Binding energy in the one-electron reductive cleavage of S-adenosylmethionine in lysine 2,3-aminomutase, a radical SAM enzyme

The common step in the actions of members of the radical SAM superfamily of enzymes is the one-electron reductive cleavage of S-adenosyl-l-methionine (SAM) into methionine and the 5'-deoxyadenosyl radical. The source of the electron is the [4Fe-4S]1+ cluster characterizing the radical SAM superfamily, to which SAM is directly ligated through its methionyl carboxylate and amino groups. The energetics of the reductive cleavage of SAM is an outstanding question in the actions of radical SAM enzymes. The energetics is here reported for the action of lysine 2,3-aminomutase (LAM), which catalyzes the interconversion of l-lysine and l-beta-lysine. From earlier work, the reduction potential of the [4Fe-4S]2+/1+ cluster in LAM is -0.43 V with SAM bound to the cluster (Hinckley, G. T., and Frey, P. A. (2006) Biochemistry 45, 3219-3225), 1.4 V higher than the reported value for trialkylsulfonium ions in solution. The midpoint reduction potential upon binding l-lysine has been estimated to be -0.6 V from the values of midpoint potentials measured with SAM bound to the cluster and l-alanine in place of l-lysine, with S-adenosyl-l-homocysteine (SAH) bound to the cluster in the presence of l-lysine, and with SAH bound to the cluster in the presence of l-alanine or of l-alanine and ethylamine in place of l-lysine. The reduction potential for SAM has been estimated to be -0.99 V from the measured value for S-3',4'-anhydroadenosyl-l-methionine. The reduction potential for the [4Fe-4S] cluster is lowered 0.17 V by the binding of lysine to LAM, and the binding of SAM to the [4Fe-4S] cluster in LAM elevates its reduction potential by 0.81 V. Thus, the binding of l-lysine to LAM contributes 4 kcal mol-1, and the binding of SAM to the [4Fe-4S] cluster in LAM contributes 19 kcal mol-1 toward lowering the barrier for reductive cleavage of SAM from 32 kcal mol-1 in solution to 9 kcal mol-1 at the active site of LAM.     

Mechanistic understanding of Pyrococcus horikoshii Dph2, a [4Fe-4S] enzyme required for diphthamide biosynthesis

Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 (EF2). The proposed biosynthesis of diphthamide involves three steps and we have recently found that in Pyrococcus horikoshii (P. horikoshii), the first step uses an S-adenosyl-L-methionine (SAM)-dependent [4Fe-4S] enzyme, PhDph2, to catalyze the formation of a C-C bond. Crystal structure shows that PhDph2 is a homodimer and each monomer contains three conserved cysteine residues that can bind a [4Fe-4S] cluster. In the reduced state, the [4Fe-4S] cluster can provide one electron to reductively cleave the bound SAM molecule. However, different from classical radical SAM family of enzymes, biochemical evidence suggest that a 3-amino-3-carboxypropyl radical is generated in PhDph2. Here we present evidence supporting that the 3-amino-3-carboxypropyl radical does not undergo hydrogen abstraction reaction, which is observed for the deoxyadenosyl radical in classical radical SAM enzymes. Instead, the 3-amino-3-carboxypropyl radical is added to the imidazole ring in the pathway towards the formation of the product. Furthermore, our data suggest that the chemistry requires only one [4Fe-4S] cluster to be present in the PhDph2 dimer.     

Role of the [2Fe-2S] cluster in recombinant Escherichia coli biotin synthase

Biotin synthase (BioB) converts dethiobiotin into biotin by inserting a sulfur atom between C6 and C9 of dethiobiotin in an S-adenosylmethionine (SAM)-dependent reaction. The as-purified recombinant BioB from Escherichia coli is a homodimeric molecule containing one [2Fe-2S](2+) cluster per monomer. It is inactive in vitro without the addition of exogenous Fe. Anaerobic reconstitution of the as-purified [2Fe-2S]-containing BioB with Fe(2+) and S(2)(-) produces a form of BioB that contains approximately one [2Fe-2S](2+) and one [4Fe-4S](2+) cluster per monomer ([2Fe-2S]/[4Fe-4S] BioB). In the absence of added Fe, the [2Fe-2S]/[4Fe-4S] BioB is active and can produce up to approximately 0.7 equiv of biotin per monomer. To better define the roles of the Fe-S clusters in the BioB reaction, M?ssbauer and electron paramagnetic resonance (EPR) spectroscopy have been used to monitor the states of the Fe-S clusters during the conversion of dethiobiotin to biotin. The results show that the [4Fe-4S](2+) cluster is stable during the reaction and present in the SAM-bound form, supporting the current consensus that the functional role of the [4Fe-4S] cluster is to bind SAM and facilitate the reductive cleavage of SAM to generate the catalytically essential 5'-deoxyadenosyl radical. The results also demonstrate that approximately (2)/(3) of the [2Fe-2S] clusters are degraded by the end of the turnover experiment (24 h at 25 degrees C). A transient species with spectroscopic properties consistent with a [2Fe-2S](+) cluster is observed during turnover, suggesting that the degradation of the [2Fe-2S](2+) cluster is initiated by reduction of the cluster. This observed degradation of the [2Fe-2S] cluster during biotin formation is consistent with the proposed sacrificial S-donating function of the [2Fe-2S] cluster put forth by Jarrett and co-workers (Ugulava et al. (2001) Biochemistry 40, 8352-8358). Interestingly, degradation of the [2Fe-2S](2+) cluster was found not to parallel biotin formation. The initial decay rate of the [2Fe-2S](2+) cluster is about 1 order of magnitude faster than the initial formation rate of biotin, indicating that if the [2Fe-2S] cluster is the immediate S donor for biotin synthesis, insertion of S into dethiobiotin would not be the rate-limiting step. Alternatively, the [2Fe-2S] cluster may not be the immediate S donor. Instead, degradation of the [2Fe-2S] cluster may generate a protein-bound polysulfide or persulfide that serves as the immediate S donor for biotin production.     

Crystal structure of the Acidaminococcus fermentans 2-hydroxyglutaryl-CoA dehydratase component A

Acidaminococcus fermentans degrades glutamate via the hydroxyglutarate pathway, which involves the syn-elimination of water from (R)-2-hydroxyglutaryl-CoA in a key reaction of the pathway. This anaerobic process is catalyzed by 2-hydroxyglutaryl-CoA dehydratase, an enzyme with two components (A and D) that reversibly associate during reaction cycles. Component A (CompA), a homodimeric protein of 2x27 kDa, contains a single, bridging [4Fe-4S] cluster and uses the hydrolysis of ATP to deliver an electron to the dehydratase component (CompD), where the electron is used catalytically. The structure of the extremely oxygen-sensitive CompA protein was solved by X-ray crystallography to 3 A resolution. The protein was found to be a member of the actin fold family, revealing a similar architecture and nucleotide-binding site. The key differences between CompA and other members of the actin fold family are: (i) the presence of a cluster binding segment, the "cluster helix"; (ii) the [4Fe-4S] cluster; and (iii) the location of the homodimer interface, which involves the bridging cluster. Possible reaction mechanisms are discussed in light of the close structural similarity to members of the actin-fold family and the functional similarity to the nitrogenase Fe- protein.     

Oxidized and reduced Azotobacter vinelandii ferredoxin I at 1.4 A resolution: conformational change of surface residues without significant change in the [3Fe-4S]+/0 cluster.

The refined structure of reduced Azotobacter vinelandii 7Fe ferredoxin FdI at 100 K and 1.4 A resolution is reported, permitting comparison of [3Fe-4S]+ and [3Fe-4S]0 clusters in the same protein at near atomic resolution. The reduced state of the [3Fe-4S]0 cluster is established by single-crystal EPR following data collection. Redundant structures are refined to establish the reproducibility and accuracy of the results for both oxidation states. The structure of the [4Fe-4S]2+ cluster in four independently determined FdI structures is the same within the range of derived standard uncertainties, providing an internal control on the experimental methods and the refinement results. The structures of the [3Fe-4S]+ and [3Fe-4S]0 clusters are also the same within experimental error, indicating that the protein may be enforcing an entatic state upon this cluster, facilitating electron-transfer reactions. The structure of the FdI [3Fe-4S]0 cluster allows direct comparison with the structure of a well-characterized [Fe3S4]0 synthetic analogue compound. The [3Fe-4S]0 cluster displays significant distortions with respect to the [Fe3S4]0 analogue, further suggesting that the observed [3Fe-4S]+/0 geometry in FdI may represent an entatic state. Comparison of oxidized and reduced FdI reveals conformational changes at the protein surface in response to reduction of the [3Fe-4S]+/0 cluster. The carboxyl group of Asp15 rotates approximately 90 degrees, Lys84, a residue hydrogen bonded to Asp15, adopts a single conformation, and additional H2O molecules become ordered. These structural changes imply a mechanism for H+ transfer to the [3Fe-4S]0 cluster in agreement with electrochemical and spectroscopic results.     

Structural studies of the interaction of S-adenosylmethionine with the [4Fe-4S] clusters in biotin synthase and pyruvate formate-lyase activating enzyme

The diverse reactions catalyzed by the radical-SAM superfamily of enzymes are thought to proceed via a set of common mechanistic steps, key among which is the reductive cleavage of S-adenosyl-L-methionine (SAM) by a reduced [4Fe-4S] cluster to generate an intermediate deoxyadenosyl radical. A number of spectroscopic studies have provided evidence that SAM interacts directly with the [4Fe-4S] clusters in several of the radical-SAM enzymes; however, the molecular mechanism for the reductive cleavage has yet to be elucidated. Selenium X-ray absorption spectroscopy (Se-XAS) was used previously to provide evidence for a close interaction between the Se atom of selenomethionine (a cleavage product of Se-SAM) and an Fe atom of the [4Fe-4S] cluster of lysine-2,3-aminomutase (KAM). Here, we utilize the same approach to investigate the possibility of a similar interaction in pyruvate formate-lyase activating enzyme (PFL-AE) and biotin synthase (BioB), two additional members of the radical-SAM superfamily. The results show that the latter two enzymes do not exhibit the same Fe-Se interaction as was observed in KAM, indicating that the methionine product of reductive cleavage of SAM does not occupy a well-defined site close to the cluster in PFL-AE and BioB. These results are interpreted in terms of the differences among these enzymes in their use of SAM as either a cofactor or a substrate.     

Cofactor dependence of reduction potentials for [4Fe-4S]2+/1+ in lysine 2,3-aminomutase

Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of l-lysine and l-beta-lysine by a free radical mechanism. The 5'-deoxyadenosyl radical derived from the reductive cleavage of S-adenosyl-l-methionine (SAM) initiates substrate-radical formation. The [4Fe-4S](1+) cluster in LAM is the one-electron source in the reductive cleavage of SAM, which is directly ligated to the unique iron site in the cluster. We here report the midpoint reduction potentials of the [4Fe-4S](2+/1+) couple in the presence of SAM, S-adenosyl-l-homocysteine (SAH), or 5'-{N-[(3S)-3-aminocarboxypropyl]-N-methylamino}-5'-deoxyadenosine (azaSAM) as measured by spectroelectrochemistry. The reduction potentials are -430 +/- 2 mV in the presence of SAM, -460 +/- 3 mV in the presence of SAH, and -497 +/- 10 mV in the presence of azaSAM. In the absence of SAM or an analogue and the presence of dithiothreitol, dihydrolipoate, or cysteine as ligands to the unique iron, the midpoint potentials are -479 +/- 5, -516 +/- 5, and -484 +/- 3 mV, respectively. LAM is a member of the radical SAM superfamily of enzymes, in which the CxxxCxxC motif donates three thiolate ligands to iron in the [4Fe-4S] cluster and SAM donates the alpha-amino and alpha-carboxylate groups of the methionyl moiety as ligands to the fourth iron. The results show the reduction potentials in the midrange for ferredoxin-like [4Fe-4S] clusters. They show that SAM elevates the reduction potential by 86 mV relative to that of dihydrolipoate as the cluster ligand. This difference accounts for the SAM-dependent reduction of the [4Fe-4S](2+) cluster by dithionite reported earlier. Analogues of SAM have a weakened capacity to raise the potential. We conclude that the midpoint reduction potential of the cluster ligated to SAM is 1.2 V less negative than the half-wave potential for the one-electron reductive cleavage of simple alkylsulfonium ions in aqueous solution. The energetic barrier in the reductive cleavage of SAM may be overcome through the use of binding energy.     

The Radical SAM Superfamily

The radical S-adenosylmethionine (SAM) superfamily currently comprises more than 2800 proteins with the amino acid sequence motif CxxxCxxC unaccompanied by a fourth conserved cysteine. The charcteristic three-cysteine motif nucleates a [4Fe-4S] cluster, which binds SAM as a ligand to the unique Fe not ligated to a cysteine residue. The members participate in more than 40 distinct biochemical transformations, and most members have not been biochemically characterized. A handful of the members of this superfamily have been purified and at least partially characterized. Significant mechanistic and structural information is available for lysine 2,3-aminomutase, pyruvate formate-lyase, coproporphyrinogen III oxidase, and MoaA required for molybdopterin biosynthesis. Biochemical information is available for spore photoproduct lyase, anaerobic ribonucleotide reductase activation subunit, lipoyl synthase, and MiaB involved in methylthiolation of isopentenyladenine-37 in tRNA. The radical SAM enzymes biochemically characterized to date have in common the cleavage of the [4Fe-4S](1 +) -SAM complex to [4Fe-4S](2 +)-Met and the 5' -deoxyadenosyl radical, which abstracts a hydrogen atom from the substrate to initiate a radical mechanism.     

Structural insights into the molecular function of human [2Fe-2S] BOLA1-GRX5 and [2Fe-2S] BOLA3-GRX5 complexes

Members of the monothiol glutaredoxin family and members of the BolA-like protein family have recently emerged as specific interacting partners involved in iron-sulfur protein maturation and redox regulation pathways. It is known that human mitochondrial BOLA1 and BOLA3 form [2Fe-2S] cluster-bridged dimeric heterocomplexes with the monothiol glutaredoxin GRX5. The structure and cluster coordination of the two [2Fe-2S] heterocomplexes as well as their molecular function are, however, not defined yet. Experimentally-driven structural models of the two [2Fe-2S] cluster-bridged dimeric heterocomplexes, the relative stability of the two complexes and the redox properties of the [2Fe-2S] cluster bound to these complexes are here presented on the basis of UV/vis, CD, EPR and NMR spectroscopies and computational protein-protein docking. While the BOLA1-GRX5 complex coordinates a reduced, Rieske-type [2Fe-2S]¹⁺ cluster, an oxidized, ferredoxin-like [2Fe-2S]²⁺ cluster is present in the BOLA3-GRX5 complex. The [2Fe-2S] BOLA1-GRX5 complex is preferentially formed over the [2Fe-2S] BOLA3-GRX5 complex, as a result of a higher cluster binding affinity. All these observed differences provide the first indications discriminating the molecular function of the two [2Fe-2S] heterocomplexes.     

Perturbation of the quinone-binding site of complex II alters the electronic properties of the proximal [3Fe-4S] iron-sulfur cluster

Succinate-ubiquinone oxidoreductase (SQR) and menaquinol-fumarate oxidoreductase (QFR) from Escherichia coli are members of the complex II family of enzymes. SQR and QFR catalyze similar reactions with quinones; however, SQR preferentially reacts with higher potential ubiquinones, and QFR preferentially reacts with lower potential naphthoquinones. Both enzymes have a single functional quinone-binding site proximal to a [3Fe-4S] iron-sulfur cluster. A difference between SQR and QFR is that the redox potential of the [3Fe-4S] cluster in SQR is 140 mV higher than that found in QFR. This may reflect the character of the different quinones with which the two enzymes preferentially react. To investigate how the environment around the [3Fe-4S] cluster affects its redox properties and catalysis with quinones, a conserved amino acid proximal to the cluster was mutated in both enzymes. It was found that substitution of SdhB His-207 by threonine (as found in QFR) resulted in a 70-mV lowering of the redox potential of the cluster as measured by EPR. The converse substitution in QFR raised the redox potential of the cluster. X-ray structural analysis suggests that placing a charged residue near the [3Fe-4S] cluster is a primary reason for the alteration in redox potential with the hydrogen bonding environment having a lesser effect. Steady state enzyme kinetic characterization of the mutant enzymes shows that the redox properties of the [3Fe-4S] cluster have only a minor effect on catalysis.     

Structural diversity in the AdoMet radical enzyme superfamily

AdoMet radical enzymes are involved in processes such as cofactor biosynthesis, anaerobic metabolism, and natural product biosynthesis. These enzymes utilize the reductive cleavage of S-adenosylmethionine (AdoMet) to afford l-methionine and a transient 5'-deoxyadenosyl radical, which subsequently generates a substrate radical species. By harnessing radical reactivity, the AdoMet radical enzyme superfamily is responsible for an incredible diversity of chemical transformations. Structural analysis reveals that family members adopt a full or partial Triose-phosphate Isomerase Mutase (TIM) barrel protein fold, containing core motifs responsible for binding a catalytic [4Fe-4S] cluster and AdoMet. Here we evaluate over twenty structures of AdoMet radical enzymes and classify them into two categories: 'traditional' and 'ThiC-like' (named for the structure of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (ThiC)). In light of new structural data, we reexamine the 'traditional' structural motifs responsible for binding the [4Fe-4S] cluster and AdoMet, and compare and contrast these motifs with the ThiC case. We also review how structural data combine with biochemical, spectroscopic, and computational data to help us understand key features of this enzyme superfamily, such as the energetics, the triggering, and the molecular mechanisms of AdoMet reductive cleavage. This article is part of a Special Issue entitled: Radical SAM Enzymes and Radical Enzymology.     

Structural and functional comparison of HemN to other radical SAM enzymes

Radical SAM enzymes have only recently been recognized as an ancient family sharing an unusual radical-based reaction mechanism. This late appreciation is due to the extreme oxygen sensitivity of most radical SAM enzymes, making their characterization particularly arduous. Nevertheless, realization that the novel apposition of the established cofactors S-adenosylmethionine and [4Fe-4S] cluster creates an explosive source of catalytic radicals, the appreciation of the sheer size of this previously neglected family, and the rapid succession of three successfully solved crystal structures within a year have ensured that this family has belatedly been noted. In this review, we report the characterization of two enzymes: the established radical SAM enzyme, HemN or oxygen-independent coproporphyrinogen III oxidase from Escherichia coli, and littorine mutase, a presumed radical SAM enzyme, responsible for the conversion of littorine to hyoscyamine in plants. The enzymes are compared to other radical SAM enzymes and in particular the three reported crystal structures from this family, HemN, biotin synthase and MoaA, are discussed.     

Redox centers of 4-hydroxybenzoyl-CoA reductase, a member of the xanthine oxidase family of molybdenum-containing enzymes

4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a key enzyme in the anaerobic metabolism of phenolic compounds. It catalyzes the reductive removal of the hydroxyl group from the aromatic ring yielding benzoyl-CoA and water. The subunit architecture, amino acid sequence, and the cofactor/metal content indicate that it belongs to the xanthine oxidase (XO) family of molybdenum cofactor-containing enzymes. 4-HBCR is an unusual XO family member as it catalyzes the irreversible reduction of a CoA-thioester substrate. A radical mechanism has been proposed for the enzymatic removal of phenolic hydroxyl groups. In this work we studied the spectroscopic and electrochemical properties of 4-HBCR by EPR and M?ssbauer spectroscopy and identified the pterin cofactor as molybdopterin mononucleotide. In addition to two different [2Fe-2S] clusters, one FAD and one molybdenum species per monomer, we also identified a [4Fe-4S] cluster/monomer, which is unique among members of the XO family. The reduced [4Fe-4S] cluster interacted magnetically with the Mo(V) species, suggesting that the centers are in close proximity, (<15 A apart). Additionally, reduction of the [4Fe-4S] cluster resulted in a loss of the EPR signals of the [2Fe-2S] clusters probably because of magnetic interactions between the Fe-S clusters as evidenced in power saturation studies. The Mo(V) EPR signals of 4-HBCR were typical for XO family members. Under steady-state conditions of substrate reduction, in the presence of excess dithionite, the [4Fe-4S] clusters were in the fully oxidized state while the [2Fe-2S] clusters remained reduced. The redox potentials of the redox cofactors were determined to be: [2Fe-2S](+1/+2) I, -205 mV; [2Fe-2S] (+1/+2) II, -255 mV; FAD/FADH( small middle dot)/FADH, -250 mV/-470 mV; [4Fe-4S](+1/+2), -465 mV and Mo(VI)/(V)/(VI), -380 mV/-500 mV. A catalytic cycle is proposed that takes into account the common properties of molybdenum cofactor enzymes and the special one-electron chemistry of dehydroxylation of phenolic compounds.     

Generation of adenosyl radical from S-adenosylmethionine (SAM) in biotin synthase

A mechanism of the C―S bond activation of S-adenosylmethionine (SAM) in biotin synthase is discussed from quantum mechanical/molecular mechanical (QM/MM) computations. The active site of the enzyme involves a [4Fe-4S] cluster, which is coordinated to the COO⁻ and NH₂ groups of the methionine moiety of SAM. The unpaired electrons on the iron atoms of the [4Fe-4S]²⁺ cluster are antiferromagnetically coupled, resulting in the S = 0 ground spin state. An electron is transferred from an electron donor to the [4Fe-4S]²⁺-SAM complex to produce the catalytically active [4Fe-4S]⁺ state. The SOMO of the [4Fe-4S]⁺-SAM complex is localized on the [4Fe-4S] moiety and the spin density of the [4Fe-4S] core is calculated to be 0.83. The C―S bond cleavage is associated with the electron transfer from the [4Fe-4S]⁺ cluster to the antibonding σ* C―S orbital. The electron donor and acceptor states are effectively coupled with each other at the transition state for the C―S bond cleavage. The activation barrier is calculated to be 16.0 kcal/mol at the QM (B3LYP/SV(P))/MM (CHARMm) level of theory and the C―S bond activation process is 17.4 kcal/mol exothermic, which is in good agreement with the experimental observation that the C―S bond is irreversibly cleaved in biotin synthase. The sulfur atom of the produced methionine molecule is unlikely to bind to an iron atom of the [4Fe-4S]²⁺ cluster after the C―S bond cleavage from the energetical and structural points of view.     

Conversion of the central [4Fe-4S] cluster into a [3Fe-4S] cluster leads to reduced hydrogen-uptake activity of the F420-reducing hydrogenase of Methanococcus voltae.

As in many other hydrogenases, the small subunit of the F420-reducing hydrogenase of Methanococcus voltae contains three iron-sulfur clusters. The arrangement of the three [4Fe-4S] clusters corresponds to the arrangement of [Fe-S] clusters in the [NiFeSe] hydrogenase of Desulfomicrobium baculatum. Many other hydrogenases contain two [4Fe-4S] clusters and one [3Fe-4S] cluster with a relatively high redox potential, which is located in the central position between a proximal and a distal [4Fe-4S] cluster. We have investigated the role of the central [4Fe-4S] cluster in M. voltae with regard to its effect on the enzyme activity and its spectroscopic properties. Using site-directed mutagenesis, we constructed a strain in which one cysteine ligand of the central [4Fe-4S] cluster was replaced by proline. The mutant protein was purified, and the [4Fe-4S] to [3Fe-4S] cluster conversion was confirmed by EPR spectroscopy. The conversion resulted in an increase in the redox potential of the [3Fe-4S] cluster by about 400 mV. The [NiFe] active site was not affected significantly by the mutation as assessed by the unchanged Ni EPR spectrum. The specific activity of the mutated enzyme did not show any significant differences with the artificial electron acceptor benzyl viologen, but its specific activity with the natural electron acceptor F420 decreased tenfold.     

NADPH inhibits [2Fe-2S] cluster protein transfer from diabetes drug target MitoNEET to an apo-acceptor protein

MitoNEET (mNT) is the founding member of the recently discovered CDGSH family of [2Fe-2S] proteins capable of [2Fe-2S] cluster transfer to apo-acceptor proteins. It is a target of the thiazolidinedione (TZD) class of anti-diabetes drugs whose binding modulate both electron transfer and cluster transfer properties. The [2Fe-2S] cluster in mNT is destabilized upon binding of NADPH, which leads to loss of the [2Fe-2S] cluster to the solution environment. Because mNT is capable of transferring [2Fe-2S] clusters to apo-acceptor proteins, we sought to determine whether NADPH binding also affects cluster transfer. We show that NADPH inhibits transfer of the [2Fe-2S] cluster to an apo-acceptor protein with an inhibition constant (K(i)) of 200 μm, which reflects that of NADPH concentrations expected under physiological conditions. In addition, we determined that the strictly conserved cluster interacting residue Asp-84 in the CDGSH domain is necessary for the NADPH-dependent inhibition of [2Fe-2S] cluster transfer. The most critical cellular function of NADPH is in the maintenance of a pool of reducing equivalents, which is essential to counteract oxidative damage. Taken together, our findings suggest that NADPH can regulate both mNT [2Fe-2S] cluster levels in the cell as well as the ability of the protein to transfer [2Fe-2S] clusters to cytosolic or mitochondrial acceptors.     

On the ATP-Dependent Activation of the Radical Enzyme (R)-2-Hydroxyisocaproyl-CoA Dehydratase

Members of the 2-hydroxyacyl-CoA dehydratase enzyme family catalyze the β,α-dehydration of various CoA-esters in the fermentation of amino acids by clostridia. Abstraction of the nonacidic β-proton of the 2-hydroxyacyl-CoA compounds is achieved by the reductive generation of ketyl radicals on the substrate, which is initiated by the transfer of an electron at low redox potentials. The highly energetic electron needed on the dehydratase is donated by a [4Fe-4S] cluster containing ATPase, termed activator. We investigated the activator of the 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile. The activator is a homodimeric protein structurally related to acetate and sugar kinases, Hsc70 and actin, and has a [4Fe-4S] cluster bound in the dimer interface. The crystal structures of the Mg-ADP, Mg-ADPNP, and nucleotide-free states of the reduced activator have been solved at 1.6-3.0 ? resolution, allowing us to define the position of Mg(2+) and water molecules in the vicinity of the nucleotides and the [4Fe-4S] cluster. The structures reveal redox- and nucleotide dependent changes agreeing with the modulation of the reduction potential of the [4Fe-4S] cluster by conformational changes. We also investigated the propensity of the activator to form a complex with its cognate dehydratase in the presence of Mg-ADP and Mg-ADPNP and together with the structural data present a refined mechanistic scheme for the ATP-dependent electron transfer between activator and dehydratase.     

Structure of a xanthine oxidase-related 4-hydroxybenzoyl-CoA reductase with an additional [4Fe-4S] cluster and an inverted electron flow

The Mo-flavo-Fe/S-dependent heterohexameric protein complex 4-hydroxybenzoyl-CoA reductase (4-HBCR, dehydroxylating) is a central enzyme of the anaerobic degradation of phenolic compounds and belongs to the xanthine oxidase (XO) family of molybdenum enzymes. Its X-ray structure was established at 1.6 A resolution. The most pronounced difference between 4-HBCR and other structurally characterized members of the XO family is the insertion of 40 amino acids within the beta subunit, which carries an additional [4Fe-4S] cluster at a distance of 16.5 A to the isoalloxazine ring of FAD. The architecture of 4-HBCR and concomitantly performed electron transfer rate calculations suggest an inverted electron transfer chain from the donor ferredoxin via the [4Fe-4S] cluster to the Mo over a distance of 55 A. The binding site of 4-hydroxybenzoyl-CoA is located in an 18 A long channel lined up by several aromatic side chains around the aromatic moiety, which are proposed to shield and stabilize the postulated radical intermediates during catalysis.