The 5'-adenylyl sulfate (APS) reductase from the marine macrophytic green alga Enteromorpha intestinalis uses reduced glutathione as the electron donor for the reduction of APS to 5'-AMP and sulfite. The E. intestinalis enzyme (EiAPR) is composed of a reductase domain and a glutaredoxin-like C-terminal domain. The enzyme contains a single [4Fe-4S] cluster as its sole prosthetic group. Three of the enzyme's eight cysteine residues (Cys166, Cys257, and Cys260) serve as ligands to the iron-sulfur cluster. Site-directed mutagenesis experiments and resonance Raman spectroscopy are consistent with the presence of a cluster in which only three of the four ligands to the cluster irons contributed by the protein are cysteine residues. Site-directed mutagenesis experiments suggest that the thiol group of Cys250, a residue found only in algal APS reductases, is not an absolute requirement for activity. The other four cysteines that do not serve as cluster ligands, all of which are required for activity, are involved in the formation of two redox-active disulfide/dithiol couples. The couple involving Cys342 and Cys345 has an E-m value at pH 7.0 of -140 mV, and the one involving Cys165 and Cys285 has an E-m value at pH 7.0 of -290 mV. The C-terminal portion of EiAPR, expressed separately, exhibits the cystine reductase activity characteristic of glutaredoxins. It is proposed that the Cys342-Cys345 disulfide provides the site for entry of electrons from reduced glutathione and that the Cys166-Cys285 disulfide may serve as a structural element that is essential for keeping the enzyme in the catalytically active conformation.

Electronic absorption and resonance Raman spectral studies of benzoylruthenocene (BRc) and 1,1'-dibenzoylruthenocene (DRc) indicate that the low-energy electronic excited states of these 4d(6) metallocenes possess metal-to-ligand charge transfer (MLCT) character. While this MLCT contribution should weaken the metal-ring bonding in the excited state, neither compound is photosensitive in nonhalogenated solvents such as methanol, acetonitrile, and cyclohexane. In contrast, irradiating BRc and DRc in the good electron-accepting solvent, carbon tetrachloride, results in ring loss via a pathway that appears to originate from a charge-transfer-to-solvent excited state. Both metallocenes function as photoinitiators for the anionic polymerization of ethyl 2-cyanoacrylate, and the kinetics and mechanism of this process have been investigated. Comparing the present results on BRc and DRc with those reported earlier for the corresponding benzoyl-substituted ferrocene compounds reveals some interesting commonalities and differences between the excited-state properties of these 3d and 4d metallocenes.

Iron, a key element in Fenton chemistry, causes oxygen-related toxicity to cells of most living organisms. Helicobacter pylori is a microaerophilic bacterium that infects human gastric mucosa and causes a series of gastric diseases. Exposure of H. pylori cells to air for 2 h elevated the level of free iron by about 4-fold as measured by electron paramagnetic resonance spectroscopy. H. pylori cells accumulated more free iron as they approached stationary phase growth, and they concomitantly suffered more DNA damage as indicated by DNA fragmentation analysis. Relationships between the intracellular free iron level, specific oxidative stress enzymes, and DNA damage were identified, and new roles for three oxidative stress-combating enzymes in H. pylori are proposed. Mutant cells defective in either catalase (KatA), in superoxide dismutase (SodB) or in alkyl hydroperoxide reductase (AhpC) were more sensitive to oxidative stress conditions; and they accumulated more free (toxic) iron; and they suffered more DNA fragmentation compared to wild type cells. A significant proportion of cells of sodB, ahpC, or katA mutant strains developed into the stress-induced coccoid form or lysed; they also contained significantly higher amounts of 8-oxo-guanine associated with their DNA, compared to wild type cells.

APS reductase from Pseudomonas aeruginosa has been shown to form a disulfide-linked adduct with mono-cysteine variants of Escherichia coli thioredoxin and Chlamydomonas reinhardtii thioredoxin h1. These adducts presumably represent trapped versions of the intermediates formed during the catalytic cycle of this thioredoxin-dependent enzyme. The oxidation–reduction midpoint potential of the disulfide bond in the P. aeruginosa APS reductase/C. reinhardtii thioredoxin h1 adduct is −280 mV. Site-directed mutagenesis and mass spectrometry have identified Cys256 as the P. aeruginosa APS reductase residue that forms a disulfide bond with Cys36 of C. reinhardtii TRX h1 and Cys32 of E. coli thioredoxin in these adducts. Spectral perturbation measurements indicate that P. aeruginosa APS reductase can also form a non-covalent complex with E. coli thioredoxin and with C. reinhardtii thioredoxin h1. Perturbation of the resonance Raman and visible-region absorbance spectra of the APS reductase [4Fe–4S] center by either APS or the competitive inhibitor 5′-AMP indicates that both the substrate and product bind in close proximity to the cluster. These results have been interpreted in terms of a scheme in which one of the redox-active cysteine residues serves as the initial reductant for APS bound at or in close proximity to the [4Fe–4S] cluster.

APS reductase from Pseudomonas aeruginosa has been shown to contain a [4Fe-4S] cluster. Thiol determinations and site-directed mutagenesis studies indicate that the single [4Fe-4S] cluster contains only three cysteine ligands, instead of the more typical arrangement in which clusters are bound to the protein by four cysteines. Resonance Raman studies in the Fe-S stretching region are also consistent with the presence of a redox-inert [4Fe-4S](2+) cluster with three cysteinate ligands and indicate that the fourth ligand is likely to be an oxygen-containing species. This conclusion is supported by resonance Raman and electron paramagnetic resonance (EPR) evidence for near stoichiometric conversion of the cluster to a [3Fe-4S](+) form by treatment with a 3-fold excess of ferricyanide. Site-directed mutagenesis experiments have identified Cys139, Cys228, and Cys231 as ligands to the cluster. The remaining two cysteines present in the enzyme, Cys140 and Cys256, form a redox-active disulfide/dithiol couple (E(m) = -300 mV at pH 7.0) that appears to play a role in the catalytic mechanism of the enzyme.

In the gastric pathogen Helicobacter pylori, catalase (KatA) and alkyl hydroperoxide reductase (AhpC) are two highly abundant enzymes that are crucial for oxidative stress resistance and survival of the bacterium in the host. Here we report a connection unidentified previously between the two stress resistance enzymes. We observed that the catalase in ahpC mutant cells in comparison with the parent strain is inactivated partially (approximately 50%). The decrease of catalase activity is well correlated with the perturbation of the heme environment in catalase, as detected by electron paramagnetic resonance spectroscopy. To understand the reason for this catalase inactivation, we examined the inhibitory effects of hydroperoxides on H. pylori catalase (either present in cell extracts or added to the purified enzyme) by monitoring the enzyme activity and the EPR signal of catalase. H. pylori catalase is highly resistant to its own substrate, without the loss of enzyme activity by treatment with a molar ratio of 1:3000 H2O2. However, it inactivated is by lower concentrations of organic hydroperoxides (the substrate of AhpC). Treatment with a molar ratio of 1:400 t-butyl hydroperoxide resulted in an inactivation of catalase by approximately 50%. UV-visible absorption spectra indicated that the catalase inactivation by organic hydroperoxides is caused by the formation of a catalytically incompetent compound II species. To further support the idea that organic hydroperoxides, which accumulate in the ahpC mutant cells, are responsible for the inactivation of catalase, we compared the level of lipid peroxidation found in ahpC mutant cells with that found in wild type cells. The results showed that the total amount of extractable lipid hydroperoxides in the ahpC mutant cells is approximately three times that in the wild type cells. Our findings reveal a novel role of the organic hydroperoxide detoxification system in preventing catalase inactivation.

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase (BSOR) catalyzes the reduction of d-biotin d-sulfoxide to biotin and contains the molybdopterin guanine dinucleotide (MGD) cofactor as its sole prosthetic group. Comparison of the primary sequences of BSOR and the closely related enzyme dimethyl sulfoxide reductase (DMSOR) indicated a number of conserved residues, including an active-site tryptophan residue (W90), which has been suggested to be involved in hydrogen bonding to the oxo group on the Mo(VI) center in BSOR. Site-directed mutagenesis has been used to replace tryptophan 90 in BSOR with phenylalanine, tyrosine, and alanine residues to examine the role of this residue in catalysis. All three BSOR mutant proteins were purified to homogeneity and contained MGD. The mutant proteins retained very limited activity toward the oxidizing substrates tested, with W90F retaining the most activity (3.4% of wild type). All three W90 mutant proteins exhibited greatly reduced kcat values compared to that of the wild-type enzyme, which was accompanied by little change in Kmapp. In addition, the mutant proteins had perturbed visible absorption and circular dichroism spectra suggesting different oxidation states of the Mo center. Purified samples of wild-type BSOR did not exhibit electron paramagnetic resonance (EPR) signals indicating a Mo(VI) center. After redox-cycling, partially reduced samples of wild-type BSOR revealed a proton-split S=1/2 Mo(V) resonance (g1,2,3=1.999, 1.981, 1.967; A1,2,3=1.40, 1.00, 1.05 mT) analogous to that observed in DMSOR. In contrast, EPR studies of the purified W90 mutant proteins revealed distinct S=1/2 Mo(V) resonances that were resistant to both oxidation and reduction, indicating that the Mo was trapped in the intermediate Mo(V) oxidation state. These results strongly suggest that W90 in BSOR plays a critical role in catalysis by serving as a hydrogen bond donor to the oxo group on the Mo(VI) center.

Analysis of the genome of the hyperthermophilic bacterium Aquifex aeolicus has revealed the presence of a previously undetected gene potentially encoding a plant- and mammalian-type [2Fe-2S] ferredoxin. Expression of that gene in Escherichia coli has yielded a novel thermostable [2Fe-2S] ferredoxin (designated ferredoxin 5) whose sequence is most similar to those of ferredoxins involved in the assembly of iron-sulfur clusters (Isc-Fd). It nevertheless differs from the latter proteins by having deletions near its N- and C-termini, and no cysteine residues other than those involved in [2Fe-2S] cluster coordination. Resonance Raman, low-temperature MCD and EPR studies show close spectral similarities between ferredoxin 5 and the Isc-Fd from Azotobacter vinelandii. Mössbauer spectra of the reduced protein were analyzed with an S = 1/2 spin Hamiltonian and interpreted in the framework of the ligand field model proposed by Bertrand and Gayda. The redox potential of A. aeolicus ferredoxin 5 (-390 mV) is in keeping with its relatedness to Isc-Fd. Unfolding experiments showed that A. aeolicus ferredoxin 5 is highly thermostable (T(m) = 106 degrees C at pH 7), despite being devoid of features (e.g., high content of charged residues) usually associated with extreme thermal stability. Searches for genes potentially encoding plant-type [2Fe-2S] ferredoxins have been performed on the sequenced genomes of hyperthermophilic organisms. None other than the two proteins from A. aeolicus were retrieved, indicating that this otherwise widely distributed group of proteins is barely represented among hyperthermophiles.

Resonance Raman spectroscopy has been employed to probe the excited-state distortions associated with the low-energy electronic transition of benzoylferrocene and 1,1'-dibenzoylferrocene. Resonance intensity enhancement of in-plane ligand modes, in general, and the carbonyl stretching mode, in particular, supports the proposal that the excited state populated by this transition contains appreciable metal-to-ligand charge transfer character. The redistribution of charge that occurs upon populating this state weakens the metal-ring bonding and facilitates the loss of a benzoylcyclopentadienide anion. This photochemical reaction has been investigated by an on-line electrospray ionization mass spectrometry technique that allows direct detection of primary and secondary products with solution lifetimes down to the millisecond range.

The roles of two evolutionarily conserved aromatic residues in the cytochrome c(1) component of the Rhodobacter capsulatus cytochrome bc(1) complex, phenylalanine 138 and tyrosine 194, were analyzed by site-directed mutagenesis, in combination with biophysical and biochemical measurements. Changing Phe138 to either alanine or valine, but not to tyrosine, results in redox heterogeneity of cytochrome c(1). Replacement of Phe138 by an aliphatic amino acid also caused changes in the EPR spectrum of the cytochrome and resulted in decreases in the steady-state V(max) for the hydroquinone/cytochrome c oxidoreductase activity of cytochrome bc(1) complexes containing the mutated cytochrome c(1). These findings indicate that the presence of an aromatic residue at position 138 is essential for maintaining the native environment of the cytochrome c(1) heme. In contrast, replacement of Tyr194 by aliphatic amino acids had no significant effect on either the E(m) of cytochrome c(1) or the steady-state activity parameters. Site-directed mutagenesis of glutamate and aspartate residues in a conserved acidic patch (region 2) on Rb. capsulatus cytochrome c(1) suggests that these negatively charged residues do not play a role in the docking of cytochrome c(2) with the cytochrome bc(1) complex.

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex molybdenum-, cytochrome b557- and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. To facilitate structure/function studies of the individual molybdenum center, we have developed bacterial expression systems for the heterologous production of the 541 residue amino-terminal, molybdenum center-containing domain of spinach nitrate reductase either as a six-histidine-tagged variant or as a glutathione-S-transferase-tagged fusion protein. Expression of the his-tagged molybdenum domain in Escherichia coli BL21(DE3) cells under anaerobic conditions yielded a 55-kDa domain with a specific activity of 1.5 μmol NO3− consumed/min/nmol enzyme and with a KmappNO3− of 8 μM. In contrast, expression of the molybdenum domain as a GST-tagged fusion protein in E. coli TP1000 (MobA− strain) cells under aerobic conditions yielded an 85-kDa fusion protein with a specific activity of 10.8 μmol NO3− consumed/min/nmol enzyme and with a KmappNO3− of 12 μM. Fluorescence analysis indicated that both forms of the molybdenum domain contained the cofactor, MPT, although the MPT content was higher in the GST-fusion domain. Inductively coupled plasma mass spectrometric analysis of both the his-tagged and GST-fusion protein domain samples indicated Mo/protein ratios of 0.44 and 0.93, respectively, confirming a very high level of Mo incorporation in the GST-fusion protein. Expression of the GST-fusion protein in TP1000 cells in the presence of elevated tungsten concentrations resulted in an 85-kDa fusion protein that contained MPT but which was devoid of nitrate-reducing activity. Partial reduction of the molybdenum domain resulted in the generation of an axial Mo(V) EPR species with g values of 1.9952, 1.9693, and 1.9665, respectively, and exhibiting superhyperfine coupling to a single exchangeable proton, analogous to that previously observed for the native enzyme. In contrast, the tungsten-substituted MPT-containing domain yielded a W(V) EPR species with g values of 1.9560, 1.9474, and 1.9271, respectively, with unresolved superhyperfine interaction. NADH:nitrate reductase activity could be reconstituted using the GST-molybdenum domain fusion protein in the presence of the recombinant forms of the spinach nitrate reductase' flavin- and heme-containing domains.

The ability to detect biomolecules in single cells is important in order to fully understand the processes by which many biochemical events occur. To that end, we have developed a bioluminescence binding assay capable of measuring the intracellular biotin content of individual cells. The assay depends on competition between an aequorin-biotin conjugate (AEQ-biotin) and free biotin within the oocytes for binding sites on the protein avidin. The assay is performed by microinjecting each component into the oocytes and following the resulting bioluminescence within the oocyte upon triggering of aequorin. Results obtained using sea urchin oocytes show that the assay performed within the cells behaves in a manner consistent with assay theory. Using the assay, the individual biotin content of the oocytes is an average of approximately 20 amol. To our knowledge, this is the first reported multicomponent binding assay to be performed inside an intact single cell.

A homogeneous binding assay for the detection of biotin in picoliter vials was developed using the photoprotein aequorin as the label. The binding assay was based on the competition of free biotin with biotinylated aequorin (AEQ-biotin) for avidin. A sequential protocol was used, and modification of the assay to reduce the number of steps was examined. Results showed that detection limits on the order of 10(-14) mol of biotin were possible. Reducing the number of steps provided similar detection limits but only if the amount of avidin used was decreased. These binding assays based on picoliter volumes have potential applications in a variety of fields, including microanalysis and single-cell analysis, where the amount of sample is limited. In addition, these assays are suitable for the high-throughput screening of biopharmaceuticals.

The electronic and redox properties of the iron and tungsten centers in the aldehyde ferredoxin oxidoreductases (AORs) from Pyrococcus furiosus (Pf) and Pyrococcus strain ES-4 (ES-4) have been investigated by the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. Parallel- and perpendicular-mode EPR studies of ES-4 AOR reveal a redox inactive ''g = 16'' resonance from an integer spin paramagnet. On the basis of the X-ray crystal structure of Pf AOR (Chan,M. K.; Mukund, S.; Kletzin, A.; Adams, M. W. W.; Rees, D. C. Science 1995, 267, 1463-1469), this resonance is attributed to a mononuclear high-spin Fe(II) ion at the subunit interface, although the possibility that this center is a carboxylate-bridged reduced diiron center in ES-4 AOR is also considered. Both enzymes have a [4Fe-4S](2+,+) cluster with unique electronic properties compared to known synthetic or biological [4Fe-4S](+) clusters, i.e. pure S = 3/2 ground state with g = 4.7, 3.4, 1.9 (E/D = 0.12 and D = +4 cm(-1)). Seven distinct W(V) EPR signals have been observed during dye-mediated redox titrations of Pf AOR, and the four major W(V) species have been rigorously identified and characterized via EPR spectral simulations of natural abundance and W-183-enriched samples (W-183, I = 1/2, 14.28% natural abundance). Both enzymes contain two major forms of W, each corresponding to approximately 20-30% of the total W. One of these is a catalytically competent W species that cycles between the W(IV)/W(V)/W(VI) states at physiologically relevant potentials (<-300 mV) and gives rise to the ''low-potential'' W(V) resonance, g similar to 1.99, 1.90, 1.86. This form of W is quantitatively and irreversibly converted into a distinct and inactive W(IV)/W(V) Species by the addition of high concentrations of glycerol or ethylene glycol at 80 degrees C and is responsible for-the ''diol-inhibited'' W(V) resonance, g similar to 1.96, 1.94, 1.89. The other major form of W gives rise to a ''high-potential'' W(V) species, g similar to 1.99, 1.96, 1.89, at nonphysiologically relevant potentials (0 mV), as a result of a one-electron redox process that is tentatively attributed to ligand based oxidation of a W(VI) species. In addition, active samples of Pf AOR, in particular, can have up to 20% of the W as an inactive W(VI)/W(V) species, with a midpoint potential close to -450 mV, and is responsible for the ''spin-coupled'' W(V) resonance. This W(V) signal exhibits a broad complex resonance spanning 600 mT due to weak spin-spin interaction with the nearby S = 3/2 [4Fe-4S](+) cluster. Structures are proposed for each; of the major W(V) species on the basis of EPR g values and W-183 A values as compared to other biological and synthetic W(V)/Mo(V) centers, VTMCD spectra, and the available X-ray crystallographic and XAS data for Pf AOR and the Mo-containing DMSO reductase from Rhodobacter sphaeroides. Comparison with the limited spectroscopic data that are available for all known tungstoenzymes suggests two major classes of enzyme with distinct active site structures.

The ground and excited state properties and ligand-binding capabilities of [ZnFe3S4](2+,+), [CoFe3S4](2+,+), and [MnFe3S4](+) clusters in Pyrococcus furiosus Fd have been investigated by the combination of EPR, variable-temperature magnetic circular dichroism (VTMCD), and MCD magnetization studies. The ground state spins, S = 5/2 for [ZnFe3S4](+), S = 2 for [ZnFe3S4](2+), S = 1 for [CoFe3S4](+), S = 1/2 for [CoFe3S4](2+), and S = 0 for [MnFe3S4](+), are consistent with a simple coupling scheme involving antiparallel interaction between the high-spin divalent metal ion and an S = 5/2 [Fe3S4](+) or S = 2 [Fe3S4](0) cluster fragment. Redox potentials (vs NHE) were determined by dye-mediated EPR titrations at pH 7.6, E(m) -241 +/- 20 mV for [ZnFe3S4](2+,+) E(m) = -163 +/- 10 mV for [CoFe3S4](2+,+), and E(m) > -100 mV for [MnFe3S4](2+,+), indicating that the potentials for [MFe(3)S(4)](2+,+) clusters in P. furiosus Fd are ordered M = Fe < Zn < Co < Mn or Ni. On the basis of changes in EPR and/or VTMCD spectra, evidence is presented for cyanide binding at the unique metal site of the [ZnFe3S4](+) and [CoFe3S4](+) clusters and for 2-mercaptoethanol binding at the Zn site of the [ZnFe3S4](+) cluster. The ground and excited state properties of the thiolate-bound [ZnFe3S4](+) cluster in P. furiosus Fd are very similar to those of the equivalent cluster in Desulfovibrio gigas FdII, indicating coordination of the Zn by the indigenous cysteinate in this protein. The site specific reactivity, intracluster magnetic interactions, and redox properties of heteronuclear cubanes in general are discussed in light of these results.

The hyperthermophilic archaeon Pyrococcus furiosus contains a novel 4Fe ferredoxin in which one Fe ion lacks cysteinyl coordination. This unique Fe ion can be easily removed to yield protein containing a [Fe3S4](0) cluster. Under reducing conditions, this cluster can bind exogenous metal dications, M(2+) (e.g., Ni2+ and Zn2+), to yield [MFe(3)S(4)](+) clusters. In this work, we have investigated the affinity of the [Fe3S4](0,+) + cluster in P. furiosus ferredoxin for the monocations Cs+ and Tl+ in the absence of reducing agents. Both of these metal ions are large and polarizable, but they differ greatly in their propensity for ionic versus covalent interactions. The structural, electronic, and magnetic properties of the [Fe3S4](0,+) + cluster in P. furiosus ferredoxin in the presence of excess Cs+ and Tl+ were studied by EPR, magnetic circular dichroism, resonance Raman, and electron-nuclear double resonance spectroscopy. Magnetic circular dichroism and resonance Raman studies indicate that Tl+ but not Cs+ is incorporated into the reduced [Fe3S4](0) cluster with retention of the S = 2 (D < 0) ground state to yield a [TlFe3S(4)](+) cluster. EPR studies provide evidence for Tl+ incorporation into the oxidized S = 1/2 [Fe3S4](+) cluster as well. The native protein exhibits a broad EPR signal as a result of the distribution of g-values from multiple cluster conformations. In the presence of excess Tl+, a much narrower axial EPR signal is observed, indicating a single cluster conformation. Furthermore,Tl-203,Tl-205 hyperfine coupling was observed at both 9 and 35 GHz. The large coupling constant, A(Tl) approximate to 370 MHz (13 mT), indicates a covalent interaction associated with the formation of [TlFe3S4](2+). In contrast, the presence of excess Cs+ does not change the EPR spectrum, nor is Cs-133 hyperfine coupling observed, indicating a failure to incorporate this ion, However, Cs-133 electron-nuclear double resonance signals were observed with hyperfine and quadrupole couplings of A(Cs) approximate to 1.2 MHz, P-z approximate to 0.7 MHz. This, in conjunction with resonance Raman data, suggests that a Cs+ ion binds to a specific residue near the oxidized cluster. This is the first report of Cs-133 ENDOR in a biological system and suggests that this readily available nucleus could provide a valuable probe for Na+ or K+ binding in paramagnetic biomolecules.