Showing papers by "Isabel Moura published in 1988"

The three classes of hydrogenases from sulfate-reducing bacteria of the genus Desulfovibrio

Abstract: Three types of hydrogenases have been isolated from the sulfate-reducing bacteria of the genus Desulfovibrio. They differ in their subunit and metal compositions, physico-chemical characteristics, amino acid sequences, immunological reactivities, gene structures and their catalytic properties. Broadly, the hydrogenases can be considered as 'iron only' hydrogenases and nickel-containing hydrogenases. The iron-sulfur-containing hydrogenase ([Fe] hydrogenase) contains two ferredoxin-type (4Fe-4S) clusters and an atypical iron-sulfur center believed to be involved in the activation of H2. The [Fe] hydrogenase has the highest specific activity in the evolution and consumption of hydrogen and in the proton-deuterium exchange reaction and this enzyme is the most sensitive to CO and NO2-. It is not present in all species of Desulfovibrio. The nickel-(iron-sulfur)-containing hydrogenases [( NiFe] hydrogenases) possess two (4Fe-4S) centers and one (3Fe-xS) cluster in addition to nickel and have been found in all species of Desulfovibrio so far investigated. The redox active nickel is ligated by at least two cysteinyl thiolate residues and the [NiFe] hydrogenases are particularly resistant to inhibitors such as CO and NO2-. The genes encoding the large and small subunits of a periplasmic and a membrane-bound species of the [NiFe] hydrogenase have been cloned in Escherichia (E.) coli and sequenced. Their derived amino acid sequences exhibit a high degree of homology (70%); however, they show no obvious metal-binding sites or homology with the derived amino acid sequence of the [Fe] hydrogenase. The third class is represented by the nickel-(iron-sulfur)-selenium-containing hydrogenases [( NiFe-Se] hydrogenases) which contain nickel and selenium in equimolecular amounts plus (4Fe-4S) centers and are only found in some species of Desulfovibrio. The genes encoding the large and small subunits of the periplasmic hydrogenase from Desulfovibrio (D.) baculatus (DSM 1743) have been cloned in E. coli and sequenced. The derived amino acid sequence exhibits homology (40%) with the sequence of the [NiFe] hydrogenase and the carboxy-terminus of the gene for the large subunit contains a codon (TGA) for selenocysteine in a position homologous to a codon (TGC) for cysteine in the large subunit of the [NiFe] hydrogenase. EXAFS and EPR studies with the 77Se-enriched D. baculatus hydrogenase indicate that selenium is a ligand to nickel and suggest that the redox active nickel is ligated by at least two cysteinyl thiolate and one selenocysteine selenolate residues.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation and characterization of rubrerythrin, a non-heme iron protein from Desulfovibrio vulgaris that contains rubredoxin centers and a hemerythrin-like binuclear iron cluster.

Abstract: A new non-heme iron protein from the periplasmic fraction of Desulfovibrio vulgaris (Hildenbourough NCIB 8303) has been purified to homogeneity, and its amino acid composition, molecular weight, redox potential, iron content, and optical, EPR, and Mossbauer spectroscopic properties have been determined. This new protein is composed of two identical subunits with subunit molecular weight of 21,900 and contains four iron atoms per molecule. The as-purified oxidized protein exhibits an optical spectrum with absorption maxima at 492, 365, and 280 nm, and its EPR spectrum shows resonances at g = 4.3 and 9.4, characteristic of oxidized rubredoxin. The Mossbauer data indicate the presence of approximately equal amounts of two types of iron; we named them the Rd-like and the Hr-like iron due to their similarity to the iron centers of rubredoxins (Rds) and hemerythrins (Hrs), respectively. For the Rd-like iron, the measured fine and hyperfine parameters (D = 1.5 cm-1, E/D = 0.26, delta EQ = -0.55 mm/s, delta = 0.27 mm/s, Axx/gn beta n = -16.5 T, Ayy/gn beta n = -15.6 T, and Azz/gn beta n = -17.0 T) are almost identical with those obtained for the rubredoxin from Clostridium pasteurianum. Redox-titration studies monitored by EPR, however, showed that these Rd-like centers have a midpoint redox potential of +230 +/- 10 mV, approximately 250 mV more positive than those reported for rubredoxins. Another unusual feature of this protein is the presence of the Hr-like iron atoms.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of two dissimilatory sulfite reductases (desulforubidin and desulfoviridin) from the sulfate-reducing bacteria. Moessbauer and EPR studies

Abstract: In this paper, the authors report a detailed Moessbauer investigation of two different sulfite reductases, namely, desulforubidin from D. baculatus and desulfoviridin from D. gigas. In order to better characterize the prosthetic groups, they have also studied the EPR spectra and determined the iron and heme contents of the /sup 57/Fe-enriched enzymes. They found that desulforubidin contains exchange-coupled siroheme-(4Fe-4S) units which are similar to those found in the hemoprotein subunit of E. coli sulfite reductase. To their surprise, they discovered that the majority of the purified desulfoviridin contains demetalized sirohydrochlorin, with only a minor portion of the sample containing siroheme. The siroheme in desulfoviridin was also found to be coupled with a (4Fe-4S) cluster.

Hydrogen Production and Deuterium-Proton Exchange Reactions Catalyzed by Desulfovibrio Nickel(II)-Substituted Rubredoxins

Abstract: The nickel tetrahedral sulfur-coordinated core formed upon metal replacement of the native iron in Desulfovibrio sp. rubredoxins is shown to mimic the reactivity pattern of nickel-containing hydrogenases with respect to hydrogen production, deuterium-proton exchange, and inhibition by carbon monoxide.

A hypothetical model of the flavodoxin-tetraheme cytochrome c3 complex of sulfate-reducing bacteria.

Abstract: A hypothetical model of the flavodoxin-tetraheme cytochrome c3 electron-transfer complex from the sulfate-reducing bacterium Desulfovibrio vulgaris has been constructed by using interactive computer graphics based on electrostatic potential field calculations and previous NMR experiments. Features of the proposed complex are (1) van der Waals contact between the flavin mononucleotide prosthetic group of flavodoxin and one heme of the cytochrome, (2) unique complementarity of electrostatic fields between the region surrounding this heme and the region surrounding the exposed portion of the flavin mononucleotide group of flavodoxin, and (3) no steric interferences between the two polypeptide chains in the complex. This complex is consistent with all structural and spectroscopic data available.

Cytochrome components of nitrate- and sulfate-respiring Desulfovibrio desulfuricans ATCC 27774.

Abstract: Three multiheme c-type cytochromes--the tetraheme cytochrome c3 (molecular weight [MW] 13,500), a dodecaheme cytochrome c (MW 40,800), and a "split-Soret" cytochrome c (MW 51,540), which is a dimer with 2 hemes per subunit (MW 26,300)--were isolated from the soluble fraction of Desulfovibrio desulfuricans (ATCC 27774) grown under nitrate- or sulfate-respiring conditions. Two of them, the dodecaheme and the split-Soret cytochromes, showed no similarities to any of the c-type cytochromes isolated from other sulfate-reducing bacteria, while the tetraheme cytochrome c3 appeared to be analogous to the cytochrome c3 found in other sulfate-reducing bacteria. For all three multiheme c-type cytochromes isolated, the homologous proteins from nitrate- and sulfate-grown cells were indistinguishable in amino acid composition, physical properties, and spectroscopic characteristics. It therefore appears that the same c-type cytochrome components are present when D. desulfuricans ATCC 27774 cells are grown under either condition. This is in contrast to the considerable difference found in Pseudomonas perfectomarina (Liu et al., J. Bacteriol. 154:278-286, 1983), a marine denitrifier, when the cells are grown on nitrate or oxygen as the terminal electron acceptor. In addition, two spectroscopy methods capable of revealing minute structural variations in proteins provided identical information about the tetraheme cytochrome c3 from nitrate-grown and sulfate-grown cells.

Assignment of individual heme EPR signals of Desulfovibrio baculatus (strain 9974) tetraheme cytochrome c3: a redox equilibria study

Abstract: An EPR redox titration was performed on the tetraheme cytochrome c3 isolated from Desulfovibrio baculatus (strain 9974), a sulfate-reducer. Using spectral differences at different poised redox states of the protein, it was possible to individualize the EPR g-values of each of the four hemes and also to determine the mid-point redox potentials of each individual heme: heme 4 (-70 mV) at gmax = 2.93, gmed = 2.26 and gmin = 1.51; heme 3 (-280 mV) at gmax = 3.41; heme 2 (-300 mV) at gmax = 3.05, gmed = 2.24 and gmin = 1.34; and heme 1 (-355 mV) at gmx = 3.18. A previously described multi-redox equilibria model used for the interpretation of NMR data of D. gigas cytochrome c3 [Santos, H., Moura, J.J.G., Moura, I., LeGall, J. & Xavier, A. V. (1984) Eur. J. Biochem. 141, 283-296] is discussed in terms of the EPR results.

Spectroscopic characterization of a high-potential monohaem cytochrome from Wolinella succinogenes, a nitrate-respiring organism. Redox and spin equilibria studies.

Abstract: When purified, a high-potential c-type monohaem cytochrome from the nitrate-respiring organism, Wollinellu succinogenes (VPI 10659), displayed a minimum molecular mass of 8.2 kDa and 0.9 mol iron and 0.95 mol haem groups/mol protein. Visible light spectroscopy suggested the presence of an equilibrium between two ligand arrangements around the haem, i. e. an absorption band at 695 nm characteristic of haem-methionine coordination (low-spin form) coexisting with a high-spin form revealed by a band at 619 nm and a shoulder at 498 nm. The mid-point redox potential measured by visible redox titration of the low-spin form was approximately + 100 mV. Binding cyanide (K, = 5 x lo5 M-’) resulted in the displacement of the methionyl axial residue, and full conversion to a low-spin, cyanide-bound form. Structural features were studied by 300-MHz ‘H-NMR spectroscopy. In the oxidized state, the pH dependence of the haein methyl resonances (pH range 5 - 10) and the magnetic susceptibility measurements (using an NMR method) were consistent with the visible light spectroscopic data for the presence of a high-spin/low-spin equilibrium with a transition pK, of 7.3. The spin equilibrium was fast on the NMR time scale. The haem methyl resonances presented large downfield chemical shifts. An unusually broad methyl resonance at around 35 ppm (pH = 7.5, 25°C) was extremely temperature-dependent [6(323K) - 6(273K) = 7.2 ppm] and was assigned to the S-CH3 group of the axial methionine. In the ferrous state only a low-spin form is present. The haem meso protons, the methyl group and the methylene protons from the axial methionine were identified in the reduced form. The resonances from the aromatic residues (three tyrosines and one phenylalanine) were also assigned. Detailed monitoring of the NMR-redox pattern of the monohaem cytochrome from the fully reduced up to the fully oxidized state revealed that the rate of the intermolecular electronic exchange process was approximately 6 x lo6 M-’ sK1 at 303 K and pH = 6.31. A dihaem cytochrome also present in the crude cell extract and purified to a homogeneous state, exhibited a molecular mass of 21 kDa and contained 2.43 mol iron and 1.89 mol haem c moieties/mol cytochrome. The absorption spectrum in the visible region exhibited no band at 695 nm, suggesting that methione is not a ligand for either of the two haems. Recovery of only small amounts of this protein prevented more detailed structural analyzes In denitrifying bacteria, nitrate and its reduction products, nitrite, nitric oxide and nitrous oxide, serve as terminal electron acceptors in anaerobic respiration [l]. The electron transport system responsible for the denitrifying process is coinplex. The different electron acceptors involved are not yet precisely placed in the electron transfer scheme. Independent or branched electron transfer chains lead the electrons towards the different nitrogen oxides during denitrification [l] and a number of b and c-type cytochromes have been detected [2]. Furthermore, certain bacteria carry out less extensive denitrification than others [3]. Some reduce nitrate incompletely to nitrous oxide. Others reduce nitrite, nitric oxide and nitrous oxide, but not nitrate to dinitrogen [3]. Different still is Wolinellu succinogenes (VPI 10659), which exhibits capability for dissimilatory reduction of nitrate

Nickel-containing hydrogenases

Abstract: Abstract Hydrogenases have been purified from different biological sources. They are highly diversified enzymes in terms of active centers constitution, although they catalyze the simplest oxidation-reduction process: H 2 ⇌ 2H + + 2e. Hydrogenases have been recognized so far to be ironsulfur proteins. Generally they contain from four to twelve atoms of non-haem iron arranged in FeS clusters representative of the known basic structures, e.g. , [2Fe-2S], 3Fe-xS], and [4Fe-4S] [1–7]. Recently, nickel joined the group of transition metals relevant in biological oxidation-reduction processes. It was shown to be a structural component of the hydrogenases isolated from Desulfovibrio gigas [1, 2], Desulfovibrio desulfuricans (ATTC 27774) [3], Desulfovibrio desulfuricans (Norway strain) [4], Methanosarcina barkeri [5], Methanobacterium thermoautotrophicum [6] and Chromatium vinosum [8]. With the exception of the last one, they were demonstrated to contain EPR nickel redox dependent signals. As an example, D. gigas hydrogenase exhibits rhombic EPR signals, with g-values 2.31, 2.23 and 2.02 (see Fig. 1). Using isotopic reconstitution by 61 Ni (nuclear magnetic moment I = 3 2 ), the EPR signal was proven arise from a nickel species [9]. The same types of experiments were reported for M. thermoautotrophicum [6] and D. desulfuricans (ATCC 27774) hydrogenases [3]. A detailed EPR study on the oxidation-reduction transition of the EPR detectable species in the presence of reductant (dithionite and hydrogen) indicates [1, 2]: 1. The reduction of the Ni EPR active species is an one-electron process (possibly associated with the redox couple Ni(III)Ni(II)). 2. No evidence was found so far for exchangeable protons in the vicinity of the nickel center in the oxidized (native) state. However, hydrogen reduced samples originate a different EPR rhombic Ni signal, which may represent an active transient species occurring during the activation of hydrogen molecules [9]. Thus, it is attractive to propose the presence of a hydride intermediate in analogy with nickel catalysts involved in hydrogenation processes [10]. 3. Although the determined mid-point redox potential (−220 mV) is more negative than that expected for nickel compounds [11] it is still more positive than that of the substrate couple H 2 /H + . The value determined was shown to be pH dependent [2].

Characterization of two dissimilatory sulfite reductases from sulfate-reducing bacteria

Abstract: Mossbauer, EPR, and biochemical techniques were used to characterize two dissimilatory sulfite reductases: desulforubidin fromDesulfovibrio baculatus strain DSM 1743 and desulfoviridin fromDesulfovibrio gigas. For each molecule of desulforubidin, there are two sirohemes and four [4Fe−4S] clusters. The [4Fe−4S] clusters are in the diamagnetic 2+ oxidation state. The sirohemes are high-spin ferric (S=5/2) and each siroheme is exchanged-coupled to a [4Fe−4S]2+ cluster. Such an exchange-coupled siroheme-[4Fe−4S] unit has also been found in the assimilatory sulfite reductase fromEscherichia coli/1/ and in a low-molecular weight sulfite reductase fromDesulfovibrio vulgaris/2/. For each molecule of defulfoviridin, there are two tetrahydroporphyrin groups and four [4Fe−4S]2+ clusters. To our surprise, we discovered that about 80% of the tetrahydroporphyrin groups, however, do not bind iron.