MUTATIONAL ANALYSIS OF THE [4FE-4S]-CLUSTER CONVERTING IRON REGULATORY FACTOR FROM ITS RNA-BINDING FORM TO CYTOPLASMIC ACONITASE

The control of cellular iron homeostasis involves the coordinate post-transcriptional regulation of ferritin mRNA translation and transferrin receptor mRNA stability. These regulatory events are mediated by a soluble cytoplasmic protein, iron regulatory factor (IRF), which binds specifically to mRNA hairpin structures, termed iron-responsive elements (IREs), in the respective mRNAs. IRF is modulated by variations of cellular iron levels and exists as either an apo-protein or a [4Fe - 4S]-cluster protein. The two conformations show distinct, mutually exclusive functions. High-affinity IRE binding is observed with the apo-form induced by iron deprivation, but is lost under high iron conditions when IRF is converted to the [4Fe - 4S]-cluster form which shows cytoplasmic aconitase activity. Moreover, IRE binding is inactivated by the sulfhydryl-oxidizing agent diamide and fully activated in vitro by 2% 2-mercaptoethanol, whereas alkylation of IRF inhibits IRE binding. In the present study, we analyzed each of the above features using site-directed mutants of recombinant human IRF. The results support the bifunctional nature of IRF. We conclude that cysteines 437, 503 and 506 anchor the [4Fe - 4S]-cluster, and are essential to the aconitase activity. Mutagenesis changing any of the cysteines to serine leads to constitutive RNA binding in 0.02% 2-mercaptoethanol. Cysteine 437 is particularly critical to the RNA - protein interaction. The spontaneous or diamide-induced formation of disulfide bonds between cysteines 437 and 503 or 437 and 506, in apo-IRF, as well as its alkylation by N-ethylmaleimide, inhibit binding to the IRE. Unlike wild-type human IRF, the cysteine mutants are not regulated in their IRE-binding activity by cellular iron levels in transiently transfected mouse L cells. Thus, our data provide direct evidence for the role of the [4Fe - 4S]-cluster insertion in modulating the RNA - protein interaction of this unique post-transcriptional regulatory system and explain the in vitro effects on IRE - IRF binding by oxidation/reduction of cysteines.