Open AccessThe mechanism of nitrogen monoxide (NO)‐mediated iron mobilization from cells
Author(s) -
Watts Ralph N.,
Richardson Des R.
Publication year - 2002
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2002.02987.x
Subject(s) - ferritin , glutathione , chemistry , biophysics , cytotoxic t cell , biochemistry , biology , enzyme , in vitro
Nitrogen monoxide (NO) is a cytotoxic effector molecule produced by macrophages that results in Fe mobilization from tumour target cells which inhibits DNA synthesis and mitochondrial respiration. It is well known that NO has a high affinity for Fe, and we showed that NO‐mediated Fe mobilization is markedly potentiated by glutathione (GSH) generated by the hexose monophosphate shunt [Watts, R.N. & Richardson, D.R. (2001) J. Biol. Chem . 276 , 4724–4732]. We hypothesized that GSH completes the coordination shell of an NO–Fe complex that is released from the cell. In this report we have extended our studies to further characterize the mechanism of NO‐mediated Fe mobilization. Native PAGE 59 Fe‐autoradiography shows that NO decreased ferritin‐ 59 Fe levels in cells prelabelled with [ 59 Fe]transferrin. In prelabelled cells, ferritin‐ 59 Fe levels increased 3.5−fold when cells were reincubated with control media between 30 and 240 min. In contrast, when cells were reincubated with NO, ferritin‐ 59 Fe levels decreased 10‐fold compared with control cells after a 240‐min reincubation. However, NO could not remove Fe from ferritin in cell lysates. Our data suggest that NO intercepts 59 Fe on route to ferritin, and indirectly facilitates removal of 59 Fe from the protein. Studies using the GSH‐depleting agent, l ‐buthionine‐( S , R )‐sulphoximine, indicated that the reduction in ferritin‐ 59 Fe levels via NO was GSH‐dependent. Competition experiments with NO and permeable chelators demonstrated that both bind a similar Fe pool. We suggest that NO requires cellular metabolism in order to effect Fe mobilization and this does not occur via passive diffusion down a concentration gradient. Based on our results, we propose a model of glucose‐dependent NO‐mediated Fe mobilization.