Molecular insights into the interaction between cytochrome c and carbon nanomaterials

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.