Molecular insights into the interaction between cytochrome c and carbon nanomaterials

Abstract

Carbon nanomaterials (CNMs) are a heterogeneous class of advanced materials. Their widespread use is associated with human safety concerns, which can be addressed by safe-by design strategies. This implies a deep knowledge of how physico-chemical properties drive biological effects. The ability of CNMs to interact with cytochrome c (cyt c), a heme-protein playing a key role in the respiratory chain, in apoptosis and in cellular redox homeostasis, has been reported in some studies. However, the consequences of this interaction on the cyt c functions are controversial. Here the mechanism of interaction of carbon nanoparticles (CNPs), chosen as model of redox-active CNMs, with cyt c has been studied with the aim to shed light into these discrepancies. The effect of CNPs on the redox state of cyt c was monitored by UV-vis spectroscopy and 1D ¹H NMR, while the effect on the primary, secondary, and tertiary cyt c structure was investigated by FIA/LC-MS and Circular Dichroism (CD). Finally, the peroxidase activity of cyt c and the involvement of superoxide radicals was evaluated by EPR spectroscopy. We demonstrate the existence of two mechanisms, one leading to the suppression of the cyt c peroxidase activity following the NADH-independent reduction of the heme-iron, and the other resulting in the irreversible protein unfolding. Overall, the results suggest that these two processes might be independently modulated by redox and surface properties respectively.

Keywords: Carbon nanomaterials; Conformational changes; Cytochrome c; Electron transfer; Heme iron; Peroxidase activity.

Graphical abstract

Fig. 1

Size of CNPs. A) Representative FESEM image; B) FESEM (red) and NTA (black) size distribution. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

A) UV–Vis spectra obtained following incubation of Fe(III) cyt c (12 μM) and CNPs (0.5 mg/mL) in PBS (10 mM, pH 7.4) for 10 min compared with the spectra recorded on 12 μM Fe(III) cyt c, 12 μM Fe(II) cyt c and 0.5 mg/mL CNPs; All solutions were stirred for 10 min in air; B) UV–Vis spectra at different time points obtained following incubation of Fe(III) cyt c (120 μM) with the solution obtained by filtrating the suspension of CNPs (5 mg/mL) through a 3 kDa dialysis membrane; C) Overlap of the 1D ¹H NMR spectra of the paramagnetic species of cyt c: green spectrum corresponds to cyt c (120 μM) in presence of CNPs (5 mg/mL), violet spectrum corresponds to Fe(II) cyt c and brown spectrum corresponds to oxidized Fe(III) cyt c. D) Amount of Fe(II) cyt c formed expressed as absorbance ratio (550 nm/540 nm) following incubation of Fe(III) cyt c (12 μM) with CNPs (0.5 mg/mL) in PBS (10 mM, pH 7.4) in air, under nitrogen atmosphere and by fluxing oxygen; The amount of Fe(II) cyt c formed with ascorbic acid in air and under oxygen flux are reported for comparison (A.A.: ascorbic acid); bars represent the mean value ± standard deviation. E) Crystal structure of cyt c. Colour indicate the coulombic electrostatic potential, red negative, white to blue positive; F) EPR spectra recorded on a) a suspension of CNPs (0.34 mg/mL) and DEPMPO (1 mM) in PBS 10 mM, pH 7.4 after 10 min of incubation; b) a solution containing xanthine 2.2 mM, xanthine oxidase 0.02 U/mL, NaOH 2.2 mM and DEPMPO 50 mM c) Simulation of the EPR spectra in b). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Effect of CNPs on the conformation of cyt c. A) CD spectrum in the far-UV region; B) CD spectrum in the visible region; the electronic spectra is reported in B′ for comparison; C) CD spectrum in the near-UV region; D) Crystal structure of cyt c, relevant amino acid residues are labelled.

Fig. 4

A) Scheme of the generation of DMPOX catalysed by cyt c; B) EPR spectra recorded on a) a solution of Fe(III) cyt c (50 μM) in the presence of 0.5 mM H₂O₂ and 22 mM DMPO; b–g: EPR spectra recorded on a solution of Fe(III) cyt c (50 μM) H₂O₂ (0.5 mM) and DMPO (22 mM) and increasing concentration of CNPs (3.1–62 μg/mL). h) Simulation of the EPR spectra in a). C) Amount of Fe(II) cyt c formed expressed as absorbance ratio (550 nm/540vnm) following 4 cycles of incubation of Fe(III) cyt c (120 μM) and CNPs (5 mg/mL). D) UV–Vis spectra obtained following incubation of Fe(III) cyt c (120 μM) and CNPs (5 mg/mL) pre-treated with serum albumin or foetal bovine serum at two different concentrations (10 and 80 %) in PBS (10 mM, pH 7.4) for 10 min.