Nitric oxide and the post- transcriptional control of cellular iron traffic Nitric oxide (NO) is a small, labile and highly reactive molecule generated in various cells by NO synthases. Several important biological functions are controlled by this messenger, and recent data suggest a novel direct role for NO in post-transcriptional gene regulation mediated by iron regulatory protein (IRP). IRP is a cytoplasmic protein that coordinates cellular iron traffic by binding to iron.responsive elements in mRNAs encoding proteins involved in iron uptake, storage and utilization. NO activates the RNA. binding activit7 of this protein and in this regard mimics the consequences of iron starvation. Cell biological and biochemical data on the function,s of NO and IRP suggest a mechanistic basis for these findings and raise the question of thetr biological implications. [I I1~ Mill Kostas Pantopoulos and Matthias Hentze are at the Gene Expression Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, D.69117 Heidelberg, Germany; and G(inter Weiss is at the Deptof Internal Medicine, University of Innsbruck, A-6020 Innsbruck, Austria. Nitric oxide (NO) Is Involved in diverse processes in various differentiated cells, rangtng from signal trans- duction in the brain and regulation of tone in the vascular system to cytotoxlclty of stimulated macro. phages In the Immune system (for reviews see Refs 1-3). The enzyme NO synthase (NOS~ catalyses NO synthesis |'tom the amino acid L-arglnlne. Different lsoforms of NOS have been characterized and cloned from various tissues, including brain neurons, the endothelium and macrophages (reviewed in Refs 2 and 3). In general, NOSs are classified Into 'consti- tutive' and 'cytoktne-lnduclble' forms ~<~, While the brain and endothelial NOSs are constitutively expressed and their activity is modulated by vari. attons In cellular Ca 2÷ concentrations, the routine macrophage enzyme Is Ca 2. Independent and tran. scriptlonally induced following treatment of animals with bacterial endotoxin or stimulation of cultured macrophages with interferon y and lipopolysac- charide. All NOSs bind haem, FMN, FAD, NADPH and calmodulin, and require tetrahydrobiopterin as a cofactor for catalysis. Biological targets of NO include m~tochondrial aconitase NO reacts with molecular oxygen 4, transition metal ions s, free radicals 6, the superoxide anion 7, thiol groups 8, and also with haem or non-haem iron in metalloproteins (for protein targets of NO see Table 1). For example, iron-nitrosyl complexes are readily formed between NO and the haem groups in haemoglobin or myoglobin. Such complexes are paramagnetic and electron paramagnetic resonance spectroscopy has proven to be a powerful tool with which to study them 9A°. Our understanding of the biological targets of NO has expanded over recent years. Proteins containing either haem iron or iron-sulphur clusters are among the best-studied examples. NO-mediated effects in the brain and in blood vessels result from the acti- vation of a guanylate cyclase by binding of NO to the haem iron of its catalytic site. This interaction is thought to cause an allosteric switch that results in cGMP synthesis and the onset of a cascade of sub- sequent reactions s. By contrast, the NO released by activated macrophages is thought to mediate their cytotoxic effects by diffusing into the target cells, and causing iron loss 11,12and shut-off of essential meta- bolic functions such as DNA synthesis 13, mitochon- drial respiration TM and the citric acid (Krebs) cycle ~s. These effects have been attributed to the inactivation of ribonucleotide reductase (the NO target is thought to be a tyrosyl radical 6) as well as to inhibition of the iron-sulphur proteins NADH:ubtqulnone oxido- reductase, NADH :succlnate oxidoreductase and mitochondrlal aconltase ~6-1~. Of this list, mltochondrlal aconitase is particularly Interesting. it catalyses the conversion of citrate to isocltrate In the Krebs cycle and thus fulfils an essen- thd cellular function. Its active site contains a 14Fe-4SI cluster, In which only three of the four Fe atoms are stably coordinated with cysteines of the polypeptlde backbone and the fourth (Fe~) is labile. The catalytic mechanism Involves direct binding of citrate to Fe a (Ref. 19) and the enzymatic activity can be modulated in vitro by lnterconverslon of the cluster between the catalytically Inactive [3Fe-4SI and the active [4Fe--4Sl form. Several observations suggest that the status of this Fe-S cluster may be directly affected by NO: (1) aconltase is Inactivated in guinea pig L10 hepatoma and murlne L1210 lym- phoblastlc leukaemla cells that are co.cultlvated with stimulated macrophages and this correlates with removal of Fe a (Ref. IS); (2) a similar effect is observed after administration of NO gas to L10 cells2" or rat hepa~ocytes2~; (3) after exposure of LI210 cells to NO from activated macrophages, iron-nitrosyl com- plexes are detected In extracts of both cell types, accompanied by loss of aconitase activlty2Z; and (4) Induction of NOS In cultured mouse pancreatic islets or treatment of the cells with NO-generating drugs results in aconitase inactivation za. A cytoplasmic homologue of mitochondrial acon- itase was identified more than 20 years ago z4, but until recently its physiological role remained a mystery. A series of experiments zs-ao has now re- vealed this enzyme to be identical to iron regulatory 82 0 1994 ElsevierScienceLtd 0962.8924/04/$07.00 TRENDS IN CELL BIOLOGY VOL. 4 MARCH 1994