Alice S. Pereira

The periplasmic hydrogenase of Desulfovibrio vulgaris (Hildenbourough) is an all Fe-containing hydrogenase. It contains two ferredoxin type [4Fe-4S] clusters, termed the F clusters, and a catalytic H cluster. Recent X-ray crystallographic studies on two Fe hydrogenases revealed that the H cluster is composed of two sub-clusters, a [4Fe-4S] cluster ([4Fe-4S]H) and-a binuclear Fe cluster ([2Fe]H), bridged by a cysteine sulfur. The aerobically purified D. vulgaris hydrogenase is stable in air. It is inactive and requires reductive activation. Upon reduction, the enzyme becomes sensitive to O(2) indicating that the reductive activation process is irreversible. Previous EPR investigations showed that upon reoxidation (under argon) the H cluster exhibits a rhombic EPR signal that is not seen in the as-purified enzyme, suggesting a conformational change in association with the reductive activation. For the purpose of gaining more information on the electronic properties of this unique H cluster and to understand further the reductive activation process, variable-temperature and variable-field Mossbauer spectroscopy has been used to characterize the Fe-S clusters in D. vulgaris hydrogenase poised at different redox states generated during a reductive titration, and in the GO-reacted enzyme. The data were successfully decomposed into spectral components corresponding to the F and H clusters,and characteristic parameters describing the electronic and magnetic properties of the F and H clusters were obtained. Consistent with the X-ray crystallographic results, the spectra of the H cluster can be understood as originating from an exchange coupled [4Fe-4S] - [2Fe] system. In particular, detailed analysis of the data reveals that the reductive activation begins with reduction of the [4Fe-4S]H cluster from the 2+ to the If state, followed by transfer of the reducing equivalent from the [4Fe-4S]H subcluster to the binuclear [2Fe]H subcluster. The results also reveal that binding of exogenous CO to the H cluster affects significantly the exchange coupling between the [4Fe-4S]H and the [2Fe]H subclusters. Implication of such a CO binding effect is discussed.

The outcome of O-2 activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122)(1) production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine)(2)-(carboxylate)(4) ligand set characteristic of the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a mu -1,2-peroxodiiron(III) intermediate,(2 3) which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H-peroxo) in O-2 activation by MMOH.(4) In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the "extra" electron that occurs in wt R2 during formation of the formally Fe(LII)Fe(IV) cluster X.(5-7) Decay of the mu1,2-peroxodiiron(III) complex in R2-W38F/D84E gives an initial brown product, which contains very little YI22(.) and which converts very slowly (t(1/2) similar to 7 h) upon incubation at 0 degreesC to an intensely purple final product. X-ray crystallographic analysis of the purple product indicates that F208 has undergone epsilon -hydroxylation and the resulting phenol has shifted significantly to become st ligand to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purple product generated with O-16(2) or O-18(2) show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from Or. Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and Mossbauer and EXAFS characterization of the brown and purple species establish that F208 hydroxylation occurs during decay of the peroxo complex and formation of the initial brown product. The slow transition to the purple Fe(LII)-phenolate species is ascribed to a ligand rearrangement in which mu -O2- is lost and the F208-derived phenolate coordinates. The reprogramming to F208 monooxygenase requires both amino acid substitutions, as very little epsilon -hydroxyphenylalanine is formed and pathways leading to Y122(.) formation predominate in both R2-D84E and R2-W48F(2-7).

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q Variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical For the catalytic process by controlling the release of the product.

Nitrous oxide (N(2)O) is a greenhouse gas, the third most significant contributor to global warming. As a key process for N(2)O elimination from the biosphere, N(2)O reductases catalyze the two-electron reduction of N(2)O to N(2). These 2 x 65 kDa copper enzymes are thought to contain a CuA electron entry site, similar to that of cytochrome c oxidase, and a CuZ catalytic center. The copper anomalous signal was used to solve the crystal structure of N(2)O reductase from Pseudomonas nautica by multiwavelength anomalous dispersion, to a resolution of 2.4 Angstrom. The structure reveals that the CuZ center belongs to a new type of metal cluster, in which four copper ions are liganded by seven histidine residues. N(2)O binds to this center via a single copper ion. The remaining copper ions might act as an electron reservoir, assuring a fast electron transfer and avoiding the formation of dead-end products.

The aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors. The protein contains six copper atoms per monomer, arranged in two centers named CUA and Cut. Cut could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)= 2.015, A(x) = 1.5 mT, g(y) = 2.071, A(y) = 2 mT, g(z) = 2.138, A(z) = 7 mT) and a strong absorption at similar to 640 nm. Cu-A can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x) g(y) = 2.021, A(x) = A(y) = 0 T, g(z) =0.178, A(z) = 4 mT) and absorption bands at 480, 540, and similar to 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the CuA center. In form A, CUA is predominantly oxidized (S = 1/2, Cu1.5+-Cu1.5+), while in form B it is mostly in the one-electron reduced state (S = 0, Cu1+-Cu1+). In both forms, Cu-Z remains reduced (S = 1/2). Complete crystallographic data at 2.4 Angstrom indicate that Cu-A is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu-Z is a novel tetracopper cluster [Brown, K., et ai. (2000) Nat. Struct. Biol. (in press)]. The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers. A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family.

Mycobacterium tuberculosis KatG is a multifunctional heme enzyme responsible for activation of the antibiotic isoniazid. A KatG(S315T) point mutation is found in >50% of isoniazid-resistant clinical isolates. Since isoniazid activation is thought to involve an oxidation reaction, the redox potential of KatG was determined using cyclic voltammetry, square wave voltammetry, and spectroelectrochemical titrations. Isoniazid activation may proceed via a cytochrome P450-like mechanism. Therefore, the possibility that substrate binding by KatG leads to an increase in the heme redox potential and the possibility that KatG(S315T) confers isoniazid resistance by altering the redox potential were examined. Effects of the heme spin state on the reduction potentials of KatG and KatG(S315T) were also determined. Assessment of the Fe3+/Fe2+ couple gave a midpoint potential of ca. -50 mV for both KatG and KatG(S315T). In contrast to cytochrome P450s, addition of substrate had no significant effect on either the KatG or KatG(S315T) redox potential. Conversion of the heme to a low-spin configuration resulted in a -150 to -200 mV shift of the KatG and KatG(S315T) redox potentials. These results suggest that isoniazid resistance conferred by KatG(S315T) is not mediated through changes in the heme redox potential. The redox potentials of isoniazid were also determined using cyclic and square wave voltammetry, and the results provide evidence that the ferric KatG and KatG(S315T) midpoint potentials are too low to promote isoniazid oxidation without formation of a high-valent enzyme intermediate such as compounds I and IT or oxyferrous KatG.

Fuscoredoxin is a unique iron containing protein of yet unknown function originally discovered in the sulfate reducers of the genus Desulfovibrio. It contains two iron-sulfur clusters: a cubane [4Fe-4S] and a mixed oxo- and sulfide-bridged 4Fe cluster of unprecedented structure. The recent determination of the genomic sequence of Escherichia coli (E. coli) has revealed a homologue of fuscoredoxin in this facultative microbe. The presence of this gene in E. coli raises interesting questions regarding the function of fuscoredoxin and whether this gene represents a structural homologue of the better-characterized Desulfovibrio proteins. In order to explore the latter, an overexpression system for the E. coli fuscoredoxin gene was devised. The gene was cloned from genomic DNA by use of the polymerase chain reaction into the expression vector pT7-7 and overexpressed in E. coli BL21(DE3) cells. After two chromatographic steps a good yield of recombinant protein was obtained (approximately 4 mg of pure protein per liter of culture). The purified protein exhibits an optical spectrum characteristic of the homologue from D. desulfuricans, indicating that cofactor assembly was accomplished. Iron analysis indicated that the protein contains circa 8 iron atoms/molecule which were shown by EPR and Mossbauer spectroscopies to be present as two multinuclear clusters, albeit with slightly altered spectroscopic features. A comparison of the primary sequences of fuscoredoxins is presented and differences on cluster coordination modes are discussed on the light of the spectroscopic data. (C) 1999 Academic Press.

An air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein, Selenium was not detected. The UV/visible absorption spectrum of D, gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both Mossbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with Fe-57 and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.

The soluble methane monooxygenase system from Methylococcus capsulatus (Bath) catalyzes the oxidation of methane to methanol and water utilizing dioxygen at a non-heme, carboxylate-bridged diiron center housed in the hydroxylase (H) component. To probe the nature of the reductive activation of dioxygen in this system, reactions of an analogous molecule, nitric oxide, with the diiron(II) form of the enzyme (H-red) Were investigated by both continuous and discontinuous kinetics methodologies using optical, EPR, and Mossbauer spectroscopy. Reaction of NO with H-red affords a dinitrosyl species, designated H-dinitrosyl, with optical spectra (lambda(max) = 450 and 620 nm) and Mossbauer parameters (delta = 0.72 mm/s, Delta E-Q = 1.55 mm/s) similar to those of synthetic dinitrosyl analogues and of the dinitrosyl adduct of the reduced ribonucleotide reductase R2 (RNR-R2) protein. The H-dinitrosyl species models features of the H-peroxo intermediate formed in the analogous dioxygen reaction. In the presence of protein B, H-dinitrosyl builds up with approximately the same rate constant as H-peroxo (similar to 26 s(-1)) at 4 degrees C. In the absence of protein B, the kinetics of H-dinitrosyl formation were best fit with a biphasic A –> B –> C model, indicating the presence of an intermediate species between H-red and H-dinitrosyl. This result contrasts with the reaction of H-red with dioxygen, in which the H-peroxo intermediate forms in measurable quantities only in the presence of protein B. These findings suggest that protein B may alter the positioning but not the availability of coordination sites on iron for exogenous ligand binding and reactivity.

Rapid freeze-quench (RFQ) Mossbauer and stopped-flow absorption spectroscopy were used to monitor the ferritin ferroxidase reaction using recombinant (apo) frog M ferritin; the initial transient ferric species could be trapped by the RFQ method using low iron loading (36 Fe2+/ferritin molecule). Biphasic kinetics of ferroxidation were observed and measured directly by the Mossbauer method; a majority (85%) of the ferrous ions was oxidized at a fast rate of similar to 80 s(-1) and the remainder at a much slower rate of similar to 1.7 s(-1). In parallel with the fast phase oxidation of the Fe2+ ions, a single transient iron species is formed which exhibits magnetic properties (diamagnetic ground state) and Mossbauer parameters (Delta E-Q = 1.08 +/- 0.03 mm/s and delta = 0.62 +/- 0.02 mm/s) indicative of an antiferromagnetically coupled peroxodiferric complex. The formation and decay rates of this transient diiron species measured by the RFQ Mossbauer method match those of a transient blue species (lambda(max) = 650 nm) determined by the stopped-flow absorbance measurement. Thus, the transient colored species is assigned to the same peroxodiferric intermediate. Similar transient colored species have been detected by other investigators in several other fast ferritins (H and M subunit types), such as the human H ferritin and the Escherichia coli ferritin, suggesting a similar mechanism for the ferritin ferroxidase step in all fast ferritins. Peroxodiferric complexes are also formed as early intermediates in the reaction of O-2 With the catalytic diiron centers in the hydroxylase component of soluble methane monooxygenase (MMOH) and in the D84E mutant of the R2 subunit of E. coli ribonucleotide reductase. The proposal that a single protein site, with a structure homologous to the diiron centers in MMOH and R2, is involved in the ferritin ferroxidation step is confirmed by the observed kinetics, spectroscopic properties, and purity of the initial peroxodiferric species formed in the frog M ferritin.

Mossbauer and EPR spectroscopies were used to characterize the Fe clusters in an Fe-S protein isolated from Desulfovibrio desulfuricans (ATCC 27774). This protein was previously thought to contain hexanuclear Fe clusters, but a recent X-ray crystallographic measurement on a similar protein isolated from Desulfovibrio vulgaris showed that the protein contains two tetranuclear clusters, a cubane-type [4Fe-4S] cluster and a mixed-ligand cluster of novel structure [Lindley et al. (1997) Abstract, Chemistry of Metals in Biological Systems, European Research Conference, Tomar, Portugal]. Three protein samples poised at different redox potentials (as-purified, 40 and 320 mV) were investigated. In all three samples, the [4Fe-4S] cluster was found to be present in the diamagnetic 2+ oxidation state and exhibited typical Mossbauer spectra. The novel-structure cluster was found to be redox active. In the 320-mV and as-purified samples, the cluster is at a redox equilibrium between its fully oxidized and one-electron reduced states. In the 40-mV sample, the cluster is in a two-electron reduced state. Distinct spectral components associated with the four Fe sites of cluster 2 in the three oxidation states were identified. The spectroscopic parameters obtained for the Fe sites reflect different ligand environments, making it possible to assign the spectral components to individual Fe sites. In the fully oxidized state, all four iron ions are high-spin ferric and antiferromagnetically coupled to form a diamagnetic S = 0 state. In the one-electron and two-electron reduced states, the reducing electrons were found to localize, consecutively, onto two Fe sites that are rich in oxygen/nitrogen ligands. Based on the X-ray structure and the Mossbauer parameters, attempts could be made to identify the reduced Fe sites. For the two-electron reduced cluster, EPR and Mossbauer data indicate that the cluster is paramagnetic with a nonzero interger spin. For the one-electron reduced cluster, the data suggest a half-integer spin of 9/2 Characteristic fine and hyperfine parameters for all four Fe sites were obtained. Structural implications and the nature of the spin-coupling interactions are discussed.

Conversion of Fe ions in solution to the solid phase in ferritin concentrates iron required for cell function. The rate of the Fe phase transition in ferritin is tissue specific and reflects the differential expression of two classes of ferritin subunits (H and L). Early stages of mineralization were probed by rapid freeze-quench Mossbauer, at strong fields (up to 8 T), and EPR spectroscopy in an H-type subunit, recombinant frog ferritin; small numbers of Fe (36 moles/mol of protein) were used to increase Fe3+ in mineral precursor forms, At 25 ms, four Fe3+-oxy species (three Fe dimers and one Fe trimer) were identified, These Fe3+-oxy species were found to form at similar rates and decay subsequently to a distinctive superparamagentic species designated the ''young core.'' The rate of oxidation of Fe2+ (1026 s(-1)) corresponded well to the formation constant for the Fe3+- tyrosinate complex (920 s(-1)) observed previously [Waldo, G. S., & Theil, E. C. (1993) Biochemistry 32, 13261] and, coupled with EPR data, indicates that several or possibly all of the Fe3+-oxy species involve tyrosine. The results, combined with previous Mossbauer studies of Y30F human H-type ferritin which showed decreases in several Fe3+ intermediates and stabilization of Fe2+ [Bauminger, E. R., et al. (1993) Biochem, J. 296, 709], emphasize the involvement of tyrosyl residues in the mineralization of H-type ferritins. The subsequent decay of these multiple Fe3+-oxy species to the superparamagnetic mineral suggests that Fe3+ species in different environments may be translocated as intact units from the protein shell into the ferritin cavity where the conversion to a solid mineral occurs.

This communication reports the isolation, purification and characterization of key enzymes involved in dissimilatory sulfate reduction of a sulfate reducing bacterium classified as Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) (Ddd NJ). The chosen strain, originally recovered from a corroding cast iron heat exchanger, was grown in large scale batch cultures. Physico-chemical and spectroscopic studies of the purified enzymes were carried out. These analyses revealed a high degree of similarity between proteins isolated from the DddNJ strain and the homologous proteins obtained from Desulfomicrobium baculatus Norway 4. In view of the results obtained, taxonomic reclassification of Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) into Desulfomicrobium baculatus (New Jersey) is proposed. (C) 1996 Academic Press, Inc.

A [2Fe-2S] cluster has been detected in mammalian ferrochelatase, the terminal enzyme of the heme biosynthetic pathway. Natural ferrochelatase, purified from mouse livers, and recombinant ferrochelatase, purified from an overproducing strain of Escherichia coli, were investigated by electron paramagnetic resonance (EPR) and Mossbauer spectroscopy. In their reduced forms, both the natural and recombinant ferrochelatases exhibited an identical EPR signal with g values (g = 2.00, 1.93, and 1.90) and relaxation properties typical of [2Fe-2S](+) cluster. Mossbauer spectra of the recombinant ferrochelatase, purified from a strain of E. coli cells transformed with a plasmid encoding murine liver ferrochelatase and grown in Fe-57-enriched medium, demonstrated unambiguously that the cluster is a [2Fe-2S] cluster. No change in the cluster oxidation state was observed during catalysis, The putative protein binding site for the Fe-S cluster in mammalian ferrochelatases is absent from the sequences of the bacterial and yeast enzymes, suggesting a possible role of the [2Fe-2S] center in regulation of mammalian ferrochelatases.