Pyruvate formate-lyase activating enzyme (PFL-AE) is a representative member of an emerging family of enzymes that utilize iron-sulfur clusters and S-adenosylmethionine (AdoMet) to initiate radical catalysis. Although these enzymes have diverse functions, evidence is emerging that they operate by a common mechanism in which a [4Fe-4S](+) interacts with AdoMet to generate a 5'-deoxyadenosyl radical intermediate. To date, however, it has been unclear whether the iron-sulfur cluster is a simple electron-transfer center or whether it participates directly in the radical generation chemistry. Here we utilize electron paramagnetic resonance (EPR) and pulsed 35 GHz electron-nuclear double resonance (ENDOR) spectroscopy to address this question. EPR spectroscopy reveals a dramatic effect of AdoMet on the EPR spectrum of the [4Fe-4S](+) of PFL-AE, changing it from rhombic (g = 2.02, 1.94, 1.88) to nearly axial (g = 2.01, 1.88, 1.87). (2)H and (13)C ENDOR spectroscopy was performed on [4Fe-4S](+)-PFL-AE (S = (1)/(2)) in the presence of AdoMet labeled at the methyl position with either (2)H or (13)C (denoted [1+/AdoMet]). The observation of a substantial (2)H coupling of approximately 1 MHz ( approximately 6-7 MHz for (1)H), as well as hyperfine-split signals from the (13)C, manifestly require that AdoMet lie close to the cluster. (2)H and (13)C ENDOR data were also obtained for the interaction of AdoMet with the diamagnetic [4Fe-4S](2+) state of PFL-AE, which is visualized through cryoreduction of the frozen [4Fe-4S](2+)/AdoMet complex to form the reduced state (denoted [2+/AdoMet](red)) trapped in the structure of the oxidized state. (2)H and (13)C ENDOR spectra for [2+/AdoMet](red) are essentially identical to those obtained for the [1+/AdoMet] samples, showing that the cofactor binds in the same geometry to both the 1+ and 2+ states of PFL-AE. Analysis of 2D field-frequency (13)C ENDOR data reveals an isotropic hyperfine contribution, which requires that AdoMet lie in contact with the cluster, weakly interacting with it through an incipient bond/antibond. From the anisotropic hyperfine contributions for the (2)H and (13)C ENDOR, we have estimated the distance from the closest methyl proton of AdoMet to the closest iron of the cluster to be approximately 3.0-3.8 A, while the distance from the methyl carbon to the nearest iron is approximately 4-5 A. We have used this information to construct a model for the interaction of AdoMet with the [4Fe-4S](2+/+) cluster of PFL-AE and have proposed a mechanism for radical generation that is consistent with these results.

Pyruvate formate-lyase activating enzyme (PFL-AE) generates the catalytically essential glycyl radical on pyruvate formate-lyase via the interaction of the catalytically active [4Fe-4S]+ cluster with S-adenosylmethionine (AdoMet). Like other members of the Fe-S/AdoMet family of enzymes, PFL-AE is thought to function via generation of an AdoMet-derived 5'-deoxyadenosyl radical intermediate; however, the mechanistic steps by which this radical is generated remain to be elucidated. While all of the members of the Fe-S/AdoMet family of enzymes appear to have a unique iron site in the [4Fe-4S] cluster, based on the presence of a conserved three-cysteine cluster binding motif, the role of this unique site has been elusive. Here we utilize 35-GHz pulsed electron nuclear double resonance (ENDOR) studies of the [4Fe-4S]+ cluster of PFL-AE in complex with isotopically labeled AdoMet (denoted [1+/AdoMet]) to show that the unique iron serves to anchor the AdoMet for catalysis. AdoMet labeled with 17O at the carboxylate shows a coupling of A = 12.2 MHz, consistent with direct coordination of the carboxylate to the unique iron of the cluster. This is supported by 13C-ENDOR with the carboxylato carbon labeled with 13C, which shows a hyperfine coupling of 0.71 MHz. AdoMet enriched with 15N at the amino position gives rise to a spectrum with A(15N) = 5.8 MHz, consistent with direct coordination of the amino group to a unique iron of the cluster. Together, the results demonstrate that the unique iron of the [4Fe-4S] cluster anchors AdoMet by forming a classical N/O chelate with the amino and carboxylato groups of the methionine fragment.

Glutaredoxin 5 (GLRX5) deficiency has previously been identified as a cause of anemia in a zebrafish model and of sideroblastic anemia in a human patient. Here we report that GLRX5 is essential for iron-sulfur cluster biosynthesis and the maintenance of normal mitochondrial and cytosolic iron homeostasis in human cells. GLRX5, a mitochondrial protein that is highly expressed in erythroid cells, can homodimerize and assemble [2Fe-2S] in vitro. In GLRX5-deficient cells, [Fe-S] cluster biosynthesis was impaired, the iron-responsive element-binding (IRE-binding) activity of iron regulatory protein 1 (IRP1) was activated, and increased IRP2 levels, indicative of relative cytosolic iron depletion, were observed together with mitochondrial iron overload. Rescue of patient fibroblasts with the WT GLRX5 gene by transfection or viral transduction reversed a slow growth phenotype, reversed the mitochondrial iron overload, and increased aconitase activity. Decreased aminolevulinate δ, synthase 2 (ALAS2) levels attributable to IRP-mediated translational repression were observed in erythroid cells in which GLRX5 expression had been downregulated using siRNA along with marked reduction in ferrochelatase levels and increased ferroportin expression. Erythroblasts express both IRP-repressible ALAS2 and non-IRP-repressible ferroportin 1b. The unique combination of IRP targets likely accounts for the tissue-specific phenotype of human GLRX5 deficiency.