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To understand the role of the [4Fe-4S](2+) cluster in controlling the activity of the Escherichia coli transcription factor FNR (fumarate nitrate reduction) during changes in O(2) availability, we have characterized a mutant FNR protein containing a substitution of Leu-28 with His (FNR-L28H) which, unlike its wild type (WT) counterpart, is functional under aerobic growth conditions. The His-28 substitution appears to stabilize the [4Fe-4S](2+) cluster of FNR-L28H in the presence of O(2) because air-exposed FNR-L28H did not undergo the rapid [4Fe-4S](2+) to [2Fe-2S](2+) cluster conversion or concomitant loss in site-specific DNA binding and dimerization, which are characteristic of WT-FNR under these conditions. This increased cluster stability was not a result of His-28 replacing the WT-FNR cluster ligands because substitution of any of these four Cys residues (cysteine 20, 23, 29, or 122) with Ser resulted in [4Fe-4S](2+) cluster-deficient preparations of FNR-L28H. The Mössbauer spectra of FNR-L28H indicated that the coordination environment of the [4Fe-4S](2+) cluster did not differ from that of WT-FNR. Whole cell Mössbauer spectroscopy showed that aerobically grown cells overexpressing FNR-L28H had levels of the FNR species containing the [4Fe-4S](2+) cluster similar to those of cells grown under anaerobic conditions. Thus, the increase in cluster stability is sufficient to allow accumulation of the [4Fe-4S](2+) cluster form of FNR-L28H under aerobic conditions and provides a reasonable explanation for why this mutant protein is functional under aerobic growth conditions. From these results, we present a model to explain how WT-FNR is normally inactivated under aerobic growth conditions.

Electric hyperfine interactions in the 57Fe nucleus of hemin were studied by Mössbauer spectroscopy using transmission geometry. It was established that the discrepancies in literature values of the quadrupole splitting were due to allotropy. Methods of obtaining reproducible quadrupole splitting were also established. The isomer shift, taken with respect to 57Fe metal, was found not to show marked variation as the quadrupole splitting - thus confirming the conclusion with regard to the variation of the latter.

We describe an alternate terminal oxidase found in the plasma membrane of Thermus thermophilus and designate it cytochrome ba3. The enzyme consists of a single approximately equal to 35-kDa polypeptide that binds one heme B molecule, one heme A molecule, and two Cu ions. Optical spectra suggest the presence of cytochrome b, cytochrome a3, and CuA in this protein. Quantitative EPR and Mössbauer studies of the oxidized protein indicate the presence of one low-spin ferric heme, which is assigned to cytochrome b. Mössbauer studies of the reduced protein show the presence of one low-spin ferrous heme, assigned to cytochrome b, and a predominant high-spin ferrous heme that reacts quantitatively with CO to yield an additional low-spin ferrous heme. The latter Fe atom is associated with the heme A and is designated cytochrome a3. The EPR spectrum of the oxidized protein also reveals the presence of a CuA-type center that accounts for half the total Cu. The remainder of the Cu would appear to be present as CuB that is magnetically coupled to the heme A. Amino acid analyses of cytochrome ba3 show the presence of eight to nine histidine residues and one cysteine residue.

Cytochrome c1aa3 from Thermus thermophilus has optical and EPR properties similar to bovine cytochrome c oxidase. We have studied 87Fe-enriched samples with Mössbauer spectroscopy in the fully oxidized and fully reduced states and in the oxidized state complexed with cyanide. The cytochromes a and c1 yielded spectra quite similar to those reported for the cytochromes c and b5; in the oxidized state the spectra reflect noninteracting, low spin ferric hemes, whereas the a- and c1-sites of the reduced enzyme are typical of low spin ferrous hemochromes. The spectra of the reduced enzyme show that reduced cytochrome a3 is high spin ferrous, with Mössbauer parameters quite similar to those of deoxymyoglobin. Upon addition of cyanide to the oxidized enzyme, the a3-site exhibits in the absence of an applied magnetic field and at temperatures down to 1.3 K a quadrupole doublet with parameters typical of low spin ferric heme-CN complexes. The low temperature spectra taken in applied magnetic fields show that the electronic ground state of the a3-CN complex has integer electronic spin, suggesting ferromagnetic coupling of the low spin ferric heme (S = 1/2) to Cu2+ (S = 1/2) to yield as S = 1 ground state. We have examined the oxidized enzyme from two different preparations. Both had good activity and identical optical and EPR spectra. The Mössbauer spectra, however, revealed that the a3-site had a substantially different electronic structure in the two preparations. Neither configuration had properties in accord with the widely accepted spin-coupling model proposed for the bovine enzyme.

Active beef heart aconitase contains a [4Fe-4S] cluster. One iron of the cluster, Fea, is labile and can be removed easily by oxidation in air to yield the [3Fe-4S]1+ cluster of inactive aconitase. We have previously shown that substrate binds to Fea. We have continued our Mössbauer studies by further investigating the active and inactive forms of the enzyme. When active aconitase, [4Fe-4S]2+, is mixed with substrate, two species (substrates or intermediates bound to Fea) labeled S1 and S2 are obtained. With the nitroanalogs of citrate and isocitrate, thought to be transition state analogs, and fluorocitrate, species S2, but not S1, is observed, suggesting that S2 represents a carbanion transition state complex. We have prepared Mössbauer samples by rapid mix/rapid freeze techniques. Using either citrate, isocitrate or cis-aconitate, the natural substrates, we have been able to detect at 0 degree C reaction intermediates in the 5-35 ms time range and, studying enzyme substrate interactions at subzero temperatures in a water/methanol/ethylene glycol solvent, we have observed new species when substrates were added at -60 degrees C. Details of these experiments are given, although in neither case can unique interpretations be offered at this time. We have also investigated reduced active aconitase ([4Fe-4S]1+; EPR at g = 1.94) in the presence of substrate with material selectively enriched with 57Fe in either Fea or the other three cluster sites. The spectra were analyzed with a spin Hamiltonian, and the results are discussed and interpreted in terms of three inequivalent Fe sites in the cluster. Finally, we have studied enzyme containing the reduced [3Fe-4S]0 cluster. There is no indication that citrate binds to the 3Fe cluster, and since no significant activity was observed, we conclude that aconitase containing a 3Fe cluster is not active in either oxidation state.

Beef heart aconitase, isolated under aerobic conditions, has been studied with Mössbauer and EPR spectroscopy. In the oxidized state, the enzyme exhibits an EPR signal at g = 2.01. The Mössbauer data show that this signal is associated with a 3Fe cluster. In dithionite-reduced aconitase, the 3Fe cluster, probably of the [3Fe-3S] type, is in a paramagnetic state of interger electronic spin (S = 2); the Mössbauer spectra exhibit al the unique features reported for proteins with 3Fe clusters. On activation of aconitase with ferrous ion, the paramagnetic 3Fe cluster of dithionite-reduced enzyme is converted into a diamagnetic (S = 0) form. Activation studies with iron enriched in either 27 Fe or 56 Fe suggest that activation transforms the 3Fe cluster into a center that has a [4Fe-4S] core. This conclusion is supported by the observation that EPR signals characteristic of reduced [4Fe-4S] clusters can be elicited under appropriate conditions. It has frequently been assumed that the activation of aconitase with Fe2+ produces an active site containing a single ferrous ion. The data reported here suggest that a ferrous ion is used to rebuild a [4Fe-4S] cluster.

Mössbauer and EPR studies of a highly active hydroxylase component of methane monooxygenase isolated from Methylosinus trichosporium OB3b are reported. The Mössbauer 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 Mössbauer 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.

The MoFe protein of nitrogenase catalyzes the six-electron reduction of dinitrogen to ammonia. It has long been believed that this protein receives the multiple electrons it requires one at a time, from the [4Fe-4S]2+/+ couple of the Fe protein. Recently an all-ferrous [4Fe-4S]0 state of the Fe protein was demonstrated suggesting instead a series of two electron steps involving the [4Fe-4S]2+/0 couple. We have examined the interactions of the [4Fe-4S]0 Fe protein with nucleotides and its ability to transfer electrons to the MoFe protein. The [4Fe-4S]0 Fe protein binds both MgATP and MgADP and undergoes the MgATP induced conformational change and then binds properly to the MoFe protein, as evidenced by the fact that the behavior of the 0 and +1 oxidation states in the chelation and chelation protection assays are indistinguishable. Nucleotide binding does not effect the distinctive UV/Vis, CD, or Mössbauer spectra exhibited by the [4Fe-4S]0 Fe protein; however, because the intensity of the g = 16.4 EPR signal of the [4Fe-4S]0 Fe protein is extremely sensitive to minor variations of the rhombicity parameter E/D, the EPR signal is sensitive to the binding of nucleotides. A 50:50 mixture of [4Fe-4S]2+ and [4Fe-4S]0 Fe protein results in electron self-exchange and 100% production of [4Fe-4S]+ Fe protein, demonstrating that the +1/0 couple is fully reversible. MgATP is absolutely required for electron transfer from the [4Fe-4S]0 Fe protein to the reduced state of the MoFe protein. In that reaction both electrons are transferred and are used to reduce substrate.

The soluble ferredoxin from Thermus thermophilus was examined by Mössbauer and EPR spectroscopies and by reductive titrations. These studies demonstrate the presence of one 3Fe center, responsible for the characteristic g = 2.02 EPR signal in the oxidized protein, and one [4Fe-4S] center which is responsible for the rhombic EPR spectrum of the fully reduced protein. These assignments should replace those made by Ohnishi et al. (Ohnishi, T., Blum, H., Sato, S., Nakazawa, K., Hon-nami, K., and Oshima, T. (1980) J. Biol. Chem. 255, 345-348) prior to the discovery of the 3Fe clusters. The amino acid composition was determined and is discussed with reference to recent structural studies of 7Fe ferredoxins.

Protocatechuate 4,5-dioxygenase from Pseudomonas testosteroni has been purified to homogeneity and crystallized. The iron containing, extradiol dioxygenase is shown to be composed of two subunit types (alpha, Mr = 17,700 and beta, Mr = 33,800) in a 1:1 ratio; such a composition has not been observed for other extradiol dioxygenases. The 4.2 K Mössbauer spectrum of native protocatechuate 4,5-dioxygenase prepared from cells grown in 57Fe-enriched media consists of a doublet with quadrupole splitting, delta EQ = 2.22 mm/s, and isomer shift delta Fe = 1.28 mm/s, demonstrating a high spin Fe2+ site. These parameters, and the temperature dependence of delta EQ, are unique among enzymes but are strikingly similar to those reported for the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26, suggesting very similar ligand environments. The Fe2+ of protocatechuate 4,5-dioxygenase can be oxidized, for instance by H2O2, to yield high spin Fe3+ with EPR g values around g = 6 (and g = 4.3). In the oxidized state, protocatechuate 4,5-dioxygenase is inactive; the iron, however, can be rereduced by ascorbate to yield active enzyme. Our data suggest that protocatechuate binds to Fe2+; the spectra indicate that the ligand binding is heterogenous. The Mössbauer spectra observed here are fundamentally different from those reported earlier (Zabinski, R., Münck, E., Champion, P., and Wood, J. M. (1972) Biochemistry 11, 3212-3219). The spectra of the earlier (reconstituted) preparations, which had substantially lower specific activities, probably reflect adventitiously bound Fe3+. We discuss here how adventitiously bound iron can be identified and removed. The Fe2+ which is present in native protocatechuate 4,5-dioxygenase and its complexes with substrates and inhibitors reacts quantitatively with nitric oxide to produce a species with electronic spin S = 3/2. The EPR and Mössbauer spectra of these complexes compare favorably with EDTA . Fe(II) . NO. We have studied the latter complex extensively and have analyzed the Mössbauer spectra with an S = 3/2 spin Hamiltonian. EPR spectra show that protocatechuate 4,5-dioxygenase-NO complexes with substrates or inhibitors are heterogeneous and consist of several well defined subspecies. The data show that NO, and presumably also O2, has access to the active site Fe2+ in the enzyme-substrate complex. The use of EPR-detectable NO complexes as a rapid and sensitive tool for the study of the EPR silent active site iron of extradiol dioxygenases is discussed.

We have purified the Rieske iron-sulfur protein from Thermus thermophilus. Chemical analyses show that the protein contains iron, labile sulfide, and cysteine in equimolar concentrations, four of each for Mr approximately 20,000. The oxidized and reduced form of the protein have been characterized by optical, EPR, CD, magnetic CD and Mössbauer spectroscopies. Our data suggest the presence of a unique iron-sulfur center. Mössbauer studies of the oxidized and reduced protein demonstrate unambiguously that the protein contains clusters with [2Fe-2S] cores. The iron analyses and the Mössbauer data, taken together, suggest that the protein has two [2Fe-2S] clusters. This is supported by the observation that two electrons are required for complete reduction of the protein and that the g = 1.94-type signal of the reduced protein has a spin concentration of one spin (S = 1/2) per 2Fe. Within the excellent resolution of the Mössbauer and EPR data, the two clusters are identical. Our results thus suggest that each [2Fe-2S] cluster is coordinated by at most two cysteine residues. The Mössbauer spectra of the reduced protein were analyzed with an S = 1/2 spin Hamiltonian. The hyperfine parameters obtained are very similar to those reported for putidaredoxin. The main difference is that the Rieske protein exhibits an increased isomer shift at the Fe2+ site, suggesting that non-cysteine ligands are coordinated to the site that becomes reduced to Fe2+ upon reduction. A comparison of our data with those reported for various NADH-dependent dioxygenases suggest that these enzymes contain a Rieske-type [2Fe-2S] center.

Cytochrome c-554 functions in the ammonia oxidizing system of Nitrosomonas europaea. We have investigated its molecular and ligand binding properties and studied the protein with optical, EPR, and Mössbauer spectroscopies in the pH range from 2 to 13. Amino acid, heme, and metal analyses show that the protein has Mr = 25,000 and that it contains four c-type hemes per molecule. Optical spectra reveal that the heme ligand structures are sensitive to the pH of the medium and that the hemes can bind small molecules such as CN-, CO, and NO under certain conditions. According to the Mössbauer and EPR studies of the ferric protein, the hemes are predominantly (75%) high spin at pH 2 and low spin (approximately equal to 100%) above pH 10. At neutral pH, Mössbauer data show that 75% of the heme is low spin and that the remainder is high spin. The EPR data, however, do not reveal any signals attributable to typical high spin or low spin species. Rather, a very complex and unusual spectrum with a main feature at g = 3.3 is observed at X-band, this feature shifts to approximately g = 3 at S-band. The EPR and Mössbauer data show clearly that the hemes are magnetically interacting, by dipolar and exchange interactions. At pH 2, the EPR spectra reveal resonances at g = 6 and 2. The Mössbauer spectra prove that all hemes are magnetically coupled at this pH. Coupling is also borne out by the observation of a half-field EPR resonance near g = 12.

During activation of aconitase a ferrous ion is incorporated into a [3Fe-4S] cluster to yield a structure with a [4Fe-4S] core. Using 57Fe or 56Fe for activation we have studied with Mössbauer spectroscopy the beef heart enzyme in the presence of citrate. Our studies show that the environment of one iron site (Fea) of the [4Fe-4S] cluster is drastically altered in the presence of citrate. Fea is the iron acquired during activation of aconitase. In the oxidized [4Fe-4S]2+ state two species with enzyme-bound substrate are observed, whereas only one is observed in the reduced [4Fe-4S]+ state. The Mössbauer parameters of Fea reveal that the site has acquired substantial high-spin ferrous character. This is most pronounced in the 1+ state where at Fea the cluster exhibits a localized valence state. The dramatic increase of the isomer shift upon substrate binding strongly suggests that the ligand environment of Fea has become at least five-coordinate and that the cluster may function as a Lewis acid. In the absence of citrate the EPR spectra of the active [4Fe-4S]+ enzyme (g1,2,3 = 2.06, 1.93, 1.86) show no hyperfine broadening in the presence of H2 17O. However, in the presence of citrate (g1,2,3 = 2.04, 1.85, 1.78) sizable transferred hyperfine interactions are observed; under the experimental conditions the hydroxyl groups of citrate and isocitrate as well as water are labeled with 17O. We did not detect broadening by 17O-labeled carboxyl groups of citrate in H2 16O. Implications for the mechanism of aconitase are discussed.

We have studied samples of oxidized (as isolated) cytochrome c1aa3 from Thermus thermophilus in the pH range 5.7 to 9.3 with Mössbauer spectroscopy. In this pH range, the spectra of cytochromes c1 and a are independent of pH, whereas the spectra of cytochrome a3 are not. Most importantly, spectra taken in applied fields up to 6.0 T reveal the presence of multiple ferric forms of cytochrome a3. At any given pH value, at least two high-spin ferric cytochrome a3 species can be distinguished; in addition, most samples contain a low-spin ferric cytochrome a3 species (less than 20% of cytochrome a3). The Mössbauer spectra show clearly that all forms of cytochrome a3 are spin coupled (to CuB). We have analyzed the high field (H greater than or equal to 1.5 tesla) spectra of a sample at pH 6.5 in the framework of a model that considers isotropic exchange-coupling, JS1.S2, between a high-spin ferric (S1 = 5/2) cytochrome a3 and cupric CuB (S2 = 1/2). In strong applied fields, the spectra can be fitted with any value for J greater than or equal to 0.5 cm-1. In the strong coupling case (J/D1 approximately greater than 3), a zero field splitting parameter D1 approximately 2.5 cm-1 is required for cytochrome a3; this value is distinctly smaller than those observed for high-spin ferric hemes (4-20 cm-1). A model assuming weak coupling magnitude of J approximately 1 cm-1, yields D1 approximately 8 cm-1 and a parameter set for cytochrome a3 quite similar to that reported for metmyoglobin. A J-value of approximately 1 cm-1 does not demand the presence of a ligand bridging between cytochrome a3 and CuB.

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). Mössbauer 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.