0.01 M PBS, pH 7.4 or 0.1 M Tris–HCl, pH 7.4). Protein concentrations were determined by the Lowry method [39].

Isolation of mitochondria from rat brain

The protocol was performed according to [40]. Briefly, 4 g of brain tissue was washed and minced in 100 ml of 0.9% KCl. The tissue was homogenized with a Potter glass homogenizer at 4 °C. The lysate was diluted ten times with 0.25 M sucrose and centrifuged at 750 × g for 10 min at 4 °C. Fat was decanted from the supernatant, and the lysate was centrifuged in 0.25 M sucrose at 12,000 × g for 15 min at 4 °C; this step was performed twice. The final mitochondrial pellet was washed in PBS buffer with sucrose and frozen quickly by immersion in liquid nitrogen.

Cytochrome oxidase activity assay

A total of 25 mg cytochrome c (Sigma-Aldrich, from horse heart) was dissolved in 1 ml of distilled water, and 1.0 ml of 0.1 M L-ascorbate (Sigma-Aldrich) was added to reduce the cytochrome. The mixture was loaded onto a 20-ml Sephadex G-15 column prewashed with 100 ml degassed 10 mM potassium phosphate, pH 7.4. The middle three-fourths of the cytochrome band was collected. The reduced cytochrome stored at − 70 °C is stable for at least 12 months.

Complex IV or cytochrome-c oxidase activity was measured by following the oxidation of reduced cytochrome c at 550 nm (extinction coefficient 19.6 mM⁻¹ cm⁻¹) [41] in a 1-ml cuvette at room temperature. First, the reduced cytochrome was added to 1 mM in 10 mM potassium phosphate, pH 7.4, and the stability of the absorbance was checked for 3 min. Then, the reaction began by adding 5 μg mitochondrial protein. The reaction was monitored as quickly as possible, and the absorbance decrease was recorded for 3 min. Specific rates were calculated either by estimation of first-order rate constants.

Statistical analyses

All tests were performed at least in triplicate in three independent experiments. Results are presented as mean ± SEM calculated using Microsoft Excel. Plots and linear regressions were generated using Origin 6.1 for Windows, OriginLab (Northampton, MA, USA).

Results and discussion

Electrostatic effects on the reactions of cytochrome c with negatively charged molecules

Cytochrome c is a water-soluble heme protein. The function of cytochrome c in the respiratory chain is an electron transfer from the respiratory complex III to the respiratory complex IV (cytochrome c oxidase) [40]. The iron atom in the cytochrome c heme group is alternately oxidized and reduced by the transfer of electrons. Quite a few amino acid residues of the cytochrome c are ionized at neutral pH [42]. The role of positive groups around the heme has been linked with the reaction of cytochrome c with its physiological redox partners. This particular charge distribution is also essential for the reaction of small charged molecules with cytochrome c [43]. Electrostatic interactions between the positively charged groups on cytochrome c and negatively charged groups on cytochrome c oxidase mediate the binding of Cyt C²⁺. Finally, cytochrome c releases the electron to the final electron carrier—cytochrome c oxidase [26]. The concave surface of cytochrome c oxidase contains CuA, which is the first loading site for electrons transferred from cytochrome c. The distance between the iron atom of heme and the copper atom of CuA is 23 Å; this creates conditions for efficient electron transfer from heme to CuA [23, 24]. As was determined in [24], the intracomplex rate constant for electron transfer from horse cytochrome c to bovine CcO is 6 × 10⁴ s⁻¹.

In the work of Witte et al. [44], quenching of Zn²⁺-cytochrome c fluorescence was used to study the effects of electrostatic charges of dendritic water soluble [60]fullerene monoadducts on their ability to bind to cytochrome c. The introduction of Zn²⁺ into the structure of the porphyrin ring leads to the appearance of effective protein fluorescence with a quantum yield of 0.04 and an excited state lifetime of 2.2 ns. Increasing concentrations of fullerene derivatives resulted in significant quenching of fluorescence, which was found to be static due to the formation of a nonfluorescent complex between cytochrome c and a fullerene derivative.

From the analysis of quenching dependences, it was found that the binding constant for anionic derivatives was in the range from 8.2 × 10⁴ M⁻¹ to 2.2 × 10⁷ M⁻¹. In contrast, for the cationic fullerene derivative, this constant is < 10² M⁻¹.

With a decrease in the number of negative charges of derivatives, the binding constant values decreased, but no clear correlation was observed. This is probably due to the difference in the spatial arrangement of positive charges on the surface of cytochrome c and anionic addends of derivatives studied in this work [44].

In our work, to assess the effect of electrostatic charges of cytochrome c and fullerene derivatives on reactions with anionic and cationic molecules in an aqueous solution, we studied the effect of quenching of excited triplet state of eosin Y by the cytochrome heme or by a set of fullerene derivatives with different anionic and cationic addends. Phosphorescence quenching experiments provided a highly sensitive and reliable assay for diffusive and static interactions between molecules in solutions at low concentration [45, 46]. The xanthene dye eosin Y, which has a high quantum yield of phosphorescence in aqueous solutions at room temperature with the lifetime about 0.5 × 10⁻³ s, was used in this work as a triplet label [16].

The effect of electrostatic interactions on the reactions of cytochrome c with an anionic molecule of eosine Y was investigated in a water solution at different ionic strength by measuring the rate of eosine Y phosphorescence quenching at different cytochrome c concentration. It is assumed that an exchange mechanism of energy transfer or electron transfer carries out the quenching of eosine Y phosphorescence by the cytochrome c heme [38, 46]. Therefore, such reactions can simulate electron transfer reactions with the participation of cytochrome c both in the dynamic and static regime.

The rate constant of quenching of eosin Y triplet state k_q was estimated from the change in the phosphorescence decay time in the absence of cytochrome c (τ₀) and upon the addition of various concentrations of cytochrome c (τ) to the solution according to the formula, where [Q] is a quencher concentration:

$$k_q=(\frac{1}{\tau }-\frac{1}{{\tau }_0})/[Q]$$ 1

The analysis of the dependence of the k_q values on the ionic strength was carried out using the Debye-Smoluchowski equation, according to which the rate constant of the bimolecular reaction k_q in the case of electrostatically charged reagents is expressed by the formula:

$$k_d=4\pi DR{\alpha }_{\mathit{el}}$$ 2

$${\alpha }_{\mathit{el}}=\frac{1}{\sigma {\int }_{\sigma }^{\infty }\mathit{exp}\left[V,(,R,),/,k,T\right]\frac{\mathit{dr}}{r^2}}$$ 3

where R represents the encounter distance taken as the sum of the molecular radii, D is the relative diffusion coefficient, V is the electrical potential energy which is a function of R, α_эл is the electrostatic factor, k is Boltzmann’s constant, and T is temperature.

At low values of the ionic strength, Eq. (2) transforms into the Bronsted equation:

$$\lg k_q=\lg k_q^0+z_1{z}_2\sqrt{\mu }$$ 4

According to the Bronsted equation, from the linear section slope of the dependence, the value of the net charge of reactant z₁ can be determined, knowing the net charge z₂ of the second reactant.

In our experiment, eosin Y, which has a net charge (− 2) at neutral pH, was used as a triplet label [47]. The ionic strength was increased by the addition of nonreactive salt NaCl to the solution.

As can be seen from Fig. 2, the dependence of log k_q on $\sqrt{\mu }$ is linear at $\sqrt{\mu }$ <0.5. At higher ionic strength values, the dependence “overcomes the limit” at the value k_q = 8 × 10⁸ M⁻¹ s⁻¹, designated as the quenching constant at “infinite” ionic strength.

Fig. 2.

Dependence of the rate constant of eosin Y phosphorescence quenching by cytochrome c (k_q) on the ionic strength (μ) of the solution

Reacting prosthetic groups are near or within van der Waals contact during the electron transfer. The observed rates of the reaction of eosin Y with cytochrome c strongly depend on ionic strength, which is consistent with the importance of electrostatic interactions for intermolecular electron transfer.

Proceeding from the fact that under the experimental conditions z₁ for eosin Y is − 2, the value of the effective charge of cytochrome c was determined from the slope of the linear section of this dependence, which was + 0.79. The obtained result shows that charges under experimental conditions create an electrostatic field equivalent to only one positive charge located in the heme region participating in the catalytic act. This approach makes it possible to assess the role of local charges of cytochrome c and charges in the active centers of other proteins—redox partners of cytochrome c—on the efficiency of their interactions.

Intermolecular interactions between eosin Y and fullerene derivatives in aqueous solution

Phosphorescence quenching experiments provided a highly sensitive and reliable assay for fullerene derivatives’ interactions with charged molecules. It is known, that fullerene cores have low-lying triplet level and are effective quenchers of other triplet-excited molecules [48]. The efficiency of phosphorescence quenching, determined by values of triplet states quenching rate constants (k_q), depends on the nature of the triplet label and the quencher, including their charges, on the solvent polarity, solvent viscosity, and temperature [45, 46]. Using this effect, we investigated the role of electrostatic charges of fullerene addends on the diffusive interaction of fullerene core with negatively charged eosine Y. Thus, the kinetics of phosphorescence decay indicated the rate of fullerene derivatives interacting with eosin Y. Comparison of the quenching rate constants in a buffer shows a clear dependence of the quenching efficiency on the electrostatic charges of eosin Y and fullerene derivatives. Derivatives 1, 2, and 4 have five negatively charged carboxylic groups. Compound 3 is a polycarboxylic derivative of [70]fullerene with eight carboxylic groups. Compound 5 is a complex mixture of sodium fullerenolates. Compound 6 represents a quaternized aminofullerene, and it has five positively-charged functional groups.

From the data given in Table 1, it can be seen that quenching of the phosphorescence of eosin Y (which has two negative charges in aqueous solutions at neutral pH) by fullerene derivatives 1–5, which also have negative charges, occurs with bimolecular rate constants on the order of ~ 10⁶–10⁷ M^−ls⁻¹, which is only two orders of magnitude slower than the diffusion-limited Smoluchowski rate.

Table 1.

Rate constants for eosin Y phosphorescence quenching by fullerene derivatives in Tris–HCl buffer (pH = 7.2, 0.02 M)

Compound	Kq × 107 M−1 s−1
1	3.1 ± 0.17
2	0.9 ± 0.04
3	0.2 ± 0.02
4	9.7 ± 0.42
5	3.9 ± 0.17
6	90 ± 3.70

It should be considered that most of the fullerene derivatives have charges located on the ends of hydrocarbon chains, attached to the fullerene core. These hydrocarbon chains attached to the fullerene core can interact both with the fullerene core and each other, thereby fixing charges either evenly around the fullerene or on one side of the core, at a distance of 8–10 Å. As a result, depending on the nature of functional groups, electrostatic repulsion on the quenching of eosin Y phosphorescence by fullerene derivatives can vary considerably, which showed good agreement with experiments.

The essential role of electrostatic interactions in the quenching of eosin phosphorescence by fullerene derivatives is also confirmed by the fact that for compound 6, which has five cationic functional groups, the quenching process differs notably. With the addition of even low concentrations of 6, the phosphorescence amplitude of eosin decreased. This effect can be explained by forming an eosin Y/6 complex due to electrostatic interactions, resulting in effective eosin Y phosphorescence quenching in the complex. As a result, the quenching rate constant k_q for derivative 6 was about 10⁹ M⁻¹ s⁻¹, close to the Smoluchowski limit.

The effect of PFDs on the electron transfer reaction between cytochrome c and cytochrome c oxidase

The electron transfer between cytochrome c and cytochrome c oxidase plays a crucial role in cellular metabolism. Cytochrome c oxidase active site has the overall negative electrostatic potential. Consequently, the positively charged lysine side chains at the binding interface of cytochrome c attracted electrostatically to the electron entry site of cytochrome c oxidase [48]. Thus, it is expected that fullerene derivatives having hydrophobic and polar fragments can modulate mitochondrial energy production.

Cytochrome c oxidase activity in mouse brain mitochondria was measured spectrophotometrically by following the oxidation of reduced cytochrome c at 550 nm (extinction coefficient 19.2 mM⁻¹ cm⁻¹) [49]. Specific rates were calculated by the estimation of first-order rate constants. Figure 3 illustrates semilogarithmic plots of the ferrocytochrome c concentration as a function of time in the presence of compounds 2 and 6.

Fig. 3.

The kinetics of oxidation of ferrocytochrome c by COX in mouse brain mitochondria were measured spectrophotometrically (at 550 nm). Linear regression relationships are shown for reactions carried out at 10 μM of compounds 2 (blue) and 6 (red). Control—probe without any tested compounds (black)

It was found that anionic fullerene derivatives 1–5 inhibit cytochrome c-oxidase at a concentration of 10 µM by 38–53% (Table 2). The addition of cationic compound 6 at the same concentration leads to complete inhibition of the enzyme. The rate constants of enzymatic oxidation of cytochrome c and the percentage of cytochrome c oxidase inhibition in the presence of studied derivatives are presented in Table 2.

Table 2.

Effect of studied compounds 1–6 on cytochrome c oxidase

Compound	k × 102 s−1	COX inhibition, %
Control	0.89 ± 0.0001	0
1	0.51 ± 0.0002	43
2	0.42 ± 0.0002	53
3	0.45 ± 0.0002	50
4	0.30 ± 0.0003	43
5	0.55 ± 0.0001	38
6	0.02 ± 0.0001	100

It should be noted that compound 6, which caused complete inhibition of the cytochrome c oxidase activity, has the highest value of the rate constant for eosin Y phosphorescence quenching. This indicates an essential role of electrostatic interactions in the interaction of 6 with an enzyme molecule.

Thus, the interaction between cytochrome c and cationic fullerene derivative 6 is accompanied by electrostatic repulsion between the positively charged chains of PFD and lysine residues on cytochrome c. In contrast, the interaction between cytochrome c and anionic fullerene derivatives 1–5 is promoted by intermolecular charge pairs formed between positively charged residues on cytochrome c and anionic chains of PFDs. The cationic fullerene derivatives used in this study represent a new class of molecules potentially able to modulate the enzymatic activity of cytochrome c oxidase.

Discussion

It is known that the functionalization of fullerenes affects their ability to form aggregates in solution. Polyfunctionalized fullerenes are more stable in solution, and their aggregation is limited [50]. For some water-soluble fullerene derivatives, such as fullerenols, cytotoxicity decreases with an increase in the number of functional groups covalently attached to the fullerene core due to a decrease in their ability to generate reactive oxygen species [51]. The toxicity of fullerene derivatives is strongly influenced by their ability to penetrate the cell membrane. At the same time, cationic derivatives, which penetrate the cell membrane easily, showed a cytotoxic effect in the micromolar concentration range, while compounds with negative charges on addends did not show any cytotoxicity at concentrations < 80 μM [52].

Cytotoxicity of the investigated compounds has been studied previously in several cell lines. The experimental CC50 value for 1 in CEM cell culture (> 63 μM) [5]. Cytotoxic concentrations of compound 1 in cell lines L1210, HeLa, and OST TK- were in the range of 6–60 μM [30]. Although the CC50 values in cell lines for compound 2 have not been published, it demonstrated quite low acute toxicity in vivo (LD50 = 300 mg/kg) [21]. It has also been shown that compound 2 did not decrease the viability of the neural stem cells (NSCs) and C6 cells at the concentration of 100 nM [10]. Compound 3 has shown relatively low cytotoxicity (> 41 μM in MDCK, HEL, HeLa, and CRFK cell lines and > 86 μM in the CEM cell line) [28]. Compound 4 had very low 1-day cytotoxicity (> 1175 μM) in Vero cells [29]. Compound 5 has shown no toxic effects in the wide range of tested cell lines (L1210, FM3A, CEM, HeLa, HEp-2, HEL, MDCK, CRFK, Vero) in concentrations of up to 0.1 mg/ml, which corresponds to the value of approximately 75 μM [30]. In contrast, compound 6 has shown relatively high in vivo acute toxicity (intraperitoneal injection in mice). The maximal tolerable dose for compound 6 was found to be around 50 mg/kg, while the median lethal dose LD50 was about 65 mg/kg [31]. The minimum cytotoxic concentration (MCC) for fullerene derivative 6 was 4 μg/ml in the Vero cell line (unpublished data obtained by Prof. J. Balzarini, KU Leuven).

In summary, the anionic fullerenes demonstrated moderate to low cytotoxicity and relatively low acute toxicity. In contrast, the cationic [60]fullerene derivative was quite cytotoxic and showed rather high acute toxicity as well.

Water-soluble fullerene derivatives are good candidates for various biological applications such as anticancer or antimicrobial therapy, cytoprotection, enzyme inhibition, and many others. Their toxicity, both in tissue culture and in vivo, is a critical characteristic for development and restriction of these applications. Here, we intend to understand how such factors as electrostatic charges of fullerene derivatives can affect their interactions with charged redox proteins like cytochrome c and cytochrome c oxidase—the terminal enzyme in the electron transfer chain. Since there is a strong electrostatic interaction between eosin Y and cytochrome c and PFDs, we suggest that electrostatic charges of studied fullerene derivatives can affect on their binding to cytochrome c. The effect of binding of fullerene derivatives to cytochrome c has already been considered as a possible cytoprotection mechanism in various diseases and toxic insults [44]. The essential role of electrostatic interactions in this process was shown: in contrast to cationic, anionic fullerene derivatives could bind to cytochrome c with high affinity [44]. When leaving the mitochondria into the surrounding cytoplasm, cytochrome c triggers a cascade of reactions ending in apoptosis [53]. The interruption of electron flow to cytochrome c oxidase increases the generation of reactive oxygen species: superoxide, H₂O₂, peroxynitrite, and hydroxyl radical. The activation of caspases via a mitochondrion-dependent pathway has been supposed in nonapoptotic differentiation programs that affect nucleated and enucleated cells [53]. Thus, cytochrome c release induces caspase activation, promoting cell death or vital processes like differentiation and proliferation. Therefore, the ability of fullerene derivatives, especially anionic PFDs, to bind cytochrome c may be essential for the normal functioning of cells.

As well it is important to consider the ability of fullerene derivatives to inhibit cytochrome c oxidase. Cytochrome oxidase is an important regulatory enzyme of the electron transport chain. The importance of cytochrome c oxidase is confirmed by the number of regulatory mechanisms of its activity. Disturbance of any of these regulatory mechanisms can lead to a pathological condition. Change in cytochrome oxidase activity directly or indirectly affects all mitochondrial functions; therefore, some diseases are associated with a reduced content or altered activity of this enzyme [54]. Under normal physiological conditions, cytochrome c oxidase acts as the rate-limiting step of the respiratory chain, and its activity is a useful indicator of the total cellular metabolic capacity [55]. Many studies suggest that cytochrome c oxidase dysfunction is associated with increased mitochondrial reactive oxygen species production and cellular toxicity. The present study shows that cationic fullerene derivative 6 inhibits cytochrome c oxidase completely, unlike anionic derivatives 1–5. It confirms the essential role of electrostatic interactions in the interaction of PFDs with an enzyme molecule. Such a phenomenon explores how cationic fullerene derivatives play a role in mitochondrial dysfunction, oxidative stress, and cytotoxicity.

Conclusions

In summary, we have shown that water-soluble polysubstituted fullerene derivatives can effectively influence the process of electron transfer to cytochrome c oxidase. It was shown that electrostatic charges of cytochrome c affect reactions with eosin Y anionic molecules in an aqueous solution. Anionic fullerene derivatives 1–5 inhibit cytochrome c oxidase by 38–53%, probably due to binding to cytochrome c. On the other hand, the cationic compound 6 at the same concentration inhibits the enzyme completely. Furthermore, compound 6 has the highest value of the rate constant for eosin Y phosphorescence quenching, which indicates an essential role of electrostatic interactions in the interaction of 6 with an enzyme molecule.

Author contribution

Conceptualization, methodology, D.A.P., A.I.K.; synthesis, O.A.K., P.A.T.; experimental investigation, D.A.P., I.I.F.; writing—original draft preparation, D.A.P.; writing—review and editing, D.A.P., R.A.K., A.I.K.; supervision, D.A.P.

Funding

The work was supported by The Ministry of Science and Higher Education of the Russian Federation, state tasks AAAA-A19-119071890015–6 and AAAA-A19-119112590105–7. Synthesis of highly water-soluble polysubstituted derivatives of [60]fullerene and [70]fullerene was supported by the Russian Science Foundation (RSF project No. 22–43-08005, https://rscf.ru/project/22-43-08005/).

Declarations

Ethics approval

All the animal experiments were performed according to the compliance with the EU Directive 2010/63/EU for animal experiments and the Russian law regulating experiments on animals and were approved by the Ethical Committee of IPCP RAS.

Consent to participate

All authors read and accepted the final version of the manuscript submitted for publication.

Conflict of interest

The authors declare no competing interests.