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

Cytochrome b(5) 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 b(5) 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 b(5) 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 b(5) and ferricyanide. Upon complex formation with cytochrome b(5) 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 b(5) 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 b(5) reductase.