Evidence for siroheme-Fe4S4 interaction in spinach ferredoxin-sulfite reductase

Spinach ferredoxin-sulfite reductase (SiR) contains one siroheme and one Fe4S4 center per polypeptide subunit. The heme is entirely in the high-spin Fe3+ state in the oxidized enzyme. When SiR is photochemically reduced with ethylenediaminetetraacetate (EDTA)-deazaflavin, the free enzyme and its CN- and CO complexes show changes in absorption spectra associated with the heme even after the heme has been reduced from the Fe3+ to the Fe2+ state. With CO- or CN--SiR, these spectral changes are associated with the appearance of a classical "g = 1.94" type of EPR spectrum characteristic of reduced Fe4S4 centers. The line shapes and exact g values of the g = 1.94 EPR spectra vary with the nature of the ligand bound to the heme Fe. Photoreduction of free SiR results in production of a novel type of EPR signal, with g = 2.48, 2.34, and 2.08 in the fully reduced enzyme; this signal accounts for 0.6 spin per heme. (A small g = 1.94 type EPR signal, representing 0.2 spin per heme, is also found.) These data suggest the presence of a strong magnetic interaction between the siroheme and Fe4S4 centers in spinach SiR, this interaction giving rise to different EPR signals depending on the spin state of the heme Fe in the reduced enzyme.     

Electron paramagnetic resonance and optical evidence for interaction between siroheme and Fe4S4 prosthetic groups in complexes of Escherichia coli sulfite reductase hemoprotein with added ligands

Janick & Siegel [Janick, P. A., & Siegel, L. M. (1982) Biochemistry 21, 3538-3547] showed that the EPR spectrum of the reduced Fe4S4 center (S = 1/2) in fully reduced native ("unligated") Escherichia coli NADPH-sulfite reductase hemoprotein subunit (SiR-HP) is perturbed by interaction with paramagnetic ferrous siroheme (S = 1 or 2) to yield several novel sets of EPR signals: one set with all g values between 2.0 and 2.8, termed "S = 1/2" type, and two sets with the lowest field g value between 4.7 and 5.4, termed "S = 3/2" type. The present study has shown that EPR spectra of fully reduced SiR-HP are nearly quantitatively converted to the classical "g = 1.94" type typical of S = 1/2 Fe4S4 clusters when the heme has been ligated by strong field ligands such as CO, CN-, S2-, and AsO2-, converting the ferroheme to S = 0. However, the exact line shapes and g values of the g = 1.94 differ markedly when different ligands are bound to the heme. Also, optical difference spectra taken between enzyme species in which the heme is kept in the same (Fe2+) oxidation state while the Fe4S4 center is reduced or oxidized show that the optical spectrum of the ligated siroheme is sensitive to the oxidation state of the Fe4S4 cluster. These results indicate that the heme-Fe4S4 interaction of native SiR-HP persists even when the heme Fe is bound to exogenous ligands. We have also found that the g values of the exchange-coupled S = 1/2 and S V 3/2 type signals of native reduced SiR-HP can be significantly shifted by addition of potential weak field heme ligands--halides and formate--or low concentrations of certain chaotropic agents--guanidinium salts and dimethyl sulfoxide--to the fully reduced enzyme. Such agents can also promote interconversion of the S = 1/2 and S = 3/2 type signals. These effects are reversed on removal of the agent. Treatment of reduced SiR-HP with relatively large concentrations of chaotropes, e.g., 60% dimethyl sulfoxide or 2 or 3 M urea, leads to abolition of the S = 1/2 and S = 3/2 EPR signals and their replacement by signals of the g = 1.94 type.     

Identification of the iron-sulfur center of spinach ferredoxin-nitrite reductase as a tetranuclear center, and preliminary EPR studies of mechanism.

EPR spectroscopic and chemical analyses of spinach nitrite reductase show that the enzyme contains one reducible iron-sulfur center, and one site for binding either cyanide or nitrite, per siroheme. The heme is nearly all in the high spin ferric state in the enzyme as isolated. The extinction coefficient of the enzyme has been revised to E386 = 7.6 X 10(4) cm-1 (M heme)-1. The iron-sulfur center is reduced with difficulty by agents such as reduced methyl viologen (equilibrated with 1 atm of H2 at pH 7.7 in the presence of hydrogenase) or dithionite. Complexation of the enzyme with CO (a known ligand for nitrite reductase heme) markedly increases the reducibility of the iron-sulfur center. New chemical analyses and reinterpretation of previous data show that the enzyme contains 6 mol of iron and 4 mol of acid-labile S2-/mol of siroheme. The EPR spectrum of reduced nitrite reductase in 80% dimethyl sulfoxide establishes clearly that the enzyme contains a tetranuclear iron-sulfur (Fe4S4) center. The ferriheme and Fe4S4 centers are reduced at similar rates (k = 3 to 4 s-1) by dithionite. The dithionite-reduced Fe4S4 center is rapidly (k = 100 s-1) reoxidized by nitrite. These results indicate a role for the Fe4S4 center in catalysis.     

Infrared evidence of cyanide binding to iron and copper sites in bovine heart cytochrome c oxidase. Implications regarding oxygen reduction

Cyanide binding to bovine heart cytochrome c oxidase at five redox levels has been investigated by use of infrared and visible-Soret spectra. A C-N stretch band permits identification of the metal ion to which the CN- is bound and the oxidation state of the metal. Non-intrinsic Cu, if present, is detected as a cyanide complex. Bands can be assigned to Cu+CN at 2093 cm-1, Cu2+CN at 2151 or 2165 cm-1, Fe3+CN at 2131 cm-1, and Fe2+CN at 2058 cm-1. Fe2+CN is found only when the enzyme is fully reduced whereas the reduced Cu+CN occurs in 2-, 3-, and 4-electron reduced species. A band for Fe3+CN is not found for the complex of fully oxidized enzyme but is for all partially reduced species. Cu2+CN occurs in both fully oxidized and 1-electron-reduced oxidase. CO displaces the CN- at Fe2+ to give a C-O band at 1963.5 cm-1 but does not displace the CN- at Cu+. Another metal site, noted by a band at 2042 cm-1, is accessible only in fully reduced enzyme and may represent Zn2+ or another Cu+. Binding of either CN- or CO may induce electron redistribution among metal centers. The extraordinary narrowness of ligand infrared bands indicates very little mobility of the components that line the O2 reduction site, a property of potential advantage for enzyme catalysis. The infrared evidence that CN- can bind to both Fe and Cu supports the possibility of an O2 reduction mechanism in which an intermediate with a mu-peroxo bridge between Fe and Cu is formed. On the other hand, the apparent independence of Fe and Cu ligand-binding sites makes a heme hydroperoxide (Fe-O-O-H) intermediate an attractive alternative to the formation an Fe-O-O-Cu linkage.     

M?ssbauer studies of Escherichia coli sulfite reductase complexes with carbon monoxide and cyanide. Exchange coupling and intrinsic properties of the [4Fe-4S] cluster

M?ssbauer studies of the hemoprotein subunit (SiR) of E. coli sulfite reductase have shown that the siroheme and the [4Fe-4S] cluster are exchange-coupled. Here we report M?ssbauer studies of SiR complexed with either CO or CN- and of SiR in the presence of the chaotropic agent dimethyl sulfoxide (Me2SO). The spectra of one-electron-reduced SiR X CN show that all five iron atoms reside in a diamagnetic environment; the ferroheme X CN complex is low spin and the [4Fe-4S] cluster is in the 2+ oxidation state. Titration with ferricyanide affords a CN- complex of oxidized SiR in which the siroheme iron is low spin ferric, with the cluster remaining in the 2+ state. At low temperatures, paramagnetic hyperfine interactions are observed for the iron sites of the cluster, suggesting that it is exchange-coupled to the heme iron. Reduction of one-electron-reduced SiR X CN and SiR X CO yields complexes with "g = 1.94"-type EPR signals showing that the second electron is accommodated by the iron-sulfur cluster. The fully reduced complexes yield well resolved M?ssbauer spectra which were analyzed in the spin Hamiltonian formalism. The analysis shows that the cluster subsites are equivalent in pairs, one pair having properties reminiscent of ferric sites whereas the other pair has features more typical of ferrous sites. The M?ssbauer spectra of oxidized SiR kept in 60% (v/v) Me2SO are virtually identical with those observed for SiR in standard buffer, implying that the coupling is maintained in the presence of the chaotrope. Fully reduced SiR displays an EPR signal with g values of g = 2.53, 2.29, and 2.07. In 60% Me2SO, this signal vanishes and a g = 1.94 signal develops; this transition is accompanied by a change in the spin state of the heme iron from S = 1 (or 2) to S = O.     

Active site structure of SoxB-type cytochrome bo3 oxidase from thermophilic Bacillus

Two-subunit SoxB-type cytochrome c oxidase in Bacillus stearothermophilus was over-produced, purified, and examined for its active site structures by electron paramagnetic resonance (EPR) and resonance Raman (RR) spectroscopies. This is cytochrome bo3 oxidase containing heme B at the low-spin heme site and heme O at the high-spin heme site of the binuclear center. EPR spectra of the enzyme in the oxidized form indicated that structures of the high-spin heme O and the low-spin heme B were similar to those of SoxM-type oxidases based on the signals at g=6.1, and g=3.04. However, the EPR signals from the CuA center and the integer spin system at the binuclear center showed slight differences. RR spectra of the oxidized form showed that heme O was in a 6-coordinated high-spin (nu3 = 1472 cm(-1)), and heme B was in a 6-coordinated low-spin (nu3 = 1500 cm(-1)) state. The Fe2+-His stretching mode was observed at 211 cm(-1), indicating that the Fe2+-His bond strength is not so much different from those of SoxM-type oxidases. On the contrary, both the Fe2+-CO stretching and Fe2+-C-O bending modes differed distinctly from those of SoxM-type enzymes, suggesting some differences in the coordination geometry and the protein structure in the proximity of bound CO in cytochrome bo3 from those of SoxM-type enzymes.     

57Fe and 1H electron-nuclear double resonance of three doubly reduced states Escherichia coli sulfite reductase

We have employed electron-nuclear double resonance (ENDOR) spectroscopy to study the 57Fe hyperfine interactions in the bridged-siroheme [4Fe-4S] cluster that forms the catalytically active center of the two-electron-reduced hemoprotein subunit of Escherichia coli NADPH-sulfite reductase (SiR2-). Previous electron paramagnetic resonance (EPR) and M?ssbauer studies have shown that this enzyme oxidation state can exist in three distinct spectroscopic forms: (1) a "g = 2.29" EPR species that predominates in unligated SiR2-, in which the siroheme Fe2+ is believed to be in an S = 1 state; (2) a "g = 4.88" type of EPR species that predominates in SiR2- in the presence of small amounts of guanidinium sulfate, in which the siroheme Fe2+ is in an S = 2 state; and (3) a classical "g = 1.94" type of EPR species that is seen in SiR2- ligated with CO, in which the siroheme Fe2+ is in an S = 0 state. In all three species, the cluster is in the [4Fe-4S]1+ state, and two distinct types of Fe site are seen in M?ssbauer spectroscopy. ENDOR studies confirm the M?ssbauer assignments for the cluster 57Fe in the g = 1.94 state, with A values of 37, 37, and 32 MHz for site I and ca. 19 MHz for site II. The hyperfine interactions are not too different on the g = 2.29 state, with site I Fe showing more anisotropic A values of 32, 24, and 20 MHz (site II was not detected).(ABSTRACT TRUNCATED AT 250 WORDS)     

M?ssbauer spectroscopic studies of Escherichia coli sulfite reductase. Evidence for coupling between the siroheme and Fe4S4 cluster prosthetic groups

Escherichia coli NADPH-sulfite reductase is a complex hemoflavoprotein with an alpha 8 beta 4 subunit structure. The beta-subunits each contain one siroheme and a tetranuclear iron-sulfur center (Fe4S4). Isolated beta-monomers can catalyze the 6-electron reduction of sulfite to sulfide. We have studied the beta-monomers with M?ssbauer and EPR spectroscopy. The data show conclusively that the siroheme and the Fe4S4 cluster are strongly exchange-coupled. This is proven by the observations that (a) the two chromophores share a single electronic spin and (b) the addition of 1 electron to oxidized sulfite reductase changes the environments of 5 iron atoms. Spin-sharing is demonstrated in oxidized and 2-electron-reduced sulfite reductase and strongly implicated in 1-electron-reduced material. Thus, sulfite reductase provides the first example of an active site where a heme and an iron-sulfur cluster are closely linked as a functional unit, probably via a common bridging ligand.     

The reduction of nitrate to ammonium by a Clostridium sp. isolated from soil

Cultures of Clostridium KDHS2 reduced 15NO3- to 15NH4+ with a concurrent increase in molar growth yield of 15.7% compared with fermentatively grown bacteria. The bacteria exhibited a Ks (NO3-) of 0.5 mM and reduced NO3- maximally at a rate of 0.1 mumol h(-1) mg dry wt)-1. A partially purified nitrate reductase was obtained which had a Km (NO3-) of 0.15 mM. The reduction of 13NO3- to 13NH4+ by resting bacteria was not inhibited by NH4+, glutamate, glutamine, methionine sulphoximine or azaserine. Glutamine synthetase affected neither the synthesis nor the activity of the NO3(-)-reducing enzymes. The results are consistent with the hypothesis that NO3- reduction to NH4+ in this Clostridium sp. is dissimilative. SO32-, but not SO42-, inhibited the reaction, apparently at the level of NO2- reduction.     

Further characterisation of the FAD and Fe2S2 redox centres of component C, the NADH:acceptor reductase of the soluble methane monooxygenase of Methylococcus capsulatus (Bath)

The absorbance contributions of the FAD and Fe2S2 redox centres of component C of the soluble methane monooxygenase complex have been resolved, using mersalyl to destroy the Fe2S2 centre. The Fe2S2 seems to be very similar to that of spinach ferredoxin, by its absorbance and electron paramagnetic resonance (EPR) spectra, and the FAD semiquinone is a neutral semiquinone. Spectrophotometry near room temperature and EPR spectroscopy near liquid-helium temperature allow the three redox couples of component C to be ordered. Component C can exist in Oe-1 (oxidised), 1e-1 (semiquinone), 2e-1 (mostly semiquinone and reduced Fe2S2), and 3e-1 forms (dihydroquinone and reduced Fe2S2), under equilibrium conditions. The ability of component C to support odd-electron forms is consistent with its proposed role as a 2e-1/1e-1 transformase, splitting electron pairs from NADH for passage to component A in one-electron steps. (The FAD appears to interact with NADH, and transfers single electrons to the Fe2S2, for donation to component A at a constant redox potential.) The mid-point potentials of component C were found using redox dyes and EPR spectroscopy and were: FAD/FAD., Em = -150 mV; Fe2S2/Fe2.S2,Em = -220 mV; FAD./FAD..,Em = -260 mV. the presence of NADH did not alter these mid-point potentials. These mid-point potentials are consistent with the role of component C as the NADH:component A reductase, passing electrons from NADH (Em = -320 mV) onto component A (Em = +150 mV and Em = -150 mV). The reducing power from NADH appears to be required by component A to activate one atom of oxygen, to insert into methane, and the reducing equivalents derived from NADH end up with the other oxygen atom, as water.     

Circular dichroism and potentiometry of FAD, heme and Mo-pterin prosthetic groups of assimilatory nitrate reductase

Oxidation-reduction midpoint potentials for flavin, heme, and molybdenum-pterin prosthetic groups of assimilatory nitrate reductase (NR) from Chlorella vulgaris were measured at room temperature by using CD and EPR potentiometry. The CD changes accompanying reduction of each prosthetic group were determined by using enzyme fragments containing either FAD or heme and molybdenum prosthetic groups, obtained by limited proteolysis, and by poising the enzyme at various redox potentials in the presence of dye mediators. Limited proteolysis did not appear to alter the environment of the prosthetic groups, as judged by their CD spectra. Also, CD potentiometric titration of FAD in intact NR (Em' = -272 mV, n = 2) gave a similar value (Em' = -286 mV) to the FAD of the flavin-containing proteolytic domain, determined by visible spectroscopy. Less than 1% of the flavin semiquinone was detected by EPR spectroscopy, indicating that Em' (FAD/FAD.-) may be more than 200 mV lower than Em' (FAD.-/FADH-). Reduction of heme resulted in splitting of both Soret and alpha CD bands into couplets. The heme Em' was -162 mV (n = 1) determined by both CD and visible spectroscopy. Reduction of Mo-pterin was followed by CD at 333 nm, and Mo(V) was monitored by room temperature EPR spectroscopy. Most of the change in the Mo-pterin CD spectrum was due to the Mo(VI)/Mo(V) transition. The Em' values determined for Mo(VI)/Mo(V) were +26 mV by CD and +16 mV by EPR, whereas Mo(V)/Mo(IV) values were -40 mV by CD and -26 mV by EPR.(ABSTRACT TRUNCATED AT 250 WORDS)     

M?ssbauer study of CO dehydrogenase from Clostridium thermoaceticum

We have studied with M?ssbauer spectroscopy the metal clusters of CO dehydrogenase from Clostridium thermoaceticum. At potentials greater than -200 mV, all of the approximately 12 irons reside in diamagnetic environments and contribute a quadrupole doublet characteristic of [Fe4S4]2+ clusters. At lower potentials a variety of components are observed. About 40% of the Fe appears to belong to one [Fe4S4]1+ cluster. We have also observed the M?ssbauer spectrum (approximately 18% of Fe) of the complex which yields EPR with g = 2.01, 1.81, and 1.65. Also present is a doublet (9% of Fe) with delta EQ = 2.90 mm/s and delta = 0.70 mm/s, values typical of a ferrous FeS4 complex. This component seems to interact with a nickel site to form an EPR-silent complex with half-integral electronic spin. We have also characterized the iron environments of the S = 1/2 NiFeC complex. This complex contributes approximately 20% of the total M?ssbauer absorption when the EPR signal has approximately 0.35 spins/12 Fe. From isomer shift comparisons in the oxidized and CO-reacted states of this center, we speculate that the NiFeC complex may consist of a nickel site exchange-coupled to a [Fe4S4]2+ cluster. Finally, the M?ssbauer and EPR data, taken together, force us to conclude that current preparations, while homogeneous according to purifications standards, are spectroscopically heterogeneous, thus rendering the development of a model of the cluster types and compositions in this enzyme premature.     

Structure and spectroscopy of the periplasmic cytochrome c nitrite reductase from Escherichia coli

The crystal structure and spectroscopic properties of the periplasmic penta-heme cytochrome c nitrite reductase (NrfA) of Escherichia coli are presented. The structure is the first for a member of the NrfA subgroup that utilize a soluble penta-heme cytochrome, NrfB, as a redox partner. Comparison to the structures of Wolinella succinogenes NrfA and Sulfospirillum deleyianum NrfA, which accept electrons from a membrane-anchored tetra-heme cytochrome (NrfH), reveals notable differences in the protein surface around heme 2, which may be the docking site for the redox partner. The structure shows that four of the NrfA hemes (hemes 2-5) have bis-histidine axial heme-Fe ligation. The catalytic heme-Fe (heme 1) has a lysine distal ligand and an oxygen atom proximal ligand. Analysis of NrfA in solution by magnetic circular dichroism (MCD) suggested that the oxygen ligand arose from water. Electron paramagnetic resonance (EPR) spectra were collected from electrochemically poised NrfA samples. Broad perpendicular mode signals at g similar 10.8 and 3.5, characteristic of weakly spin-coupled S = 5/2, S = 1/2 paramagnets, titrated with E(m) = -107 mV. A possible origin for these are the active site Lys-OH(2) coordinated heme (heme 1) and a nearby bis-His coordinated heme (heme 3). A rhombic heme Fe(III) EPR signal at g(z) = 2.91, g(y) = 2.3, g(x) = 1.5 titrated with E(m) = -37 mV and is likely to arise from bis-His coordinated heme (heme 2) in which the interplanar angle of the imidazole rings is 21.2. The final two bis-His coordinated hemes (hemes 4 and 5) have imidazole interplanar angles of 64.4 and 71.8. Either, or both, of these hemes could give rise to a "Large g max" EPR signal at g(z)() = 3.17 that titrated at potentials between -250 and -400 mV. Previous spectroscopic studies on NrfA from a number of bacterial species are considered in the light of the structure-based spectro-potentiometric analysis presented for the E. coli NrfA.     

The effect of Fe2+ on the activity of human plasma protein kinase

A Fe2+ stimulatory protein kinase in human plasma is demonstrated. This enzyme was partial purified from the 100,000 X g supernatant of human plasma by using (NH4)2SO4 fractionation. The activity of this protein kinase existed in 30-50% fraction. The effect of various compounds on the activity of this partial purified protein kinase was studied. The Fe2+ stimulates 5 to 10-fold of the protein kinase activity. The maximum concentration of Fe2+ to stimulate the activity of this plasma protein kinase was 2 mM. Double reciprocal plot for the stimulation showed that Fe2+ increased the Vm but not the Km for ATP. Other divalent compounds such as MgCl2, Mg(Oac)2, MnCl2 and BaCl2 were less effective.     

Binding of 17O-labeled substrate and inhibitors to protocatechuate 4,5-dioxygenase-nitrosyl complex. Evidence for direct substrate binding to the active site Fe2+ of extradiol dioxygenases

Pseudomonas testosteroni protocatechuate 4,5-dioxygenase catalyzes extradiol-type oxygenolytic cleavage of the aromatic ring of its substrate. The essential active site Fe2+ binds nitric oxide (NO) to produce an EPR active complex with an electronic spin of S = 3/2. Hyperfine broadening of the EPR resonances of the nitrosyl complex of the enzyme by protocatechuate (3,4-(OH)2-benzoate, PCA) enriched specifically with 17O (I = 5/2) in either the 3 or the 4 hydroxyl group shows that both groups can bind directly to the Fe2+ in the ternary complex. Analogous results are obtained for PCA binding to catechol 2,3-dioxygenase-NO complex suggesting that substrate binding by the Fe2+ may be a general property of extradiol dioxygenases. The protocatechuate 4,5-dioxygenase inhibitor, 4-17OH-benzoate binds directly to the Fe of the nitrosyl adduct of the enzyme through the OH group. Since previous studies have shown that water also is bound to the Fe in this ternary complex, but not in the ternary complex with PCA, the data strongly imply that there are 3 sites in the Fe coordination which can be occupied by exogenous ligands. 3-17OH-benzoate is an inhibitor of the enzyme but does not elicit detectable hyperfine broadening in the EPR spectrum of the nitrosyl adduct suggesting that it binds to the enzyme, but not to the Fe. The EPR spectra of ternary enzyme-NO complexes with PCA or 4-OH-benzoate labeled with 17O exclusively in the carboxylate substituent are not broadened, suggesting that this moiety does not bind to the Fe.     

Electron transfer from HiPIP to the photooxidized tetraheme cytochrome subunit of Allochromatium vinosum reaction center: new insights from site-directed mutagenesis and computational studies

The kinetics of electron transfer from reduced high-potential iron-sulfur protein (HiPIP) to the photooxidized tetraheme cytochrome c subunit (THC) bound to the photosynthetic reaction center (RC) from the purple sulfur bacterium Allochromatium vinosum were studied under controlled redox conditions by flash absorption spectroscopy. At ambient redox potential Eh = +200 mV, where only the high-potential (HP) hemes of the THC are reduced, the electron transfer from HiPIP to photooxidized HP heme(s) follows second-order kinetics with rate constant k = (4.2 +/- 0.2) 10(5) M(-1) s(-1) at low ionic strength. Upon increasing the ionic strength, k increases by a maximum factor of ca. 2 at 640 mM KCl. The role of Phe48, which lies on the external surface of HiPIP close to the [Fe4S4] cluster and presumably on the electron transfer pathway to cytochrome heme(s), was investigated by site-directed mutagenesis. Substitution of Phe48 with arginine, aspartate, and histidine completely prevents electron donation. Conversely, electron transfer is still observed upon substitution of Phe48 with tyrosine and tryptophan, although the rate is decreased by more than 1 order of magnitude. These results suggest that Phe48 is located on a key protein surface patch essential for efficient electron transfer, and that the presence of an aromatic hydrophobic residue on the putative electron-transfer pathway plays a critical role. This conclusion was supported by protein docking calculations, resulting in a structural model for the HiPIP-THC complex, which involves a docking site close to the LP heme farthest from the bacteriochlorophyll special pair.     

The displacement of copper by iron at the specific binding sites of ovotransferrin

We have examined the kinetics and mechanism by which iron can displace copper at the specific metal-binding sites of ovotransferrin. Fe2+ was added to Cu2+-ovotransferrin-CO3(2-) in the presence of NaHCO3 and ambient O2. The reaction has been followed by standard and stopped-flow spectrophotometry, EPR spectroscopy and analysis of chromogen-reactive Fe2+. The reaction is best described as triphasic. An initial jump in absorbance takes place in the first 2 s. In the next minute there is a further increase in absorbance and shift in the spectral maximum from 440 to 446 nm. The third phase is complex. The bulk of the spectrophotometric change, a decrease in absorbance with a shift to a maximum of 453 nm, lasts approx. 3 min. Minor spectral and EPR changes, however, take place over the next several hours. Chromogenic analysis of Fe2+ indicates that approx. 1 min is required to oxidize the Fe2+. EPR spectra reveal the formation of an Fe3+-ovotransferrin complex within the first 20 s; however, this lacks the characteristic doublet of specific Fe3+-ovotransferrin-CO3(2-). The simultaneous presence of specific Cu2+-ovotransferrin-CO3(2-) and Fe3+-ovotransferrin-CO3(2-) signals suggests a period in which the protein specifically binds both metal ions perhaps resulting from a differential reactivity of the two metal-binding sites. The addition of Cu(NO3)2 to Fe3+-ovotransferrin-CO3(2-) resulted in a complex with specific Fe3+ and non-specific Cu2+. The EPR spectrum of this complex and the final product of our displacement reaction were virtually identical. Distinct parallels in reaction of Cu2+-ovotransferrin-CO3(2-) with Fe(NH4)2(SO4)2, Fe(NO3)3 and Fe3+-nitrilotriacetic acid were observed. A reaction sequence involving the binding and oxidation of non-specific Fe2+ followed by Cu2+ displacement by Fe3+ at the specific sites and binding of non-specific Cu2+ is suggested.     

EPR and redox characterization of iron-sulfur centers in nitrate reductases A and Z from Escherichia coli. Evidence for a high-potential and a low-potential class and their relevance in the electron-transfer mechanism.

The redox properties of the iron-sulfur centers of the two nitrate reductases from Escherichia coli have been investigated by EPR spectroscopy. A detailed study of nitrate reductase A performed in the range +200 mV to -500 mV shows that the four iron-sulfur centers of the enzyme belong to two classes with markedly different redox potentials. The high-potential group comprises a [3Fe-4S] and a [4Fe-4S] cluster whose midpoint potentials are +60 mV and +80 mV, respectively. Although these centers are magnetically isolated, they are coupled by a significant anticooperative redox interaction of about 50 mV. The [4Fe-4S]1+ center occurs in two different conformations as shown by its composite EPR spectrum. The low-potential group contains two [4Fe-4S] clusters with more typical redox potentials (-200 mV and -400 mV). In the fully reduced state, the three [4Fe-4S]1+ centers are magnetically coupled, leading to a broad featureless spectrum. The redox behaviour of the high-pH EPR signal given by the molybdenum cofactor was also studied. The iron-sulfur centers of the second nitrate reductase of E. coli, nitrate reductase Z, exhibit essentially the same characteristics than those of nitrate reductase A, except that the midpoint potentials of the high-potential centers appear negatively shifted by about 100 mV. From the comparison between the redox centers of nitrate reductase and of dimethylsulfoxide reductase, a correspondence between the high-potential iron-sulfur clusters of the two enzymes can be proposed.     

The hybrid-cluster protein ('prismane protein') from Escherichia coli. Characterization of the hybrid-cluster protein, redox properties of the [2Fe-2S] and [4Fe-2S-2O] clusters and identification of an associated NADH oxidoreductase containing FAD and [2Fe-2S].

Hybrid-cluster proteins ('prismane proteins') have previously been isolated and characterized from strictly anaerobic sulfate-reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe-4S] cubane cluster with spin-admixed S = 3/2 ground-state paramagnetism and a novel type of hybrid [4Fe-2S-2O] cluster, which can attain four redox states. Genomic sequencing reveals that genes encoding putative hybrid-cluster proteins are present in a range of bacterial and archaeal species. In this paper we describe the isolation and spectroscopic characterization of the hybrid-cluster protein from Escherichia coli. EPR spectroscopy shows the presence of a hybrid cluster in the E. coli protein with characteristics similar to those in the proteins of anaerobic sulfate reducers. EPR spectra of the reduced E. coli hybrid-cluster protein, however, give evidence for the presence of a [2Fe-2S] cluster instead of a [4Fe-4S] cluster. The hcp gene encoding the hybrid-cluster protein in E. coli and other facultative anaerobes occurs, in contrast with hcp genes in obligate anaerobic bacteria and archaea, in a small operon with a gene encoding a putative NADH oxidoreductase. This NADH oxidoreductase was also isolated and shown to contain FAD and a [2Fe-2S] cluster as cofactors. It catalysed the reduction of the hybrid-cluster protein with NADH as an electron donor. Midpoint potentials (25 degrees C, pH 7.5) for the Fe/S clusters in both proteins indicate that electrons derived from the oxidation of NADH (Em NADH/NAD+ couple: -320 mV) are transferred along the [2Fe-2S] cluster of the NADH oxidoreductase (Em = -220 mV) and the [2Fe-2S] cluster of the hybrid-cluster protein (Em = -35 mV) to the hybrid cluster (Em = -50, +85 and +365 mV for the three redox transitions). The physiological function of the hybrid-cluster protein has not yet been elucidated. The protein is only detected in the facultative anaerobes E. coli and Morganella morganii after cultivation under anaerobic conditions in the presence of nitrate or nitrite, suggesting a role in nitrate-and/or nitrite respiration.     

The iron-sulfur centers of the soluble [NiFeSe] hydrogenase, from Desulfovibrio baculatus (DSM 1743). EPR and M?ssbauer characterization

The soluble (cytoplasmic plus periplasmic) Ni/Fe-S/Se-containing hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from cells grown in an 57Fe-enriched medium, and its iron-sulfur centers were extensively characterized by M?ssbauer and EPR spectroscopies. The data analysis excludes the presence of a [3Fe-4S] center, either in the native (as isolated) or in the hydrogen-reduced states. In the native state, the non-heme iron atoms are arranged as two diamagnetic [4Fe-4S]2+ centers. Upon reduction, these two centers exhibit distinct and unusual M?ssbauer spectroscopic parameters. The centers were found to have similar mid-point potentials (approximately -315 mV) as determined by oxidation-reduction titratins followed by EPR.