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.     

Evidence for participation of iron in lipoxygenase reaction from optical and electron spin resonance studies.

Optical and EPR studies indicate that the iron present in lipoxygenase participates in catalysis. Addition of linoleic acid hydroperoxide to lipoxygenase 1 causes an increase in abosrbance over the range of 350 to 650 nm which is reversed when linoleic acid hydroperoxide is destroyed upon the addition of linoleic acid under anaerobic conditions. Lipoxygenase 1 alone exhibits no EPR signal but upon addition of linoleic acid hydroperoxide or linoleic acid several signals appear. Addition of linoleic acid hydroperoxide results in an EPR signal at g approximately equal to 6 accompanied by a small but relatively sharp signal at g approximately equal to 2. Under anaerobic conditions the latter is replaced by a broad anisotropic signal around g approximately equal to 2. The appearance of the EPR signal at g approximately equal to 6 coincides with the change in the optical spectrum of the enzyme. When linoleic acid is added under anaerobic conditions a broad anisotropic EPR signal around g approximately equal to 2 is observed. Thus it appears that lipoxygenase can exist in two forms: (a) a resting form with a very weak absorbance in the visible range of the light spectrum and no EPR signal and (b) an active form (after addition of linoleic acid hydroperoxide) with an increased optical absorbance and EPR signal at g approximately equal to 6. This observation may be related to the earlier discovery that the lipoxygenase reaction occurs with a lag which can be overcome by addition of product hydroperoxide. The EPR experiments indicate that lipoxygenase in the active form contains high spin ferric ion. Although EPR signals in the g approximately equal to 6 region are frequently observed with heme proteins, the only nonheme protein, other than lipoxygenase, reported to show an EPR signal in this region is the phenolytic dioxygenase, protocatechuate 3,4-dioxygenase (Peisach, J., Fujisawa, H., Blumberg, W. E., and Hayaishi, O. (1972) Fed. Proc. 31, 448).     

X-ray absorption spectroscopy of soybean lipoxygenase-1. Influence of lipid hydroperoxide activation and lyophilization on the structure of the non-heme iron active site.

X-ray absorption spectra at the Fe K-edge of the non-heme iron site in Fe(II) as well as Fe(III) soybean lipoxygenase-1, in frozen solution or lyophilized, are presented; the latter spectra were obtained by incubation of the Fe(II) enzyme with its product hydroperoxide. An edge shift of about 2-3 eV to higher energy occurs upon oxidation of the Fe(II) enzyme to the Fe(III) species, corresponding to the valence change. The extended X-ray absorption fine structure shows clear differences in active-site structure as a result of this conversion. Curve-fitting on the new data of the Fe(II) enzyme, using the EXCURV88 program, leads to a coordination sphere that is in agreement with the active-site structure proposed earlier (6 +/- 1 N/O ligands at 0.205-0.209 nm with a maximum variance of 0.009 nm, including 4 +/- 1 imidazole ligands) [Navaratnam, S., Feiters, M. C., Al-Hakim, M., Allen, J. C., Veldink, G. A. and Vliegenthart, J. F. G. (1988) Biochim. Biophys. Acta 956, 70-76], while for the Fe(III) enzyme a shortening in ligand distances occurs (6 +/- 1 N/O ligands at 0.200-0.203 nm with maximum variance of 0.008 nm) and one imidazole is replaced by an oxygen ligand of unknown origin. Lyophilization does not lead to any apparent differences in the iron coordination of either species and gives a much better signal/noise ratio, allowing analysis of a larger range of data.     

Simultaneous determination of Fe(III) and Fe(II) in water solutions and tissue homogenates using desferal and 1,10-phenanthroline

At neutral pH values 1,10-phenanthroline forms a colored complex with Fe(II), but it does not form such a complex with Fe(III). On the contrary, only Fe(III) forms with desferal a yellow complex with a g = 4.3 electron paramagnetic resonance (EPR) signal, but Fe(II) is rapidly oxidized by desferal to Fe(III), which gives then a yellow complex. On the basis of these facts, a method for simultaneous determination of both Fe(II) and Fe(III) ions was elaborated using a desferal-phenanthroline mixture. Two ways of detecting Fe(II) and Fe(III) have been suggested: (1) the spectrophotometric method for transparent water solutions, and (2) the EPR-spectrometric method for turbid solutions and tissue homogenates. The latter method was applied for determination of free and weakly bound iron in rat liver. The Fe(II) amount in intact liver was 22.2 ± 7.6 nmol/g of wet tissue; free Fe(III) was not found.     

Coordination changes and auto-hydroxylation of FIH-1: uncoupled O2-activation in a human hypoxia sensor

Hypoxia sensing is the generic term for pO₂-sensing in humans and other higher organisms. These cellular responses to pO₂ are largely controlled by enzymes that belong to the Fe(II) α-ketoglutarate (αKG) dependent dioxygenase superfamily, including the human enzyme called the factor inhibiting HIF (FIH-1), which couples O₂-activation to the hydroxylation of the hypoxia inducible factor α (HIFα). Uncoupled O₂-activation by human FIH-1 was studied by exposing the resting form of FIH-1 (αKG + Fe)FIH-1, to air in the absence of HIFα. Uncoupling lead to two distinct enzyme oxidations, one a purple chromophore (λ_max = 583 nm) arising from enzyme auto-hydroxylation of Trp²⁹⁶, forming an Fe(III)-O-Trp²⁹⁶ chromophore [Y.-H. Chen, L.M. Comeaux, S.J. Eyles, M.J. Knapp, Chem. Commun. (2008), doi:10.1039/B809099H]; the other a yellow chromophore due to Fe(III) in the active site, which under some conditions also contained variable levels of an oxygenated surface residue (oxo)Met²⁷⁵. The kinetics of purple FIH-1 formation were independent of Fe(II) and αKG concentrations, however, product yield was saturable with increasing [αKG] and required excess Fe(II). Yellow FIH-1 was formed from (succinate + Fe)FIH-1, or by glycerol addition to (αKG + Fe)FIH-1, suggesting that glycerol could intercept the active oxidant from the FIH-1 active site and prevent hydroxylation. Both purple and yellow FIH-1 contained high-spin, rhombic Fe(III) centers, as shown by low temperature EPR. XAS indicated distorted octahedral Fe(III) geometries, with subtle differences in inner-shell ligands for yellow and purple FIH-1. EPR of Co(II)-substituted FIH-1 (αKG + Co)FIH-1, indicated a mixture of 5-coordinate and 6-coordinate enzyme forms, suggesting that resting FIH-1 can readily undergo uncoupled O₂-activation by loss of an H₂O ligand from the metal center.     

Spectroscopic studies on the interaction of phosphate with uteroferrin

The effect of phosphate on the binuclear iron center of pink (reduced) uteroferrin was examined by magnetic resonance and optical spectroscopy. The purple (oxidized) protein, which contains 1 mol of tightly bound phosphate per mol of enzyme at isolation, does not give rise to a 31P NMR signal. Phosphate binding to phosphate-stripped pink uteroferrin is indistinguishable from that in the native purple phosphoprotein. As measured by EPR and optical spectroscopy, the rate of reaction between phosphate and pink uteroferrin is pH-dependent, decreasing as the pH increases. Phosphate is capable of binding to the reduced protein between pH 3 and 7.8, resulting in formation of the purple uteroferrin-phosphate complex. Evans susceptibility measurements at pH 4.9 indicate that the EPR silent species with a maximum absorption at 535 nm, generated upon phosphate addition to pink uteroferrin, is diamagnetic. Moreover, phosphate causes disappearance of the hyperfine-shifted resonances in the 1H NMR spectra of the reduced protein. We therefore have not been able to identify the paramagnetic "purple reduced enzyme-phosphate complex" reported by Pyrz et al. (Pyrz, J. W., Sage, J. T., Debrunner, P. G., and Que, Jr., L. (1986) J. Biol Chem. 261, 11015-11020) using Mossbauer spectroscopy and dithionite-reduced 57Fe-reconstituted uteroferrin. Our present data with native unmodified enzyme are in accord with our earlier results (Antanaitis, B. C., and Aisen, P. (1985) J. Biol. Chem. 260, 751-756) and with the results of Burman et al. (Burman, S., Davis, J. C., Weber, M. J., and Averill, B. A. (1986) Biochem. Biophys. Res. Commun. 136, 490-497) on bovine spleen phosphatase, suggesting that phosphate binding to reduced protein rapidly induces oxidation of the binuclear iron center.     

Single turnover of substrate-bound ferric cysteine dioxygenase with superoxide anion: enzymatic reactivation, product formation, and a transient intermediate

Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the O(2)-dependent oxidation of L-cysteine (Cys) to produce cysteine sulfinic acid (CSA). In this study we demonstrate that the catalytic cycle of CDO can be "primed" by one electron through chemical oxidation to produce CDO with ferric iron in the active site (Fe(III)-CDO, termed 2). While catalytically inactive, the substrate-bound form of Fe(III)-CDO (2a) is more amenable to interrogation by UV-vis and EPR spectroscopy than the 'as-isolated' Fe(II)-CDO enzyme (1). Chemical-rescue experiments were performed in which superoxide (O(2)(?-)) anions were introduced to 2a to explore the possibility that a Fe(III)-superoxide species represents the first intermediate within the catalytic pathway of CDO. In principle, O(2)(?-) can serve as a suitable acceptor for the remaining 3-electrons necessary for CSA formation and regeneration of the active Fe(II)-CDO enzyme (1). Indeed, addition of O(2)(?-) to 2a resulted in the rapid formation of a transient species (termed 3a) observable at 565 nm by UV-vis spectroscopy. The subsequent decay of 3a is kinetically matched to CSA formation. Moreover, a signal attributed to 3a was also identified using parallel mode X-band EPR spectroscopy (g ~ 11). Spectroscopic simulations, observed temperature dependence, and the microwave power saturation behavior of 3a are consistent with a ground state S = 3 from a ferromagnetically coupled (J ~ -8 cm(-1)) high-spin ferric iron (S(A) = 5/2) with a bound radical (S(B) = 1/2), presumably O(2)(?-). Following treatment with O(2)(?-), the specific activity of recovered CDO increased to ~60% relative to untreated enzyme.     

Oxidase reaction of cytochrome cd(1) from Paracoccus pantotrophus

Cytochrome cd(1) (cd(1)NIR) from Paracoccus pantotrophus, which is both a nitrite reductase and an oxidase, was reduced by ascorbate plus hexaamineruthenium(III) chloride on a relatively slow time scale (hours required for complete reduction). Visible absorption spectroscopy showed that mixing of ascorbate-reduced enzyme with oxygen at pH = 6.0 resulted in the rapid oxidation of both types of heme center in the enzyme with a linear dependence on oxygen concentration. Subsequent changes on a longer time scale reflected the formation and decay of partially reduced oxygen species bound to the d(1) heme iron. Parallel freeze-quench experiments allowed the X-band electron paramagnetic resonance (EPR) spectrum of the enzyme to be recorded at various times after mixing with oxygen. On the same millisecond time scale that simultaneous oxidation of both heme centers was seen in the optical experiments, two new EPR signals were observed. Both of these are assigned to oxidized heme c and resemble signals from the cytochrome c domain of a "semi-apo" form of the enzyme for which histidine/methionine coordination was demonstrated spectroscopically. These observations suggests that structural changes take around the heme c center that lead to either histidine/methionine axial ligation or a different stereochemistry of bis-histidine axial ligation than that found in the as prepared enzyme. At this stage in the reaction no EPR signal could be ascribed to Fe(III) d(1) heme. Rather, a radical species, which is tentatively assigned to an amino acid radical proximal to the d(1) heme iron in the Fe(IV)-oxo state, was seen. The kinetics of decay of this radical species match the generation of a new form of the Fe(III) d(1) heme, probably representing an OH(-)-bound species. This sequence of events is interpreted in terms of a concerted two-electron reduction of oxygen to bound peroxide, which is immediately cleaved to yield water and an Fe(IV)-oxo species plus the radical. Two electrons from ascorbate are subsequently transferred to the d(1) heme active site via heme c to reduce both the radical and the Fe(IV)-oxo species to Fe(III)-OH(-) for completion of a catalytic cycle.     

On the anomalous temperature behaviour of the EPR signal of monovalent nickel in hydrogenase

The dependence on temperature in the range between 4.2 K and 20 K was measured for the EPR signal of monovalent nickel in H2-reduced hydrogenase from Chromatium vinosum and from Methanobacterium thermoautotrophicum. In accordance with measurements on the hydrogenase from Desulfovibrio gigas [Teixeira, M., Moura, I., Xavier, A. V., Huynh, B. H., DerVartanian, D. V., Peck, H. D., Jr, LeGall, J. and Moura, J. J. G. (1985) J. Biol. Chem. 260, 8942-8950; and Cammack, R., Patil, D. S. and Fernandez, V. M. (1985) Biochem. Soc. Trans. 13, 572-578], the enzyme from C. vinosum showed a distinct transformation of the EPR signal of nickel in this temperature region. The light sensitivity did not change. EPR spectra recorded at 9 GHz and at 35 GHz showed that the transformation of the spectrum at 4.2 K is caused by spin coupling to an unknown paramagnet. No coupling was apparent at temperatures above 20 K. At 4.2 K, additional, very broad signals in the region g= 1.2-3, as well as a signal around g = 5, were detected In the enzyme from C. Vinosum, both in the H2-reduced state and in the Ar-reoxidised state. The possible origin of the paramagnetic species responsible for these signals is discussed. The EPR signal of monovalent nickel in the enzyme from M. thermoautotrophicum showed no significant changes in line shape between 4.2 K and 70 K, nor were any additional signals detected. This suggests that in the reduced form of this enzyme similar paramagnetic species might be absent or not reduced.     

Nitrous oxide reductase from Pseudomonas stutzeri. Redox properties and spectroscopic characterization of different forms of the multicopper enzyme

The oxidation-reduction and spectroscopic properties of various forms of nitrous oxide reductase from Pseudomonas stutzeri were investigated. The high-activity form I of the enzyme (purple, 8 Cu, Mr 140,000) was reduced by a large variety of cationic, anionic and photochemically generated agents. The blue form III was the only product found in these experiments under anaerobic conditions. Reductive (dithionite) and oxidative (ferricyanide) titrations showed that the conversion of the purple form I to the blue species III was fully reversible in the absence of dioxygen. Two kinetically different phases of the reaction of form I with a stoichiometric amount of dithionite (1e- -equivalent/Cu) were detected: in the fast phase (seconds), the purple chromophore with lamba max at 540 nm disappeared almost completely, whereas in the slower phase (minutes) the blue species with lambda max around 650 nm was generated. Irrespective of the nature of the reductant the blue species did not react even at large excess of reductant. It was reoxidized by ferricyanide, hydrogen peroxide and nitric oxide. A new, catalytically inactive derivative of nitrous oxide reductase (form V, 2 Cu, Mr 140,000) was isolated from a transposon Tn5-induced mutant with defective chromophore biosynthesis. The pink color of the mutant protein faded almost completely after addition of 0.5e- -equivalent/Cu. In this case no blue species was found, similar to earlier observations for the regenerated, catalytically inactive protein. Varying with the sample and the pH, 50-80% of the total copper of form I was in an electron-paramagnetic-resonance-(EPR)-silent state as compared to 47% in the mutant protein. The broad, featureless EPR signal recorded at 9.32 GHz for the blue, reduced form III of nitrous oxide reductase represented approximately 20% of the total copper. For the blue species no resolution enhancement was achieved at 34 GHz. At this frequency both forms I and V showed similar EPR signals with apparent g-values at 2.16 and 1.99. At 9.32 GHz, form V had an EPR signal with gII at 2.18, AII = 3.55 mT (4 or 5 lines, in contrast to form I) and gI at 2.03. Above 100 K the splitting of the gII region into seven equidistant lines in the EPR signal of the high-activity form I and the hyperfine structure of the perpendicular transition disappeared. Carbon monoxide and nitric oxide, but not nitrous oxide, had marked effects on the spectroscopic properties of the purple form I. Marked effects were also obtained for the exogenous ligands nitrite, azide, cyanate and thiocyanate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Physical and chemical studies on bacterial superoxide dismutases. Purification and some anion binding properties of the iron-containing protein of Escherichia coli B.

Highly purified iron superoxide dismutase was obtained from Escherichia coli B using a modification of the procedure of Yost and Jridovich (Yost, F. J., Jr., and Fridovich, I. (1973) J. Biol. Chem. 248, 4905-4908). The protein contained 1.8 +/- 0.2 atoms of iron per 38,700 g of protein. We have found that cyanide does not bind to the Fe3+ ion of iron dismutase but fluoride and azide have moderately large binding constants. Optical and electron paramagnetic resonance (EPR) measurements suggested that 2 fluoride ions could associate with each iron atom with the first having an association constant of approximately 520 M-1 and the second with an estimated value of 24 M-1. Activity measurements yielded an inhibition constant for fluoride of 30 M-1. At room temperature only one azide binds to the Fe3+ (K = 760 M-1) and this does not interfere with superoxide dismutase activity. Upon freezing solutions of iron superoxide dismutase in the presence of excess azide their color changes from yellow to pink. Combined EPR and optical titrations with azide suggest the presence of two binding sites on Fe3+ with only the first being occupied at room temperature and the second binding azide only upon freezing the solution. The results suggest that each Fe3+ ion of this superoxide dismutase has two coordination positions available for interaction with solute molecules but only one is necessary for catalysis of the superoxide dismutation reaction. The EPR, optical, and circular dichroism spectra of the native protein and the various fluoride and azide complexes are presented.     

Unincorporated iron pool is linked to oxidative stress and iron levels in Caenorhabditis elegans

Free radicals or reactive oxygen species (ROS) are relatively short-lived and are difficult to measure directly; so indirect methods have been explored for measuring these transient species. One technique that has been developed using Escherichia coli and Saccharomyces cerevisiae systems, relies on a connection between elevated superoxide levels and the build-up of a high-spin form of iron (Fe(III)) that is detectable by electron paramagnetic resonance (EPR) spectroscopy at g?=?4.3. This form of iron is referred to as "free" iron. EPR signals at g?=?4.3 are commonly encountered in biological samples owing to mononuclear high-spin (S?=?5/2) Fe(III) ions in sites of low symmetry. Unincorporated iron in this study refers to this high-spin Fe(III) that is captured by desferrioxamine which is detected by EPR at g value of 4.3. Previously, we published an adaptation of Fe(III) EPR methodology that was developed for Caenorhabditis elegans, a multi-cellular organism. In the current study, we have systematically characterized various factors that modulate this unincorporated iron pool. Our results demonstrate that the unincorporated iron as monitored by Fe(III) EPR at g?=?4.3 increased under conditions that were known to elevate steady-state ROS levels in vivo, including: paraquat treatment, hydrogen peroxide exposure, heat shock treatment, or exposure to higher growth temperature. Besides the exogenous inducers of oxidative stress, physiological aging, which is associated with elevated ROS and ROS-mediated macromolecular damage, also caused a build-up of this iron. In addition, increased iron availability increased the unincorporated iron pool as well as generalized oxidative stress. Overall, unincorporated iron increased under conditions of oxidative stress with no change in total iron levels. However, when total iron levels increased in vivo, an increase in both the pool of unincorporated iron and oxidative stress was observed suggesting that the status of the unincorporated iron pool is linked to oxidative stress and iron levels.     

EPR detection of protein-derived radicals in the reaction of H(2)O(2) with Fe bound in mitochondrial F(1)ATPase.

A severe inactivation is obtained upon the addition of H(2)O(2) to bovine heart F(1)ATPase samples containing Fe(III) in the nucleotide-independent site, and Fe(II) in the ATP-dependent site. EPR spectra at 4.9 K of these samples indicate that H(2)O(2) produces the complete oxidation of Fe(II) to Fe(III) and the concomitant appearance of two protein-derived radical species. The two signals (g = 2.036 and g = 2.007) display a different temperature dependence and saturation behavior. The relaxation properties of the radical at g = 2.036 suggest magnetic interaction with one of the two iron centers. Such events are not observed when H(2)O(2) is added either to native F(1)ATPase containing a high amount of Fe(II) and low amount of Fe(III) or to F(1)ATPase deprived of endogenous Fe and subsequently loaded with only Fe(III) in both sites. It is hypothesized that in F(1)ATPase samples containing both Fe(III) and Fe(II), intramolecular long-range electron transfer may occur from Fe(II) to a high oxidation state species of Fe formed in the nucleotide-independent site upon oxidation of Fe(III) by H(2)O(2).     

Optical and EPR spectroscopic studies of demetallation of hemin by L-chain apoferritins

Earlier crystallographic and spectroscopic studies had shown that horse spleen apoferritin was capable of removing the metal ion from hemin (Fe(III)-protoporphyrin IX) [G. Précigoux, J. Yariv, B. Gallois, A. Dautant, C. Courseille, B. Langlois d'Estaintot, Acta Cryst. D50 (1994) 739-743; R.R. Crichton, J.A. Soruco, F. Roland, M.A. Michaux, B. Gallois, G. Précigoux, J.-P. Mahy, D. Mansuy, Biochemistry 36 (1997) 15049-15054]. We have carried out a detailed re-analysis of this phenomenon using both horse spleen and recombinant horse L-chain apoferritins, by electron paramagnetic resonance spectroscopy (EPR) to unequivocally distinguish between heme and non-heme iron. On the basis of site-directed mutagenesis and chemical modification of carboxyl residues, our results show that the UV-visible difference spectroscopic method that was used to establish the mechanism of demetallation is not representative of hemin demetallation. EPR spectroscopy does establish, as in the initial crystallographic investigation, that hemin demetallation occurs, but it is much slower. The signal at g=4.3 corresponding to high spin non-heme-iron (III) increases while the signal at g=6 corresponding to heme-iron decreases. Demetallation by the mutant protein, while slower than the wild-type, still occurs, suggesting that the mechanism of demetallation does not only involve the cluster of four glutamate residues (Glu 53, 56, 57, 60), proposed in earlier studies. However, the mutant protein had lost its capacity to incorporate iron, as had the native protein in which the four Glu residues had been chemically modified. Interestingly, a signal at g=1.94 is also observed. This signal most likely corresponds to a mixed-valence Fe(II)-Fe(III) cluster suggesting that a redox reaction may also be involved in the mechanism of demetallation.     

Evidence for a mu-oxo-bridged binuclear iron cluster in the hydroxylase component of methane monooxygenase. M?ssbauer and EPR studies

M?ssbauer and EPR studies of a highly active hydroxylase component of methane monooxygenase isolated from Methylosinus trichosporium OB3b are reported. The M?ssbauer spectra of the oxidized (as isolated) hydroxylase show iron in a diamagnetic cluster containing an even number of Fe3+ sites. The parameters are consistent with an antiferromagnetically coupled binuclear cluster similar to those of hemerythrin and purple acid phosphatases. Upon partial reduction of the hydroxylase, an S = 1/2 EPR spectrum with g values at 1.94, 1.86, and 1.75 (gav = 1.85) is observed. Such spectra are characteristic of oxo-bridged iron dimers in the mixed valent Fe(II).Fe(III) state. Further reduction leads to the appearance of a novel EPR resonance at g = 15. Comparison with an inorganic model compound for mu-oxo-bridged binuclear iron suggests that the g = 15 signal is characteristic of the doubly reduced state of the cluster in the protein. In this state, the M?ssbauer spectra exhibit two quadrupole doublets typical of high spin Fe2+, consistent with the Fe(II).Fe(II) form of the cluster. The spectral features of the iron center of the hydroxylase in three oxidation states are all similar to those reported for mu-oxo (or mu-hydroxo)-bridged binuclear iron clusters. Since no known monooxygenase contains such a cluster, a new oxygenase mechanism is suggested. Three different preparative methods yielded hydroxylases spanning a 9-fold range in specific activity, yet the same cluster concentration and spectral characteristics were observed. Thus, other parameters than those measured here have a major influence on the activity.     

Nitrous oxide reductase from denitrifying Pseudomonas perfectomarina. Purification and properties of a novel multicopper enzyme

Nitrous oxide reductase from the denitrifying bacterium Pseudomonas perfectomarina has been isolated and purified to homogeneity. The enzyme contained about eight copper atoms/120 kDa and was composed of two presumably identical subunits. The isoelectric point was 5.1. Several spectroscopically distinct forms of the enzyme were identified. A 'pink' form of the enzyme was obtained when the purification was done aerobically. The specific activity of this species was around 30 nkat/mg protein as measured by the nitrous-oxide-dependent oxidation of photochemically reduced benzyl viologen. A 'purple' form of the enzyme, whose catalytic activity was 2-5-fold higher, was obtained when the purification was done anaerobically. The activity of both forms of the enzyme was substantially increased by dialyzing the protein against 2-(N-cyclohexylamino)ethanesulfonate buffer at pH approximately equal to 10. A maximal activity of 1000 nkat/mg protein has been obtained for the purple form using this procedure. A 'blue', enzymatically inactive form of the enzyme resulted when either the pink or the purple species was exposed to excess dithionite or ascorbate. Anaerobic, potentiometric titrations of both the purple and the pink form of the enzyme gave a Nernst factor, n540, of 0.95 and a midpoint potential, E'0,540 of +260 mV (vs SHE, 25 degrees C, Tris/HCl buffer, pH 7.5). Electron paramagnetic resonance (EPR) and optical spectra of N2O reductase suggested the presence of an unusual type 1 copper center. Type 2 copper was absent. The hyperfine splitting in the g parallel region consisted of a seven-line pattern. In the presence of excess of reductant, a broad EPR signal with g values at 2.18 and 2.06 was observed. The EPR spectra of the pink and purple forms of the enzyme were similar; however, the spectrum of the purple form was better resolved with g parallel = 2.18 (A parallel = 3.83 mT) and g perpendicular = 2.03 (A perpendicular = 2.8 mT). Most of the copper in N2O reductase was removed by anaerobic dialysis against KCN. Reaction of the apoprotein with Cu(en)2SO4 partially regenerated the optical and EPR spectra of the holoprotein; the resulting protein was enzymatically inactive. Monospecific antibodies against the copper protein strongly inhibited the N2O reductase activity of purified samples and cell-free extracts.     

Hydrogen peroxide dependent cis-dihydroxylation of benzoate by fully oxidized benzoate 1,2-dioxygenase

Rieske dioxygenases catalyze the reductive activation of O2 for the formation of cis-dihydrodiols from unactivated aromatic compounds. It is known that O2 is activated at a mononuclear non-heme iron site utilizing electrons supplied by a nearby Rieske iron sulfur cluster. However, it is controversial whether the reactive species is an Fe(III)-(hydro)peroxo or an Fe(II)-(hydro)peroxo (or electronically equivalent species formed by breaking the O-O bond). Here it is shown that benzoate 1,2 dioxygenase oxygenase component (BZDO) prepared in a form with the Rieske cluster oxidized and the mononuclear iron in the Fe(III) state can utilize H2O2 as a source of reduced oxygen to form the correct cis-dihydrodiol product from benzoate. The reaction approaches stoichiometric yield relative to the mononuclear Fe(III) concentration, being limited to a single turnover by inefficient product release from the Fe(III)-product complex. EPR and M?ssbauer studies show that the iron remains ferric throughout this single turnover "peroxide shunt" reaction. These results strongly support Fe(III)-(hydro)peroxo (or Fe(V)-oxo-hydroxo) as the reactive species because there is no source of additional reducing equivalents to form the Fe(II)-(hydro)peroxo state. This conclusion could be further tested in the case of BZDO because the peroxide shunt occurs very slowly compared with normal turnover, allowing the reactive intermediate to be trapped for spectroscopic analysis. We attribute the slow reaction rate to a forced change in the normally strict order of the substrate binding and enzyme reduction steps that regulate the catalytic cycle. The reactive intermediate is a high-spin ferric species exhibiting an unusual negative zero field splitting and other EPR and M?ssbauer spectroscopic properties reminiscent of previously characterized side-on-bound peroxide adducts of Fe(III) model complexes. If the species in BZDO is a similar adduct, its isomer shift is most consistent with an Fe(III)-hydroperoxo reactive state.     

Benzoate 1,2-dioxygenase from Pseudomonas putida: single turnover kinetics and regulation of a two-component Rieske dioxygenase

The benzoate 1,2-dioxygenase system (BZDOS) from Pseudomonas putida mt-2 catalyzes the NADH-dependent oxidation of benzoate to 1-carboxy-1,2-cis-dihydroxycyclohexa-3,5-diene. Both the oxygenase (BZDO) and reductase (BZDR) components of BZDOS have been purified and characterized kinetically and by optical, EPR, and M?ssbauer spectroscopies. BZDO has an (alpha beta)(3) subunit structure in which each alpha subunit contains a Rieske [2Fe-2S] cluster and a mononuclear iron site. Two different purification protocols were developed for BZDO allowing the mononuclear iron to be stabilized in either the Fe(III) or the Fe(II) state for spectroscopic characterization. Using single turnover reactions, it is shown that fully reduced BZDO alone is capable of yielding the cis-diol product in high yield at rates that exceed the BZDOS turnover number. At the conclusion of turnover, quantification of each oxidation state of the metal sites by EPR and M?ssbauer spectroscopies shows that the Rieske cluster and mononuclear iron are each oxidized in amounts equal to the product yield, suggesting that the two electrons required for catalysis derive from the two metal centers. These results are in agreement with our previous study of naphthalene 1,2-dioxygenase [Wolfe, M. D., Parales, J. V., Gibson, D. T., and Lipscomb, J. D. (2001) J. Biol. Chem. 276, 1945-1953], which belongs to a different Rieske dioxygenase subclass, suggesting that it is a universal characteristic of Rieske dioxygenases that oxygen activation and substrate oxidation are catalyzed by the oxygenase component alone. The EPR spectrum of the Fe(III) center after a single turnover is distinct from either of those of substrate-free or substrate-bound enzyme. The complex with this spectrum is not formed by addition of cis-diol product to the resting Fe(III) form of the enzyme but is observed when the Fe(II) form is oxidized in the presence of product. Together, these results suggest that product exchange occurs only when the mononuclear iron is reduced. Stopped-flow and rapid scan analyses monitoring the oxidation of the Rieske cluster during the single turnover reaction show that it occurs in three phases that are kinetically competent for catalysis. The rate of each phase was found to be dependent on the type of substrate present, suggesting that the substrate influences the rate of electron transfer between the metal clusters. The participation of substrate in the oxygen activation reaction suggests a new aspect of the mechanism of this process by the Rieske dioxygenase class.     

4-Nitrocatechol as a colorimetric probe for non-heme iron dioxygenases.

4-Nitrocatechol is examined as an active site probe for non-heme iron dioxygenases and found to be of value, particularly with those containing iron in the Fe(II) oxidation state. 4-Nitrocatechol is astrong competitive inhibitor of substrate oxygenation by protocatechuate 3,4-dioxygenase, forming a reversible complex with this enzyme, and by pyrocatechase. The number of binding sites per enzyme molecule titrated spectrophotometrically with 4-nitrocatechol agrees with results from previous studies with either the principal substrate or other analogues, as expected of an effective probe. Despite these facts and the observation that both enzymes cleave the same substrates at the same carbon-carbon bond, the optical and electron paramagnetic resonance (EPR) spectra of their 4-nitrocatechol complexes are remarkably different. The 4-nitocatechol-protocatechuate 3,4-dioxygenase optical spectra resemble that of the 4-nitrocatecholate ion shifted 20 to 30 nm to longer wavelength. Concomitant with this change the EPR signal centered at g equal 4.28 shows increased rhombicity (g values at 4.74, 4.28, and 3.74).In contrast, the spectrum of the 4-nitrocatechol-pyrocatechase complex has a maximum at the same wavelength as that of a 1:1 solution of free Fe(II) and 4-nitrocatechol in the absence of enzyme after titration of the catecholic protons with base and the g equal 4.28 EPR signal is not resolved at liquid N-2 temperature. These changes are interpreted as resulting in part from a pronounced change in the ligand fields about the irons at the active sites which in the case of protocatechuate 3,4-dioxygenase leads to enzyme inactivation. The results also are the first indication that substrate analogues change their ionization form upon complexation with Fe (III) dioxygenases. The interaction of the probe with metapyrocatechase, an Fe(III) containing dioxygenase, and with several additional oxygenases and hydroperoxidases is also briefly examined. The probe is not specific for any particular class of non-heme iron dioxygenases.     

The effect of substrate, dihydrobiopterin, and dopamine on the EPR spectroscopic properties and the midpoint potential of the catalytic iron in recombinant human phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin (BH(4)) and non-heme iron-dependent enzyme that hydroxylates L-Phe to L-Tyr. The paramagnetic ferric iron at the active site of recombinant human PAH (hPAH) and its midpoint potential at pH 7.25 (E(m)(Fe(III)/Fe(II))) were studied by EPR spectroscopy. Similar EPR spectra were obtained for the tetrameric wild-type (wt-hPAH) and the dimeric truncated hPAH(Gly(103)-Gln(428)) corresponding to the "catalytic domain." A rhombic high spin Fe(III) signal with a g value of 4.3 dominates the EPR spectra at 3.6 K of both enzyme forms. An E(m) = +207 +/- 10 mV was measured for the iron in wt-hPAH, which seems to be adequate for a thermodynamically feasible electron transfer from BH(4) (E(m) (quinonoid-BH(2)/BH(4)) = +174 mV). The broad EPR features from g = 9.7-4.3 in the spectra of the ligand-free enzyme decreased in intensity upon the addition of L-Phe, whereas more axial type signals were observed upon binding of 7,8-dihydrobiopterin (BH(2)), the stable oxidized form of BH(4), and of dopamine. All three ligands induced a decrease in the E(m) value of the iron to +123 +/- 4 mV (L-Phe), +110 +/- 20 mV (BH(2)), and -8 +/- 9 mV (dopamine). On the basis of these data we have calculated that the binding affinities of L-Phe, BH(2), and dopamine decrease by 28-, 47-, and 5040-fold, respectively, for the reduced ferrous form of the enzyme, with respect to the ferric form. Interestingly, an E(m) value comparable with that of the ligand-free, resting form of wt-hPAH, i.e. +191 +/- 11 mV, was measured upon the simultaneous binding of both L-Phe and BH(2), representing an inactive model for the iron environment under turnover conditions. Our findings provide new information on the redox properties of the active site iron relevant for the understanding of the reductive activation of the enzyme and the catalytic mechanism.