Biomedical subjects

B H Huynh

Publications and source records attributed to B H Huynh.

At least 37 records · Page 2Linked to original sources

PubMed1995

Mössbauer characterization of Paracoccus denitrificans cytochrome c peroxidase. Further evidence for redox and calcium binding-induced heme-heme interaction.

Mössbauer and electron paramagnetic resonance (EPR) spectroscopies were used to characterize the diheme cytochrome c peroxidase from Paracoccus denitrificans (L.M.D. 52.44). The spectra of the oxidized enzyme show two distinct spectral components characteristic of low spin ferric hemes (S = 1/2), revealing different heme environments for the two heme groups. The Paracoccus peroxidase can be non-physiologically reduced by ascorbate. Mössbauer investigation of the ascorbate-reduced peroxidase shows that only one heme (the high potential heme) is reduced and that the reduced heme is diamagnetic (S = 0). The other heme (the low potential heme) remains oxidized, indicating that the enzyme is in a mixed valence, half-reduced state. The EPR spectrum of the half-reduced peroxidase, however, shows two low spin ferric species with gmax = 2.89 (species I) and gmax = 2.78 (species II). This EPR observation, together with the Mössbauer result, suggests that both species are arising from the low potential heme. More interestingly, the spectroscopic properties of these two species are distinct from that of the low potential heme in the oxidized enzyme, providing evidence for heme-heme interaction induced by the reduction of the high potential heme. Addition of calcium ions to the half-reduced enzyme converts species II to species I. Since calcium has been found to promote peroxidase activity, species I may represent the active form of the peroxidatic heme.

Animals↗

PubMed1995

Recombinant Desulfovibrio vulgaris rubrerythrin. Isolation and characterization of the diiron domain.

The gene encoding Desulfovibrio (D.) vulgaris rubrerythrin (Prickril, B. C., Kurtz, D. M., Jr., LeGall, J., & Voordouw, G. (1991) Biochemistry 30, 1118), a protein of unknown function containing both FeS4 and (mu-oxo)diiron sites, was cloned and overexpressed in Escherichia coli. Upon cell lysis, the overexpressed protein was found in an insoluble form deficient in iron. Iron was incorporated in vitro by dissolving the protein in 3 M guanidinium chloride, adding Fe(II) anaerobically and diluting the denaturant. This recombinant rubrerythrin was found to have properties very similar to those of rubrerythrin isolated from D. vulgaris, except that the recombinant rubrerythrin contained six rather than four (or five) iron atoms per 44 kDa homodimer. Analyses of UV-vis, Mössbauer, and EPR spectra showed that the six iron atoms in recombinant rubrerythrin are organized as two FeS4 and two (mu-oxo/hydroxo)diiron sites. In order to allow examination of the diiron sites in the absence of the FeS4 sites, a truncated gene encoding the N-terminal 152 residues of D. vulgaris rubrerythrin was also cloned and overexpressed as an insoluble protein in E. coli, and iron was incorporated by a procedure analogous to that for recombinant rubrerythrin. This so-called "chopped" rubrerythrin (CRr) was found to consist of an approximately 35 kDa homodimer containing four iron atoms. Spectroscopic characterization indicated that the four iron atoms in CRr are organized as two diiron sites, the majority of which closely resemble the (mu-oxo)diiron(III) sites in E. coli ribonucleotide reductase R2 protein, and a minor fraction of which resemble the mixed-valent diiron(II,III) site in methane monooxygenase hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗

PubMed1995

Structure and function of ferrochelatase.

Ferrochelatase is the terminal enzyme of the heme biosynthetic pathway in all cells. It catalyzes the insertion of ferrous iron into protoporphyrin IX, yielding heme. In eukaryotic cells, ferrochelatase is a mitochondrial inner membrane-associated protein with the active site facing the matrix. Decreased values of ferrochelatase activity in all tissues are a characteristic of patients with protoporphyria. Point-mutations in the ferrochelatase gene have been recently found to be associated with certain cases of erythropoietic protoporphyria. During the past four years, there have been considerable advances in different aspects related to structure and function of ferrochelatase. Genomic and cDNA clones for bacteria, yeast, barley, mouse, and human ferrochelatase have been isolated and sequenced. Functional expression of yeast ferrochelatase in yeast strains deficient in this enzyme, and expression in Escherichia coli and in baculovirus-infected insect cells of different ferrochelatase cDNAs have been accomplished. A recently identified (2Fe-2S) cluster appears to be a structural feature shared among mammalian ferrochelatases. Finally, functional studies of ferrochelatase site-directed mutants, in which key amino acids were replaced with residues identified in some cases of protoporphyria, will be summarized in the context of protein structure.

Amino Acid Sequence↗

PubMed1995

Use of rapid kinetics methods to study the assembly of the diferric-tyrosyl radical cofactor of E. coli ribonucleotide reductase.

The SF-Abs, RFQ-EPR, and RFQ-Möss data on the R2 reconstitution reaction are all consistent with the mechanism of Scheme I, in which the intermediate X is the immediate precursor to the product cofactor, and illustrate how the continuous SF approach and the discontinuous RFQ methods can be complementary. Given the inherent differences in the methods, it should not be taken for granted that data from the two will be consistent. A number of problems can be associated with the RFQ approach. For example, isopentane could conceivably interfere with or alter the chemistry to be studied. A second potential problem involves temperature-dependent equilibria among different intermediate species. This problem has been encountered by Dooley et al. with the 6-hydroxydopa-requiring protein, plasma amine oxidase and was previously observed with the adenosylcobalamin-dependent ribonucleotide reductase by Blakley and co-workers. This potential complication should be considered when discrepancies arise between SF and RFQ data and in low temperature structural studies of reactive intermediates in general. Each of the three methods employed can yield time-resolved quantitation of reaction components. In this regard, SF-Abs has the disadvantage of poor resolution, such that quantitation of individual components most often requires sophisticated mathematical analysis. Obvious advantages to the RFQ-Möss method are the presence of an internal standard (the known amount of 57Fe being proportional to the total absorption area) and the spectroscopic activity of all reaction components which contain iron. In our hands, quantitation by RFQ-EPR was most problematic and least reproducible. This irreproducibility most likely relates to heterogeneity among samples in terms of volume and density. As discussed in detail by Ballou and Palmer, the packing factor, which relates to the fraction of a sample made up by the reaction solution (the remainder being frozen isopentane), is dependent on the investigator. Given this caveat, it is not surprising that the RFQ-EPR data had the greatest uncertainty in our hands. Placing a chemically unreactive, EPR active standard in each reaction mixture could help alleviate this problem. Time-resolved Möss methods can be extremely powerful if excellent, nonoverlapping reference spectra of starting materials, products, and intermediates are available. All of the iron centers can be examined simultaneously. The problems associated with Möss arise from its extreme insensitivity. It takes millimolar solutions of proteins and several days for data collection of each time point.(ABSTRACT TRUNCATED AT 400 WORDS)

Coenzymes↗

PubMed1994

Mössbauer characterization of the metal clusters in Azotobacter vinelandii nitrogenase VFe protein.

The VFe protein of alternative nitrogenase, isolated from Azotobacter vinelandii, strain LS15 and designated as Av1', has been investigated by Mössbauer spectroscopy. The Mössbauer spectrum of the dithionite-reduced Av1', recorded at 4.2 K with a 60-millitesla magnetic field applied parallel to the gamma-beam, is a superposition of three spectral components: 1) a complex spectrum (the M component) with magnetic hyperfine structures attributed to the paramagnetic FeV cofactor, 2) a component (the P component) consisting of three quadrupole doublets identifiable as the Fe2+, D, and S doublets similar to those observed for the P cluster pairs in MoFe proteins, and 3) a minor (4% of total absorption) quadrupole doublet attributable to adventitiously bound iron. The observed 4.2-K parameters for the Fe2+ (delta EQ = 2.99 mm/s and delta = 0.64 mm/s), D (delta EQ = 0.75 mm/s and delta = 0.63 mm/s), and S (delta EQ = 1.2 mm/s and delta = 0.65 mm/s) iron sites and their temperature dependence are very similar to those observed for the P cluster pairs in the conventional MoFe proteins. Similar to those of the MoFe protein, strong field spectra indicate that these doublets are associated with a diamagnetic system. Their percent absorption intensities (Fe(2+)/D/S = 13.0:32.2:6.8) determined at 4.2 K after the removal of the contributions from the adventitiously bound iron are comparable to those of the P cluster pairs in MoFe proteins. These observations established that Av1' also contains P cluster pairs that are identical, in both composition and quantity, to those of the MoFe proteins; i.e. each molecule contains two P cluster pairs and each pair is formed by two Fe2+, five D, and one S iron sites. Considering that 52% absorption of the P component corresponding to two 8Fe clusters, the remaining 48% absorption determined for the M component is consistent with two 7Fe-containing FeV cofactors/molecule of Av1'. The fact that both P cluster pairs are found in the diamagnetic states implies that the S = 3/2 and S = 1/2 signals detected in earlier EPR measurements are associated with the FeV cofactor and suggests a spin mixture for the FeV cofactor in the dithionite-reduced Av1'.

Azotobacter vinelandii↗

PubMed1994

Spectroscopic properties of desulfoferrodoxin from Desulfovibrio desulfuricans (ATCC 27774).

Desulfoferrodoxin, a non-heme iron protein, was purified previously from extracts of Desulfovibrio desulfuricans (ATCC 27774) (Moura, I., Tavares, P., Moura, J. J. G., Ravi, N., Huynh, B. H., Liu, M.-Y., and LeGall, J. (1990) J. Biol. Chem. 265, 21596-21602). The as-isolated protein displays a pink color (pink form) and contains two mononuclear iron sites in different oxidation states: a ferric site (center I) with a distorted tetrahedral sulfur coordination similar to that found in desulforedoxin from Desulfovibrio gigas and a ferrous site (center II) octahedrally coordinated with predominantly nitrogen/oxygen-containing ligands. A new form of desulfoferrodoxin which displays a gray color (gray form) has now been purified. Optical, electron paramagnetic resonance (EPR), and Mössbauer data of the gray desulfoferrodoxin indicate that both iron centers are in the high-spin ferric states. In addition to the EPR signals originating from center I at g = 7.7, 5.7, 4.1, and 1.8, the gray form of desulfoferrodoxin exhibits a signal at g = 4.3 and a shoulder at g = 9.6, indicating a high-spin ferric state with E/D approximately 1/3 for the oxidized center II. Redox titrations of the gray form of the protein monitored by optical spectroscopy indicate midpoint potentials of +4 +/- 10 and +240 +/- 10 mV for centers I and II, respectively. Mössbauer spectra of the gray form of the protein are consistent with the EPR finding that both centers are high-spin ferric and can be analyzed in terms of the EPR-determined spin Hamiltonian parameters. The Mössbauer parameters for both the ferric and ferrous forms of center II are indicative of a mononuclear high spin iron site with octahedral coordination and predominantly nitrogen/oxygen-containing ligands. Resonance Raman studies confirm the structural similarity of center I and the distorted tetrahedral FeS4 center in desulforedoxin and provide evidence for one or two cysteinyl-S ligands for center II. On the basis of the resonance Raman results, the 635 nm absorption band that is responsible for the gray color of the oxidized protein is assigned to a cysteinyl-S-->Fe(III) charge transfer transition localized on center II. The novel properties and possible function of center II are discussed in relation to those of mononuclear iron centers in other enzymes.

Desulfovibrio↗

PubMed1994

Mammalian ferrochelatase, a new addition to the metalloenzyme family.

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 Mössbauer 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. Mössbauer spectra of the recombinant ferrochelatase, purified from a strain of E. coli cells transformed with a plasmid encoding murine liver ferrochelatase and grown in 57Fe-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.

Amino Acid Sequence↗

PubMed1993

Characterization of D. desulfuricans (ATCC 27774) [NiFe] hydrogenase EPR and redox properties of the native and the dihydrogen reacted states.

Redox intermediates of D. desulfuricans ATCC 27774 [NiFe] hydrogenase were generated under dihydrogen. Detailed redox titrations, coupled to EPR measurements, give access to the mid-point redox potentials of the iron-sulfur centers and of the Nickel-B signal that represents the ready form of the enzyme. The interaction between the dihydrogen molecule and the nickel centre was probed by the observation of an isotopic effect on the EPR signals detected in turnover conditions, by comparison of the H2O/H2 and D2O/D2-reacted samples.

Binding Sites↗

PubMed1993

Spectroscopic characterization of 57Fe-reconstituted rubrerythrin, a non-heme iron protein with structural analogies to ribonucleotide reductase.

Rubrerythrin, a contraction of rubredoxin and hemerythrin, is the trivial name given to a non-heme iron protein isolated from Desulfovibrio vulgaris (Hildenborough). This protein, whose physiological function is unknown, was first characterized by J. LeGall et al. [(1988) Biochemistry 28, 1636] as being a homodimer of subunit M(r) = 21,900 with four Fe per homodimer distributed as two rubredoxin-type FeS4 centers and one hemerythrin-type diiron cluster. Subsequent analysis of the amino acid sequence of the rubrerythrin gene [Kurtz, D. M., Jr., & Prickril, B.C. (1991) Biochem. Biophys. Res. Commun. 181, 137] revealed an internal homology which suggested that each subunit can accommodate one diiron cluster. Here, we report a procedure for reconstitution of the as-isolated D. vulgaris rubrerythrin with 57Fe. The reconstituted protein was characterized by optical, electron paramagnetic resonance, and Mössbauer spectroscopies. The results indicate successful incorporation of 57Fe into the two types of sites and strongly suggest that each subunit of rubrerythrin can indeed accommodate one diiron cluster as well as one rubredoxin-type center. Combined with amino acid sequence analysis, the spectroscopic characterization further suggests that the rubrerythrin subunit contains a diiron site whose structure is more closely related to that in ribonucleotide reductase than to that in hemerythrin.

Bacterial Proteins↗

PubMed1992

Direct spectroscopic evidence for the presence of a 6Fe cluster in an iron-sulfur protein isolated from Desulfovibrio desulfuricans (ATCC 27774)

A novel iron-sulfur protein was purified from the extract of Desulfovibrio desulfuricans (ATCC 27774) to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a monomer of 57 kDa molecular mass. It contains comparable amounts of iron and inorganic labile sulfur atoms and exhibits an optical spectrum typical of iron-sulfur proteins with maxima at 400, 305, and 280 nm. Mössbauer data of the as-isolated protein show two spectral components, a paramagnetic and a diamagnetic, of equal intensity. Detailed analysis of the paramagnetic component reveals six distinct antiferromagnetically coupled iron sites, providing direct spectroscopic evidence for the presence of a 6Fe cluster in this newly purified protein. One of the iron sites exhibits parameters (delta EQ = 2.67 +/- 0.03 mm/s and delta = 1.09 +/- 0.02 mm/s at 140 K) typical for high spin ferrous ion; the observed large isomer shift indicates an iron environment that is distinct from the tetrahedral sulfur coordination commonly observed for the iron atoms in iron-sulfur clusters and is consistent with a penta- or hexacoordination containing N and/or O ligands. The other five iron sites are most probably high spin ferric. Three of them show parameters characteristic for tetrahedral sulfur coordination. In correlation with the EPR spectrum of the as-purified protein which shows a resonance signal at g = 15.3 and a group of signals between g = 9.8 and 5.4, this 6Fe cluster is assigned to an unusual spin state of 9/2 with zero field splitting parameters D = -1.3 cm-1 and E/D = 0.062. Other EPR signals attributable to minor impurities are also observed at the g = 4.3 and 2.0 regions. The diamagnetic Mössbauer component represents a second iron cluster, which, upon reduction with dithionite, displays an intense S = 1/2 EPR signal with g values at 2.00, 1.83, and 1.31. In addition, an EPR signal of the S = 3/2 type is also observed for the dithionite-reduced protein.

Amino Acid Sequence↗

PubMed1992

Mössbauer study of the native, reduced and substrate-reacted Desulfovibrio gigas aldehyde oxido-reductase.

The Desulfovibrio gigas aldehyde-oxido-reductase contains molybdenum and iron-sulfur clusters. Mössbauer spectroscopy was used to characterize the iron-sulfur clusters. Spectra of the enzyme in its oxidized, partially reduced and benzaldehyde-reacted states were recorded at different temperatures and applied magnetic fields. All the iron atoms in D. gigas aldehyde oxido-reductase are organized as [2Fe-2S] clusters. In the oxidized enzyme, the clusters are diamagnetic and exhibit a single quadrupole doublet with parameters (delta EQ = 0.62 +/- 0.02 mm/s and delta = 0.27 +/- 0.01 mm/s) typical for the [2Fe-2S]2+ state. Mössbauer spectra of the reduced clusters also show the characteristics of a [2Fe-2S]1+ cluster and can be explained by a spin-coupling model proposed for the [2Fe-2S] cluster where a high-spin ferrous ion (S = 2) is antiferromagnetically coupled to a high-spin ferric ion (S = 5/2) to form a S = 1/2 system. Two ferrous sites with different delta EQ values (3.42 mm/s and 2.93 mm/s at 85 K) are observed for the reduced enzyme, indicating the presence of two types of [2Fe-2S] clusters in the D. gigas enzyme. Taking this observation together with the re-evaluated value of iron content (3.5 +/- 0.1 Fe/molecule), it is concluded that, similar to other Mo-hydroxylases, the D. gigas aldehyde oxido-reductase also contains two spectroscopically distinguishable [2Fe-2S] clusters.

Aldehyde Oxidoreductases↗

PubMed1992

Mössbauer characterization of the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Spectral deconvolution of the heme components.

Mössbauer spectroscopy was used to study the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Samples with different degrees of reduction were prepared using a redoxtitration technique. In the reduced cytochrome c3, all four hemes are reduced and exhibit diamagnetic Mössbauer spectra typical for low-spin ferrous hemes (S = 0). In the oxidized protein, the hemes are low-spin ferric (S = 1/2) and exhibit overlapping magnetic Mössbauer spectra. A method of differential spectroscopy was applied to deconvolute the four overlapping heme spectra and a crystal-field model was used for data analysis. Characteristic Mössbauer spectral components for each heme group are obtained. Hyperfine and crystal-field parameters for all four hemes are determined from these deconvoluted spectra.

Cytochrome c Group↗

PubMed1991

Mechanism of assembly of the tyrosyl radical-dinuclear iron cluster cofactor of ribonucleotide reductase.

Incubation of the apoB2 subunit of Escherichia coli ribonucleotide reductase with Fe2+ and O2 produces native B2, which contains the tyrosyl radical-dinuclear iron cluster cofactor required for nucleotide reduction. The chemical mechanism of this reconstitution reaction was investigated by stopped-flow absorption spectroscopy and by rapid freeze-quench EPR (electron paramagnetic resonance) spectroscopy. Two novel intermediates have been detected in the reaction. The first exhibits a broad absorption band centered at 565 nanometers. Based on known model chemistry, this intermediate is proposed to be a mu-peroxodiferric complex. The second intermediate exhibits a broad absorption band centered at 360 nanometers and a sharp, isotropic EPR signal with g = 2.00. When the reaction is carried out with 57Fe2+, this EPR signal is broadened, demonstrating that the intermediate is an iron-coupled radical. Variation of the ratio of Fe2+ to B2 in the reaction and comparison of the rates of formation and decay of the intermediates to the rate of formation of the tyrosyl radical (.Y122) suggest that both intermediates can generate .Y122. This conclusion is supported by the fact that both intermediates exhibit an increased lifetime in a mutant B2 subunit (B2-Y122F) lacking the oxidizable Y122. Based on these kinetic and spectroscopic data, a mechanism for the reaction is proposed. Unlike reactions catalyzed by heme-iron peroxidases, oxygenases, and model complexes, the reconstitution reaction appears not to involve high-valent iron intermediates.

Binding Sites↗

PubMed1990

Purification and characterization of desulfoferrodoxin. A novel protein from Desulfovibrio desulfuricans (ATCC 27774) and from Desulfovibrio vulgaris (strain Hildenborough) that contains a distorted rubredoxin center and a mononuclear ferrous center.

A new type of non-heme iron protein was purified to homogeneity from extracts of Desulfovibrio desulfuricans (ATCC 27774) and Desulfovibrio vulgaris (strain Hildenborough). This protein is a monomer of 16-kDa containing two iron atoms per molecule. The visible spectrum has maxima at 495, 368, and 279 nm and the EPR spectrum of the native form shows resonances at g = 7.7, 5.7, 4.1 and 1.8 characteristic of a high-spin ferric ion (S = 5/2) with E/D = 0.08. Mössbauer data indicates the presence of two types of iron: an FeS4 site very similar to that found in desulforedoxin from Desulfovibrio gigas and an octahedral coordinated high-spin ferrous site most probably with nitrogen/oxygen-containing ligands. Due to this rather unusual combination of active centers, this novel protein is named desulfoferrodoxin. Based on NH2-terminal amino acid sequence determined so far, the desulfoferrodoxin isolated from D. desulfuricans (ATCC 27774) appears to be a close analogue to a recently discovered gene product from D. vulgaris (Brumlik, M.J., and Voordouw, G. (1989) J. Bacteriol. 171, 49996-50004), which was suggested to be a rubredoxin oxidoreductase. However, reduced pyridine nucleotides failed to reduce the desulforedoxin-like center of this new protein.

Amino Acid Sequence↗

PubMed1990

Hexaheme nitrite reductase from Desulfovibrio desulfuricans. Mössbauer and EPR characterization of the heme groups.

Mössbauer and EPR spectroscopy were used to characterize the heme prosthetic groups of the nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774), which is a membrane-bound multiheme cytochrome capable of catalyzing the 6-electron reduction of nitrite to ammonia. At pH 7.6, the as-isolated enzyme exhibited a complex EPR spectrum consisting of a low-spin ferric heme signal at g = 2.96, 2.28, and 1.50 plus several broad resonances indicative of spin-spin interactions among the heme groups. EPR redox titration studies revealed yet another low-spin ferric heme signal at g = 3.2 and 2.14 (the third g value was undetected) and the presence of a high-spin ferric heme. Mössbauer measurements demonstrated further that this enzyme contained six distinct heme groups: one high-spin (S = 5/2) and five low-spin (S = 1/2) ferric hemes. Characteristic hyperfine parameters for all six hemes were obtained through a detailed analysis of the Mössbauer spectra. D. desulfuricans nitrite reductase can be reduced by chemical reductants, such as dithionite or reduced methyl viologen, or by hydrogenase under hydrogen atmosphere. Addition of nitrite to the fully reduced enzyme reoxidized all five low-spin hemes to their ferric states. The high-spin heme, however, was found to complex NO, suggesting that the high-spin heme could be the substrate binding site and that NO could be an intermediate present in an enzyme-bound form.

Desulfovibrio↗

PubMed1990

The iron-sulfur centers of the soluble [NiFeSe] hydrogenase, from Desulfovibrio baculatus (DSM 1743). EPR and Mössbauer 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 Mössbauer 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 Mössbauer spectroscopic parameters. The centers were found to have similar mid-point potentials (approximately -315 mV) as determined by oxidation-reduction titratins followed by EPR.

Desulfovibrio↗

PubMed1990

The active centers of adenylylsulfate reductase from Desulfovibrio gigas. Characterization and spectroscopic studies.

In order to utilize sulfate as the terminal electron acceptor, sulfate-reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO4) reductase plays one of the major roles, reducing AdoPSO4 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovibrio gigas. The protein is composed of two non-identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form (approximately 400 kDa). It is an iron-sulfur, flavin-containing protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa. Low-temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered at g = 2.02 and only detectable below 20 K. This signal represented a minor species (0.10-0.25 spins/mol) and showed line broadening in the enzyme isolated from 57Fe-grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal at g values 2.096, 1.940 and 1.890 (reduced Fe-S center I) observable below 30 K and a concomitant decrease in intensity of the g = 2.02 signal were detected. Effects of chemical reductants (ascorbate, H2/hydrogenase-reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modified g values at 2.079, 1.939 and 1.897), and the complete disappearance of the g = 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin-spin-coupled nature (observable below 20 K), indicating the presence of at least two iron-sulfur centers, (centers I and II). Mössbauer studies on 57Fe-enriched D. gigas AdoPSO4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential less than or equal to 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin-type [4Fe-4S] cluster. Center I exhibits spectra with atypical Mössbauer parameters in its reduced state and has a midpoint potential around 0 mV, which is distinct from that of a ferredoxin-type [4Fe-4S] cluster, suggesting a different structure and/or a distinct cluster-ligand environment.

Adenosine Monophosphate↗

PubMed1989

Redox intermediates of Desulfovibrio gigas [NiFe] hydrogenase generated under hydrogen. Mössbauer and EPR characterization of the metal centers.

The hydrogenase (EC 1.2.2.1) of Desulfovibrio gigas is a complex enzyme containing one nickel center, one [3Fe-4S] and two [4Fe-4S] clusters. Redox intermediates of this enzyme were generated under hydrogen (the natural substrate) using a redox-titration technique and were studied by EPR and Mössbauer spectroscopy. In the oxidized states, the two [4Fe-4S]2+ clusters exhibit a broad quadrupole doublet with parameters (apparent delta EQ = 1.10 mm/s and delta = 0.35 mm/s) typical for this type of cluster. Upon reduction, the two [4Fe-4S]1+ clusters are spectroscopically distinguishable, allowing the determination of their midpoint redox potentials. The cluster with higher midpoint potential (-290 +/- 20 mV) was labeled Fe-S center I and the other with lower potential (-340 +/- 20 mV), Fe-S center II. Both reduced clusters show atypical magnetic hyperfine coupling constants, suggesting structural differences from the clusters of bacterial ferredoxins. Also, an unusually broad EPR signal, labeled Fe-S signal B', extending from approximately 150 to approximately 450 mT was observed concomitantly with the reduction of the [4Fe-4S] clusters. The following two EPR signals observed at the weak-field region were tentatively attributed to the reduced [3Fe-4S] cluster: (i) a signal with crossover point at g approximately 12, labeled the g = 12 signal, and (ii) a broad signal at the very weak-field region (approximately 3 mT), labeled the Fe-S signal B. The midpoint redox potential associated with the appearance of the g = 12 signal was determined to be -70 +/- 10 mV. At potentials below -250 mV, the g = 12 signal began to decrease in intensity, and simultaneously, the Fe-S signal B appeared. The transformation of the g = 12 signal into the Fe-S signal B was found to parallel the reduction of the two [4Fe-4S] clusters indicating that the [3Fe-4S]o cluster is sensitive to the redox state of the [4Fe-4S] clusters. Detailed redox profiles for the previously reported Ni-signal C and the g = 2.21 signal were obtained in this study, and evidence was found to indicate that these two signals represent two different oxidation states of the enzyme. Finally, the mechanistic implications of our results are discussed.

Desulfovibrio↗