Human erythrocytes exposure to juglone leads to an increase of superoxide anion production associated with cytochrome b5 reductase uncoupling

Cytochrome b5 reductase is an enzyme with the ability to generate superoxide anion at the expenses of NADH consumption. Although this activity can be stimulated by cytochrome c and could participate in the bioenergetic failure accounting in apoptosis, very little is known about other molecules that may uncouple the function of the cytochrome b5 reductase. Naphthoquinones are redox active molecules with the ability to interact with electron transfer chains. In this work, we made an inhibitor screening against recombinant human cytochrome b5 reductase based on naphthoquinone properties. We found that 5-hydroxy-1,4-naphthoquinone (known as juglone), a natural naphthoquinone extracted from walnut trees and used historically in traditional medicine with ambiguous health and toxic outcomes, had the ability to uncouple the electron transfer from the reductase to cytochrome b5 and ferricyanide. Upon complex formation with cytochrome b5 reductase, juglone is able to act as an electron acceptor leading to a NADH consumption stimulation and an increase of superoxide anion production by the reductase. Our results suggest that cytochrome b5 reductase could contribute to the measured energetic failure in the erythrocyte apoptosis induced by juglone, that is concomitant with the reactive oxygen species produced by cytochrome b5 reductase.This work was supported by the Associate Laboratory for Green Chemistry-LAQV which is financed by national funds from FCT/MCTES (UID/QUI/50006/2019). Experimental work was also supported by funding from Ayuda a Grupos de la Junta de Extremadura (Group GR18118) co-financed by the European Funds for Structural Development (FEDER). The authors also thank FCT for funds to GHTM (UID/Multi/04413/2013) and AKSA acknowledges FCT/MCTES for their “Investigador Doutorado” contracts' funding and signed with FCT/UNL in accordance with DL 57/2016 e Lei 57/2017.Peer reviewe