Roselite from the Aghbar Mine, Morocco, [Ca2(Co2+,Mg)(AsO4)2 2H2O], was investigated by X-ray Photoelectron and Raman spectroscopy. X-ray Photoelectron Spectroscopy revealed a cobalt to magnesium ratio of 3:1. Magnesium, cobalt and calcium showed single bands associated with unique crystallographic positions. The oxygen 1s spectrum displayed two bands associated with the arsenate group and crystal water. Arsenic 3d exhibited bands with a ratio close to that of the cobalt to magnesium ratio, indicative of the local arsenic environment being sensitive to the substitution of magnesium for cobalt. The Raman arsenate symmetric and antisymmetric modes were all split with the antisymmetric modes observed around 865 and 818 cm−1, while the symmetric modes were found around 980 and 709 cm−1. An overlapping water-libration mode was observed at 709 cm−1. The region at 400–500 cm−1 showed splitting of the arsenate antisymmetric mode with bands at 499, 475, 450 and 425 cm−1. The 300–400 cm−1 region showed the corresponding symmetric bending modes at 377, 353, 336 and 304 cm−1. The bands below 300 cm−1 were assigned to lattice modes.

Layered clay systems intercalated with inorganic and organic compounds were analyzed to highlight how XPS can provide information on the different environments surrounding a particular atom as well as provide discernments on the size, coordination, and structural and oxidative transformations of the intercalating/pillaring compounds. XPS data on the intercalation of urea and K-acetate in low- and high-defect kaolinite revealed the interaction of the intercalating group NH2 with the siloxane functional groups in the interlayer surface. The intercalation of HDTMA in Mt demonstrated the use of XPS in monitoring the change in conformation assumed by alkylammonium intercalating compounds in Mt with increasing CEC. Studies on the pillaring of Mt by Al13 and Ga13 by XPS allowed determination of the coordination of the pillaring compound within the Mt layer. Lastly, the intercalation of hexacyanoferrate in hydrotalcite demonstrated the capability of XPS in following changes in the oxidation state of the iron compound. These were gleaned from interpretation of the shifts in binding energies and presence of multiplet splitting in the XPS of the component elements of the minerals or the intercalating compounds.

Electron Paramagnetic Resonance (EPR) and Electron Nuclear Double Resonance (ENDOR) spectroscopies are extremely powerful and versatile methods for the characterization of paramagnetic systems in biology, chemistry and physics. For iron centers in the radical SAM enzymes however, Mossbauer spectroscopy has proven to be both powerful and useful as a complementary spectroscopic technique in determining not just the oxidation states but also the type of iron species present in the catalytic center. The cluster content of the radical SAM protein, Pyruvate Formate-Lyase-Activating Enzyme (PFL-AE), was characterized using EPR and Mossbauer techniques while additional ENDOR analysis helped determine the novel interaction of the co-substrate, SAdenosylmethionine (SAM or AdoMet) with the Fe-S cluster of PFL-AE. The anchoring role of the Fe-S cluster to the co-substrate derived from the spectroscopic data supports the mechanism where a SAM-based radical species is involved during catalysis.

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 delta, 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.

The H-cluster is a complex bridged metal assembly at the active site of [FeFe]-hydrogenases that consists of a [4Fe-4S] subcluster bridged to a 2Fe-containing subcluster with unique nonprotein ligands, including carbon monoxide, cyanide, and a dithiolate ligand of unknown composition. Specific biosynthetic gene products (HydE, HydF, andHydG) responsible for the biosynthesis of the H-cluster and the maturation of active [FeFe]-hydrogenase have previously been identified and shown to be required for the heterologous expression of active [FeFe]-hydrogenase [Posewitz, M. C., et al. (2004) J. Biol. Chem. 279, 25711-25720]. The precise roles of the maturation proteins are unknown; the most likely possibility is that they are directed at the synthesis of the entire 6Fe-containing H-cluster, the 2Fe subcluster, or only the unique ligands of the 2Fe subcluster. The spectroscopic and biochemical characterization of HydA (the [FeFe]-hydrogenase structural protein expressed in the absence of the maturation machinery) reported here indicates that a [4Fe-4S] cluster is incorporated into the H-cluster site. The purified protein in a representative preparation contains Fe (3.1 ( 0.5 Fe atoms per HydA) and S (1.8 ( 0.5 S atoms per HydA) and exhibits UV-visible spectroscopic features characteristic of iron-sulfur clusters, including a bleaching of the visible chromophore upon addition of dithionite. The reduced protein gave rise to an axial S= /2 EPR signal (g= 2.04 and 1.91) characteristic of a reduced [4Fe-4S] cluster. M€ ossbauer spectroscopic characterization of Fe-enriched HydA provided further evidence of the presence of a redox active [4Fe-4S] cluster. Iron K-edge EXAFS data provided yet further support for the presence of a [4Fe-4S] cluster in HydA. These spectroscopic studies were combined with in vitro activation studies that demonstrate that HydA can be activated by the specific maturases only when a [4Fe-4S] cluster is present in the protein. In sum, this work supports a model in which the role of the maturation machinery is to synthesize and insert the 2Fe subcluster and/or its ligands and not the entire 6Fe-containing H-cluster bridged assembly. The [NiFe]and [FeFe]-hydrogenases are widely distributed in nature and efficiently catalyze the reversible oxidation of molecular hydrogen (H2 T 2H + + 2e). The [NiFe]-hydrogenases, present in archaea and bacteria, generally function to oxidize molecular H2 and provide reducing equivalents for metabolic processes, while the [FeFe]-hydrogenases, present in bacteria and eukarya, functionmore broadly to catalyze both proton reduction andH2 oxidation (2, 3).Recently, there has been a growing interest in these metalloenzymes because of their inherent applicability in the development of renewable H2-based energy technology. The active sites for both [NiFe]and [FeFe]-hydrogenases have been determined by X-ray crystallography and are united by the presence of π acceptor CO and CN ligands, which are not common in biology. These diatomic ligands stabilize low-spin and low-valent oxidation states of the metal centers at the active sites. For the [NiFe]-hydrogenase, the active sites from a variety of different sulfate-reducing bacterial sources have been determined to consist of a Ni atom coordinated to an Fe atom via two thiolate ligands and a bridging oxygen species (4). The Ni atom is further coordinated by two cysteine ligands from the protein, while the Fe atom is coordinated to two terminal CN ligands and one terminal CO ligand. In comparison, the [FeFe]-hydrogenase active site contains a 6Fe-containing complex cluster termed the H-cluster, as determined for This work was supported by AFOSR Multidisciplinary University Research Initiative Award FA9550-05-01-0365 (J.W.P.), NASA Astrobiology Institute FundedAstrobiologyBiogeocatalysisResearchCenter Grant NNA08C-N85A (J.W.P., J.B.B., and R.K.S.), National Institutes of Health Grant GM47295 (B.H.), and National Science Foundation Grant NSF0755676 (R.K.S.). *To whom correspondence should be addressed. Phone: (406) 9947211. Fax: (406) 994-7212. E-mail: john.peters@chemistry.montana. edu. Abbreviations: CO, carbon monoxide; CN, cyanide; Fe-S, ironsulfur; HydA, HydA expressed in a genetic background devoid of HydE, HydF, and HydG; LB, Luria-Bertani; IPTG, isopropyl β-D-1thiogalactopyranoside; PMSF, phenylmethanesulfonyl fluoride; DTT, dithiothreitol; DT, sodium dithionite; EPR, electron paramagnetic resonance; EXAFS, extended X-ray absorption fine structure analysis; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; EDTA, ethylenediaminetetraacetic acid; XAS, X-ray absorption spectroscopy; HydF*, HydF containing the ligand modified 2Fe subcluster. D ow nl oa de d by A IS T I on A ug us t 4 , 2 00 9 Pu bl is he d on M ay 1 2, 2 00 9 on h ttp :// pu bs .a cs .o rg | do i: 10 .1 02 1/ bi 90 00 56 3 Article Biochemistry, Vol. 48, No. 26, 2009 6241 Clostridium pasteurianum (CpI) (5, 6) and Desulfovibrio desulfuricans (7, 8). The H-cluster consists of a [4Fe-4S] subcluster coordinated to a 2Fe subcluster via a cysteine thiolate ligand. The two Fe centers in the 2Fe subcluster are bridged via a five-atom dithiolate ligand and a CO ligand. The chemical composition of the dithiolate ligand has not yet been determined unambiguously and has been proposed to be dithiomethylether (6), propane dithiolate (7), or dithiomethylamine (8). In addition, both Fe centers contain terminal CO and CN ligands. For the presumed oxidized state of CpI, a water molecule is present at the distal Fe center in the proximity of the [4Fe-4S] subcluster. Biosynthesis and maturation of the [NiFe]-hydrogenases have been thoroughly studied, including identification of at least six gene products involved in formation of active [NiFe]-hydrogenases and the interactions between gene products during maturation. The metabolic source of diatomic CN ligands has been identified to be carbamoyl phosphate (9), whereas the metabolic source for the CO ligand is still in question. In contrast, relatively little is known concerning the biosynthesis and maturation of [FeFe]-hydrogenases. By analysis of several mutant strains of Chlamydomonas reinhardtii that are unable to produce hydrogen, the genes hydEF and hydG were discovered to be required for maturation of [FeFe]-hydrogenases (1). Subsequent expression studies revealed that formation of an active [FeFe]-hydrogenase was achieved only whenHydAwas heterologously expressed in a background of coexpressed gene products HydEF and HydG in Escherichia coli (1). In most organisms, hydEF exists as two separate genes, hydE and hydF (1), and it has been shown that the coexpression in E. coli of HydE, HydF, and HydG from Clostridium acetobutylicum with the [FeFe]-hydrogenase structural gene product from various algal and bacterial sources is sufficient to effect expression of active [FeFe]-hydrogenase (10). Deduced amino acid sequence analysis of HydE, HydF, and HydGgene products revealsHydE andHydG tobe radical-SAM Fe-S enzymes as they both have the C-X3-C-X2-C radical-SAM signature motif and HydF to be a GTPase (1, 9). Also, preliminary biochemical characterization of HydE and HydG has revealed associated SAMcleavage activity (11). In addition, it has been shown that upon reconstitution HydF binds an Fe-S cluster and exhibits GTPase activity (12). The involvement of HydE, HydF, and HydG maturation enzymes in the biosynthesis of the H-cluster was further elaborated when it was shown that HydA, expressed in a genetic background devoid of HydE, HydF, and HydG (HydA), is a stable protein capable of being activated in vitro by the aforementioned proteins (13). It was also determined that HydF behaves as a scaffold protein in which an H-cluster precursor is assembled and can be subsequently transferred to HydA, resulting in the formation of an active [FeFe]-hydrogenase (14). Both in vitro activation studies imply that cluster biosynthesis does not take place on the structural protein (HydA) and that a chemical precursor to the H-cluster is synthesized in the absence ofHydA that upon transfer toHydA results in its activation. Although no chemical precursors or intermediates to the H-cluster have yet been characterized or even identified, it can be hypothesized that HydE, HydF, and HydG are directed toward the synthesis of (1) the entire 6Fe-containing H-cluster, (2) the 2Fe subcluster of the H-cluster, or (3) the biologically unique ligands of the 2Fe subcluster of the H-cluster. The characterization of HydA provides a critical link in our understanding of this fascinating process by defining the substrate for the Hyd maturation proteins. In this study, we present spectroscopic and biochemical characterizations of HydA from C. reinhardtii to provide insights into the [FeFe]-hydrogenase maturation and Hcluster biosynthesis. HydA from the eukaryotic green algae C. reinhardtii contains only the H-cluster binding domains and represents the simplest [FeFe]-hydrogenase known. Unlike [FeFe]-hydrogenases fromCl. pasteurianum andD. desulfuricans, the [FeFe]-hydrogenases from eukaryotic green algae do not contain additional accessory Fe-S clusters with plant-type ferredoxin domains that would complicate spectroscopic characterization of the Fe-S clusters present at the active site (15-17). Our characterization of HydA from C. reinhardtii indicates that a [4Fe-4S] cluster is present in HydA and is required for in vitro activation by the HydE, HydF, and HydG maturation enzymes. Accordingly, it follows that the aforementioned maturation enzymes are not directed at the synthesis of the entire 6Fe-containing H-cluster. EXPERIMENTAL PROCEDURES Cloning and Cell Growth Conditions. HydA from C. reinhardtii was cloned into a pET Duet vector as described previously (10) and modified for the presence of an N-terminal six-histidine tag. HydA from C. reinhardtii was expressed in E. coli BL21(DE3) cells and cultivated in either 2 L flasks with a 1 L medium v

The H-cluster is a complex bridged metal assembly at the active site of [FeFe]-hydrogenases that consists of a [4Fe-4S] subcluster bridged to a 2Fe-containing subduster with unique nonprotein ligands, including carbon monoxide, cyanide, and a dithiolate ligand of unknown composition. Specific biosynthetic gene products (HydE, HydF, and HydG) responsible for the biosynthesis of the H-cluster and the maturation of active [FeFe]-hydrogenase have previously been identified and shown to be required for the heterologous expression of active [FeFe]-hydrogenase [Posewitz, M. C., et al. (2004). J. Biol. Chem. 279, 25711-25720]. The precise roles of the maturation proteins are unknown; the most likely possibility is that they are directed at the synthesis of the entire 6Fe-containing H-cluster, the 2Fe subcluster, or only the unique ligands of the 2Fe subduster. The spectroscopic and biochemical characterization of HydA(Delta EFG) (the[FeFe]-hydrogenase structural protein expressed in the absence of the maturation machinery) reported here indicates that a [4Fe-4S] cluster is incorporated into the H-cluster site. The purified protein in a representative preparation contains Fe (3.1 +/- 0.5 Fe atoms per HydA(Delta EFG)) and S2- (1.8 +/- 0.5 S2- atoms per HydA(Delta EFG)) and exhibits UV-visible spectroscopic features characteristic of iron-sulfur clusters, including a bleaching of the visible chromophore upon addition of dithionite. The reduced protein gave rise to an axial S = 1/2 EPR signal (g = 2.04 and 1.91) characteristic of a reduced [4Fe-4S](+) cluster. Mossbauer spectroscopic characterization of Fe-57-enriched HydA(Delta EFG) provided further evidence of the presence of a redox active [4Fe-4S](2+)/(+) cluster. Iron K-edge EXAFS data provided yet further support for the presence of a [4Fe-4S] cluster in HydA(Delta EFG). These spectroscopic studies were combined with in vitro activation studies that demonstrate that HydA(Delta EFG) can be activated by the specific maturases only when a [4Fe-4S] cluster is present in the protein. In sum, this work supports a model in which the role of the maturation machinery is to synthesize and insert the 2Fe subduster and/or its ligands and not the entire 6Fe-containing H-cluster bridged assembly.

Pyruvate formate-lyase activating enzyme (PFL-AE) catalyzes the generation of a catalytically essential glycyl radical on pyruvate formate-lyase (PFL). Purified PFL-AE contains an oxygen-sensitive, labile [4Fe-4S] cluster that undergoes cluster interconversions in vitro, with only the [4Fe-4S](+) cluster state being catalytically active. Such cluster interconversions could play a role in regulating the activity of PFL-AE, and thus of PFL, in response to oxygen levels in vivo. Here we report a Mossbauer investigation on whole cells overexpressing PFL-AE following incubation under aerobic and/or anaerobic conditions and provide evidence that PFL-AE undergoes cluster interconversions in vivo. After 2 h aerobic induction of PFL-AE expression, approximately 44% of the total iron is present in [4Fe-4S](2+) clusters, 6% in [2Fe-2S](2+) clusters, and the remainder as noncluster Fe(III) (29%) and Fe(II) (21%) species. Subsequent anaerobic incubation of the culture results in approximately 75% of the total iron being present as [4Fe-4S](2+) clusters, with no detectable [2Fe-2S](2+). Ensuing aerobic incubation of the culture converts the iron species nearly back to the original composition (42% [4Fe-4S](2+), 10% [2Fe-2S](2+), 19% Fe(III), and 29% Fe(II)). The results provide evidence for changes in cluster composition of PFL-AE in response to the redox state of the cell. Furthermore, the Mossbauer spectra reveal that the [4Fe-4S](2+) cluster of PFL-AE in whole cells contains a valence-localized Fe(III)Fe(II) pair which has not been previously observed in the purified enzyme. Addition of certain small molecules containing adenosyl moieties, including 5'-deoxyadenosine, AMP, ADP, and methylthioadenosine, to purified PFL-AE reproduces the valence-localized state of the [4Fe-4S](2+) cluster. It is speculated that the [4Fe-4S](2+) cluster of PFL-AE in whole cells may be coordinated by a small molecule, probably AMP, and that such coordination may protect this labile cluster from oxidative damage.

We report the generation and characterization of a diiron(III) intermediate formed during reaction with dioxygen of the reduced hydroxylase component of toluene/o-xylene monooxygenase from Pseudomonas sp. OX1. The decay rate of this species is accelerated upon mixing with phenol, a substrate for this system. Under steady-state conditions, hydrogen peroxide was generated in the absence of substrate. The oxidized hydroxylase also decomposed hydrogen peroxide to liberate dioxygen in the absence of reducing equivalents. This activity suggests that dioxygen activation may be reversible. The linear free energy relationship determined from hydroxylation of para-substituted phenols under steady-state turnover has a negative slope. A value of rho < 0 is consistent with electrophilic attack by the oxidizing intermediate on the aromatic substrates. The results from these steady and pre-steady-state experiments provide compelling evidence that the diiron(III) intermediate is the active oxidant in the toluene/o-xylene monooxygenase system and is a peroxodiiron(III) transient, despite differences between its optical and Mössbauer spectroscopic parameters and those of other peroxodiiron(III) centers.

Electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and Mössbauer spectroscopies and other physical methods have provided important new insights into the radical-SAM superfamily of proteins, which use iron-sulfur clusters and S-adenosylmethionine to initiate H atom abstraction reactions. This remarkable chemistry involves the generation of the extremely reactive 5'-deoxyadenosyl radical, the same radical intermediate utilized in B12-dependent reactions. Although early speculation focused on the possibility of an organometallic intermediate in radical-SAM reactions, current evidence points to novel chemistry involving a site-differentiated [4Fe-4S] cluster. The focus of this forum article is on one member of the radical-SAM superfamily, pyruvate formate-lyase activating enzyme, and how physical methods, primarily EPR and ENDOR spectroscopies, are contributing to our understanding of its structure and mechanism. New ENDOR data supporting coordination of the methionine moiety of SAM to the unique site of the [4Fe-4S]2+/+ cluster are presented.

Paramagnetic resonance studies have provided critical insight into the structural and mechanistic properties of the "radical-SAM" enzymes, a superfamily of enzymes which utilize iron-sulfur clusters and S-adenosylmethionine (SAM or AdoMet) to initiate radical catalysis. Here we report recent studies of pyruvate formate-lyase activating enzyme (PFL-AE), which functions to generate the catalytically essential glycyl radical of pyruvate formate-lyase (PFL). Quantitative EPR studies show that the [4Fe-4S](+) cluster on PFL-AE is the electron source required for generation of the glycyl radical on PFL. Electron-nuclear double resonance (ENDOR) studies show that the co-substrate AdoMet sits close to the [4Fe-4S](+) cluster of PFL-AE in the ES complex, with the methyl carbon approximately 4-5 Angstrom, and the methyl hydrogen approximately 3-4 Angstrom, from the closest iron of the cluster. This close association of AdoMet and the Fe-S cluster has significant implications regarding the catalytic mechanism.

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.

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.