17O electron nuclear double resonance characterization of substrate binding to the [4Fe-4S]1+ cluster of reduced active aconitase

To characterize the binding of substrate to aconitase, we have made 17O electron nuclear double resonance (ENDOR) measurements on reduced active ([4Fe-4S]1+) beef heart aconitase, both in H216O and H217O, in the presence of substrate and the inhibitors, tricarballylate, trans-aconitate, and 1-hydroxy-2-nitro-1, 3-propanedicarboxylate, referred to here as nitroisocitrate; the hydroxyl of the latter also was isotypically labeled with 17O. The hydroxyl oxygen of citrate and isocitrate is exchanged with solvent water by aconitase, but the hydroxyl of nitroisocitrate is not. Thus, the isotopic composition of nitroisocitrate can be chemically controlled, allowing direct identification of any 17O ENDOR signal associated with it. 17O ENDOR signals were observed from Hx17O (mean = 1 or 2) bound to the [4Fe-4S]1+ cluster in samples prepared with trans-aconitate and unlabeled nitroisocitrate. 17O-Labeled nitroisocitrate in H216O bound to the cluster showed a signal from the 17OH group; in H217O it showed 17O ENDOR resonances due to both Hx17O and 17OH of substrate. This result demonstrates that the cluster participates in substrate binding and can simultaneously coordinate the hydroxyl of a substrate (or analogue) and water (or hydroxyl). The sample with citrate in H217O showed only the Hx17O signal, although aconitase exchanges the hydroxyl of substrate with solvent water. The mechanism of action of aconitase is discussed in light of this observation. Comparison shows the ENDOR study to be in agreement with previous M?ssbauer and EPR spectroscopic results.     

Characterization of the [4Fe-4S]+ cluster at the active site of aconitase by 57Fe, 33S, and 14N electron nuclear double resonance spectroscopy

57Fe, 33S, and 14N electron nuclear double resonance (ENDOR) studies have been performed to characterize the [4Fe-4S]+ cluster at the active site of aconitase. Q-band 57Fe ENDOLR of isotopically enriched enzyme, both substrate free and in the enzyme-substrate complex, reveals four inequivalent iron sites. In agreement with M?ssbauer studies [Kent et al. (1985) J. Biol. Chem. 260, 6371-6881], one of the iron ions, Fea, which is easily removed by oxidation to yield the [3Fe-4S]+ cluster of inactive aconitase, shows a dramatic change in the presence of substrate. The remaining iron sites, Feb1,2,3, show minor changes when substrate is bound. Methods devised by us for analyzing and simulating ENDOR spectra of a randomly oriented paramagnet have been used to determine the principal values and orientation relative to the g tensor for the hyperfine tensors of three of the four inequivalent iron sites of the [4Fe-4S]+ cluster, Fea, Feb2, and Feb3, in the substrate-free enzyme and the enzyme-substrate complex. The full tensor for the fourth site, Feb1, could not be obtained because its signal is seen only over a limited range of the EPR envelope. 33S ENDOR data for the enzyme-substrate complex using enzyme reconstituted with 33S show that the four inorganic bridging sulfide ions of the [4Fe-4S]+ cube have isotropic hyperfine couplings of A(S) less than 12 MHz, and analysis indicates that they can be divided into two pairs, one with couplings of A(S1) approximately less than 1 MHz and the other with A(S2) approximately 6-12 MHz; the analysis further places these pairs within the cube relative to the iron sites. 33S data for substrate-free enzyme is qualitatively similar and can be completely simulated by two types of S2- ion, with A(S1) approximately 7.5 and A(S2) approximately 9 MHz; the full hyperfine tensors have been determined. The hyperfine values for the two enzyme forms correspond to surprisingly small unpaired spin density on S2-. 14N ENDOR at Q-band reveals a nitrogen signal that does not change upon substrate binding.     

Potentiometric and electron nuclear double resonance properties of the two spin forms of the [4Fe-4S]+ cluster in the novel ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus.

Pyrococcus furiosus ferredoxin contains a single [4Fe-4S] that exists in both S = 1/2 (20%) and S = 3/2 (80%) ground states in the reduced protein. We report here on the temperature-dependent potentiometric properties of the two spin forms, their stability, and on the structural features that differentiate them. The midpoint potential (Em) of the cluster in either spin state was determined at -365 mV (30 degrees C, pH 8.0). By rapidly freezing samples for EPR analyses, it was shown that the Em values of both spin states appear to change by -1.7 mV/degrees C over the range 20 degrees-80 degrees C, and by -6 mV/degrees C between 80 and 89 degrees C. The Em values and the relative amounts of the S = 1/2 and S = 3/2 forms of the cluster were unaffected by pH (6.8-10.5), even at 85 degrees C, and were unchanged by the presence of NaCl (1.0 M), sodium dodecyl sulfate (10%, w/v) or ethylene glycol (50%, v/v), even at 80 degrees C. The S = 1/2 form of the [4Fe-4S]+ cluster was found to exhibit a strongly coupled 1H ENDOR resonance (A = 22 MHz) that was exchangeable with the solvent. Such a large coupling has not been observed in any other iron-sulfur protein. Since a unique feature of this 4Fe-ferredoxin is that only 3 cysteinyl residues appear to be coordinated to the [4Fe-4S] cluster, the ENDOR data are consistent with an H2O molecule being a ligand to the unique Fe site. The S = 3/2 form of the [4Fe-4S]+ cluster exhibited a similar, strongly coupled 1H ENDOR resonance, but in this spin state it was not exchangeable with the solvent. This suggests that the [4Fe-4S]+ cluster exhibiting the S = 3/2, but not the S = 1/2 ground state, is "shielded" from the solvent, presumably by neighboring amino acid residues. In view of the pH dependence of the midpoint potential of the two spin states, the fourth ligand to the cluster and the source of the strongly coupled 1H ENDOR resonance is probably an OH- rather than H2O molecule.     

Impact of mutations on the midpoint potential of the [4Fe-4S]+1,+2 cluster and on catalytic activity in electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO)

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulfur flavoprotein that accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the Q-pool. ETF-QO contains a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. Mutations were introduced by site-directed mutagenesis of amino acids in the vicinity of the iron-sulfur cluster of Rhodobacter sphaeroides ETF-QO. Y501 and T525 are equivalent to Y533 and T558 in the porcine ETF-QO. In the porcine protein, these residues are within hydrogen-bonding distance of the Sgamma of the cysteine ligands to the iron-sulfur cluster. Y501F, T525A, and Y501F/T525A substitutions were made to determine the effects on midpoint potential, activity, and EPR spectral properties of the cluster. The integrity of the mutated proteins was confirmed by optical spectra, EPR g-values, and spin-lattice relaxation rates, and the cluster to flavin point-dipole distance was determined by relaxation enhancement. Potentiometric titrations were monitored by changes in the CW EPR signals of the cluster and semiquinone. Single mutations decreased the midpoint potentials of the iron-sulfur cluster from +37 mV for wild type to -60 mV for Y501F and T525A and to -128 mV for Y501F/T525A. Lowering the midpoint potential resulted in a decrease in steady-state ubiquinone reductase activity and in ETF semiquinone disproportionation. The decrease in activity demonstrates that reduction of the iron-sulfur cluster is required for activity. There was no detectable effect of the mutations on the flavin midpoint potentials.     

Evidence for the formation of a linear [3Fe-4S] cluster in partially unfolded aconitase

Beef heart aconitase, as isolated under aerobic conditions, is inactive and contains a [3Fe-4S]1+ cluster. On incubation at pH greater than 9.5 (or treatment with 4-8 M urea) the color of the protein changes from brown to purple. This purple form is stable and can be converted back in good yield to the active [4Fe-4S]2+ form by reduction in the presence of iron. Active aconitase is converted to the purple form at alkaline pH only after oxidative inactivation. The Fe/S2- ratio of purple aconitase is 0.8, indicating the presence of [3Fe-4S] clusters. The number of SH groups readily reacting with 5,5'-dithiobis(2-nitrobenzoic acid) is increased from approximately 1 in the enzyme as isolated to 7-8 in the purple form, indicating a partial unfolding of the protein. On conversion of inactive aconitase to the purple form, the EPR signal at g = 2.01 (S = 1/2) is replaced by signals at g = 4.3 and 9.6 (S = 5/2). M?ssbauer spectroscopy shows that purple aconitase has high-spin ferric ions, each residing in a tetrahedral environment of sulfur atoms. The three iron sites are exchange-coupled to yield a ground state with S = 5/2. Analysis of the data within a spin coupling model shows that J13 congruent to J23 and 2 J12 less than J13, where the Jik describe the antiferromagnetic (J greater than 0) exchange interactions among the three iron pairs. Comparison of our data with those reported for synthetic Fe-S clusters (Hagen, K. S., Watson, A. D., and Holm, R. H., (1983) J. Am. Chem. Soc. 105, 3905-3913) shows that purple aconitase contains a linear [3Fe-4S]1+ cluster, a structural isomer of the S = 1/2 cluster of inactive aconitase. Our studies also show that protein-bound [2Fe-2S] clusters can be generated under conditions where partial unfolding of the protein occurs.     

Resonance Raman studies of the [4Fe-4S] to [2Fe-2S] cluster conversion in the iron protein of nitrogenase

Resonance Raman spectroscopy has been used to investigate the Fe-S stretching modes of the [4Fe-4S]2+ cluster in the oxidized iron protein of Clostridium pasteurianum nitrogenase. The results are consistent with a cubane [4Fe-4S] cluster having effective Td symmetry with cysteinyl coordination for each iron. In accord with previous optical and EPR studies [(1984) Biochemistry 23, 2118-2122], treatment with the iron chelator alpha, alpha'-dipyridyl in the presence of MgATP is shown to effect cluster conversion to a [2Fe-2S]2+ cluster. Resonance Raman data also indicate that partial conversion to a [2Fe-2S]2+ cluster is induced by thionine-oxidation in the presence of MgATP in the absence of an iron chelator. This result suggests new explanations for the dramatic change in the CD spectrum that accompanies MgATP-binding to the oxidized Fe protein and the anomalous resonance Raman spectra of thionine-oxidized Clostridium pasteurianum bidirectional hydrogenase.     

Iron-sulfur cluster interconversions in biotin synthase: dissociation and reassociation of iron during conversion of [2Fe-2S] to [4Fe-4S] clusters

Biotin synthase catalyzes the insertion of a sulfur atom into the saturated C6 and C9 carbons of dethiobiotin. This reaction has long been presumed to occur through radical chemistry, and recent experimental results suggest that biotin synthase belongs to a family of enzymes that contain an iron-sulfur cluster and reductively cleave S-adenosylmethionine, forming an enzyme or substrate radical, 5'-deoxyadenosine, and methionine. Biotin synthase (BioB) is aerobically purified as a dimer of 38 kDa monomers that contains two [2Fe-2S](2+) clusters per dimer. Maximal in vitro biotin synthesis requires incubation of BioB with dethiobiotin, AdoMet, reductants, exogenous iron, and crude bacterial protein extracts. It has previously been shown that reduction of BioB with dithionite in 60% ethylene glycol produces one [4Fe-4S](2+/1+) cluster per dimer. In the present work, we use UV/visible and electron paramagnetic resonance spectroscopy to show that [2Fe-2S] to [4Fe-4S] cluster conversion occurs through rapid dissociation of iron from the protein followed by rate-limiting reassociation. While in 60% ethylene glycol the product of dithionite reduction is one [4Fe-4S](2+) cluster per dimer, the product in water is one [4Fe-4S](1+) cluster per dimer. Further, incubation with excess iron, sulfide, and dithiothreitol produces protein that contains two [4Fe-4S](2+) clusters per dimer; subsequent reduction with dithionite produces two [4Fe-4S](1+) clusters per BioB dimer. BioB that contains two [4Fe-4S](2+/1+) clusters per dimer is rapidly and reversibly reduced and oxidized, suggesting that this is the redox-active form of the iron-sulfur cluster in the anaerobic enzyme.     

Electron paramagnetic resonance and electron nuclear double resonance spectroscopic identification and characterization of the tyrosyl radicals in prostaglandin H synthase 1

The tyrosyl radicals generated in reactions of ethyl hydrogen peroxide with both native and indomethacin-pretreated prostaglandin H synthase 1 (PGHS-1) were examined by low-temperature electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies. In the reaction of peroxide with the native enzyme at 0 degrees C, the tyrosyl radical EPR signal underwent a continuous reduction in line width and lost intensity as the incubation time increased, changing from an initial, 35-G wide doublet to a wide singlet of slightly smaller line width and finally to a 25-G narrow singlet. The 25-G narrow singlet produced by self-inactivation was distinctly broader than the 22-G narrow singlet obtained by indomethacin treatment. Analysis of the narrow singlet EPR spectra of self-inactivated and indomethacin-pretreated enzymes suggests that they reflect conformationally distinct tyrosyl radicals. ENDOR spectroscopy allowed more detailed characterization by providing hyperfine couplings for ring and methylene protons. These results establish that the wide doublet and the 22-G narrow singlet EPR signals arise from tyrosyl radicals with different side-chain conformations. The wide-singlet ENDOR spectrum, however, is best accounted for as a mixture of native wide-doublet and self-inactivated 25-G narrow-singlet species, consistent with an earlier EPR study [DeGray et al. (1992) J. Biol. Chem. 267, 23583-23588]. We conclude that a tyrosyl residue other than the catalytically essential Y385 species is most likely responsible for the indomethacin-inhibited, narrow-singlet spectrum. Thus, this inhibitor may function by redirecting radical formation to a catalytically inactive side chain. Either radical migration or conformational relaxation at Y385 produces the 25-G narrow singlet during self-inactivation. Our ENDOR data also indicate that the catalytically active, wide-doublet species is not hydrogen bonded, which may enhance its reactivity toward the fatty-acid substrate bound nearby.     

IscU as a scaffold for iron-sulfur cluster biosynthesis: sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU

Iron-sulfur cluster biosynthesis in both prokaryotic and eukaryotic cells is known to be mediated by two highly conserved proteins, termed IscS and IscU in prokaryotes. The homodimeric IscS protein has been shown to be a cysteine desulfurase that catalyzes the reductive conversion of cysteine to alanine and sulfide. In this work, the time course of IscS-mediated Fe-S cluster assembly in IscU was monitored via anaerobic anion exchange chromatography. The nature and properties of the clusters assembled in discrete fractions were assessed via analytical studies together with absorption, resonance Raman, and M?ssbauer investigations. The results show sequential cluster assembly with the initial IscU product containing one [2Fe-2S](2+) cluster per dimer converting first to a form containing two [2Fe-2S](2+) clusters per dimer and finally to a form that contains one [4Fe-4S](2+) cluster per dimer. Both the [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU are reductively labile and are degraded within minutes upon being exposed to air. On the basis of sequence considerations and spectroscopic studies, the [2Fe-2S](2+) clusters in IscU are shown to have incomplete cysteinyl ligation. In addition, the resonance Raman spectrum of the [4Fe-4S](2+) cluster in IscU is best interpreted in terms of noncysteinyl ligation at a unique Fe site. The ability to assemble both [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU supports the proposal that this ubiquitous protein provides a scaffold for IscS-mediated assembly of clusters that are subsequently used for maturation of apo Fe-S proteins.     

Simulations of the (1)H electron spin echo-electron nuclear double resonance and (2)H electron spin echo envelope modulation spectra of exchangeable hydrogen nuclei coupled to the S(2)-state photosystem II manganese cluster

The pulsed EPR methods of electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) are used to investigate the proximity of exchangeable hydrogens around the paramagnetic S(2)-state Mn cluster of the photosystem II oxygen-evolving complex. Although ESEEM and ESE-ENDOR are both pulsed electron paramagnetic resonance techniques, the specific mechanisms by which nuclear spin transitions are observed are quite different. We are able to generate good simulations of both (1)H ESE-ENDOR and (2)H ESEEM signatures of exchangeable hydrogens at the S(2)-state cluster. The convergence of simulation parameters for both methods provides a high degree of confidence in the simulations. Several exchangeable protons-deuterons with strong dipolar couplings are observed. In the simulations, two of the close ( approximately 2.5 A) hydrogen nuclei exhibit strong isotropic couplings and are therefore most probably associated with direct substrate ligation to paramagnetic Mn. Another two of the close ( approximately 2.7 A) hydrogen nuclei show no isotropic couplings and are therefore most probably not contained in Mn ligands. We suggest that these proximal hydrogens may be associated with a Ca(2+)-bound substrate, as indicated in recent mechanistic proposals for O(2) formation.     

Dynamics of the [4Fe-4S] cluster in Pyrococcus furiosus D14C ferredoxin via nuclear resonance vibrational and resonance Raman spectroscopies, force field simulations, and density functional theory calculations

We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study oxidized and reduced forms of the [4Fe-4S] cluster in the D14C variant ferredoxin from Pyrococcus furiosus (Pf D14C Fd). To assist the normal-mode assignments, we conducted NRVS with D14C ferredoxin samples with (36)S substituted into the [4Fe-4S] cluster bridging sulfide positions, and a model compound without ligand side chains, (Ph(4)P)(2)[Fe(4)S(4)Cl(4)]. Several distinct regions of NRVS intensity are identified, ranging from "protein" and torsional modes below 100 cm(-1), through bending and breathing modes near 150 cm(-1), to strong bands from Fe-S stretching modes between 250 and ～400 cm(-1). The oxidized ferredoxin samples were also investigated by resonance Raman (RR) spectroscopy. We found good agreement between NRVS and RR frequencies, but because of different selection rules, the intensities vary dramatically between the two types of spectra. The (57)Fe partial vibrational densities of states for the oxidized samples were interpreted by normal-mode analysis with optimization of Urey-Bradley force fields for local models of the [4Fe-4S] clusters. Full protein model calculations were also conducted using a supplemented CHARMM force field, and these calculations revealed low-frequency modes that may be relevant to electron transfer with Pf Fd partners. Density functional theory (DFT) calculations complemented these empirical analyses, and DFT was used to estimate the reorganization energy associated with the [Fe(4)S(4)](2+/+) redox cycle. Overall, the NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.     

Detection of a [3Fe-4S] cluster intermediate of cytosolic aconitase in yeast expressing iron regulatory protein 1. Insights into the mechanism of Fe-S cluster cycling.

Interconversion of iron regulatory protein 1 (IRP1) with cytosolic aconitase (c-aconitase) occurs via assembly/disassembly of a [4Fe-4S] cluster. Recent evidence implicates oxidants in cluster disassembly. We investigated H(2)O(2)-initiated Fe-S cluster disassembly in c-aconitase expressed in Saccharomyces cerevisiae. A signal for [3Fe-4S] c-aconitase was detected by whole-cell EPR of aerobically grown, aco1 yeast expressing wild-type IRP1 or a S138A-IRP1 mutant (IRP1(S138A)), providing the first direct evidence of a 3Fe intermediate in vivo. Exposure of yeast to H(2)O(2) increased this 3Fe c-aconitase signal up to 5-fold, coincident with inhibition of c-aconitase activity. Untreated yeast expressing IRP1(S138D) or IRP1(S138E), which mimic phosphorylated IRP1, failed to give a 3Fe signal. H(2)O(2) produced a weak 3Fe signal in yeast expressing IRP1(S138D). Yeast expressing IRP1(S138D) or IRP1(S138E) were the most sensitive to inhibition of aconitase-dependent growth by H(2)O(2) and were more responsive to changes in media iron status. Ferricyanide oxidation of anaerobically reconstituted c-aconitase yielded a strong 3Fe EPR signal with wild-type and S138A c-aconitases. Only a weak 3Fe signal was obtained with S138D c-aconitase, and no signal was obtained with S138E c-aconitase. This, paired with loss of c-aconitase activity, strongly argues that the Fe-S clusters of these phosphomimetic c-aconitase mutants undergo more complete disassembly upon oxidation. Our results demonstrate that 3Fe c-aconitase is an intermediate in H(2)O(2)-initiated Fe-S cluster disassembly in vivo and suggest that cluster assembly/disassembly in IRP1 is a dynamic process in aerobically growing yeast. Further, our results support the view that phosphorylation of IRP1 can modulate its response to iron through effects on Fe-S cluster stability and turnover.     

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.     

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.     

Human ferrochelatase: crystallization, characterization of the [2Fe-2S] cluster and determination that the enzyme is a homodimer

Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX to form protoheme IX. Previously we have demonstrated that the mammalian enzyme is associated with the inner surface of the inner mitochondrial membrane and contains a nitric oxide sensitive [2Fe-2S] cluster that is coordinated by four Cys residues whose spacing in the primary sequence is unique to animal ferrochelatase. We report here the characterization and crystallization of recombinant human ferrochelatase with an intact [2Fe-2S] cluster. Gel filtration chromatography and dynamic light scattering measurements revealed that the purified recombinant human ferrochelatase in detergent solution is a homodimer. EPR redox titrations of the enzyme yield a midpoint potential of -453+/-10 mV for the [2Fe-2S] cluster. The form of the protein that was crystallized has a single Arg to Leu substitution. This mutation has no detectable effect on enzyme activity but is critical for crystallization. The crystals belong to the space group P2(1)2(1)2(1) and have unit cell constants of a=93.5 A, b=87.7 A, and c=110.2 A. There are two molecules in the asymmetric unit and the crystals diffract to better than 2.0 A resolution. The Fe to Fe distance of the [2Fe-2S] cluster is calculated to be 2.7 A based upon the Bijvoet difference Patterson map.     

Noncysteinyl coordination to the [4Fe-4S]2+ cluster of the DNA repair adenine glycosylase MutY introduced via site-directed mutagenesis. Structural characterization of an unusual histidinyl-coordinated cluster

The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine-2'-deoxyadenosine (OG*A) mismatches in DNA. MutY prevents DNA mutations caused by the misincorporation of A opposite OG by catalyzing the deglycosylation of the aberrant adenine. MutY is representative of a unique subfamily of DNA repair enzymes that also contain a [4Fe-4S]2+ cluster, which has been implicated in substrate recognition. Previously, we have used site-directed mutagenesis to individually replace the cysteine ligands to the [4Fe-4S]2+ cluster of E. coli MutY with serine, histidine, or alanine. These experiments suggested that histidine coordination to the iron-sulfur cluster may be accommodated in MutY at position 199. Purification and enzymatic analysis of C199H and C199S forms indicated that these forms behave nearly identical to the WT enzyme. Furthermore, introduction of the C199H mutation in a truncated form of MutY (C199HT) allowed for crystallization and structural characterization of the modified [4Fe-4S] cluster coordination. The C199HT structure showed that histidine coordinated to the iron cluster although comparison to the structure of the WT truncated enzyme indicated that the occupancy of iron at the modified position had been reduced to 60%. Electron paramagnetic resonance (EPR) spectroscopy on samples of C199HT indicates that a significant percentage (15-30%) of iron clusters were of the [3Fe-4S]1+ form. Oxidation of the C199HT enzyme with ferricyanide increases the amount of the 3Fe cluster by approximately 2-fold. Detailed kinetic analysis on samples containing a mixture of [3Fe-4S]1+ and [4Fe-4S]2+ forms indicated that the reactivity of the [3Fe-4S]1+ C199HT enzyme does not differ significantly from that of the WT truncated enzyme. The relative resistance of the [4Fe-4S]2+ cluster toward oxidation, as well as the retention of activity of the [3Fe-4S]1+ form, may be an important aspect of the role of MutY in repair of DNA damage resulting from oxidative stress.