Crystal structure of the zinc-, cobalt-, and iron-containing adenylate kinase from Desulfovibrio gigas: a novel metal-containing adenylate kinase from Gram-negative bacteria.

Adenylate kinases (AK) from Gram-negative bacteria are generally devoid of metal ions in their LID domain. However, three metal ions, zinc, cobalt, and iron, have been found in AK from Gram-negative bacteria. Crystal structures of substrate-free AK from Desulfovibrio gigas with three different metal ions (Zn(2+), Zn-AK; Co(2+), Co-AK; and Fe(2+), Fe-AK) bound in its LID domain have been determined by X-ray crystallography to resolutions 1.8, 2.0, and 3.0 Å, respectively. The zinc and iron forms of the enzyme were crystallized in space group I222, whereas the cobalt-form crystals were C2. The presence of the metals was confirmed by calculation of anomalous difference maps and by X-ray fluorescence scans. The work presented here is the first report of a structure of a metal-containing AK from a Gram-negative bacterium. The native enzyme was crystallized, and only zinc was detected in the LID domain. Co-AK and Fe-AK were obtained by overexpressing the protein in Escherichia coli. Zn-AK and Fe-AK crystallized as monomers in the asymmetric unit, whereas Co-AK crystallized as a dimer. Nevertheless, all three crystal structures are very similar to each other, with the same LID domain topology, the only change being the presence of the different metal atoms. In the absence of any substrate, the LID domain of all holoforms of AK was present in a fully open conformational state. Normal mode analysis was performed to predict fluctuations of the LID domain along the catalytic pathway.

Analysis of the activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase through an electron transfer chain.

The activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase (CCP) was probed through the mediated electrochemical catalysis by its physiological electron donor, P. stutzeri cytochrome c-551. A comparative study was carried out, by performing assays with the enzyme in the resting oxidized state as well as in the mixed-valence activated form, using cyclic voltammetry and a pyrolytic graphite membrane electrode. In the presence of both the enzyme and hydrogen peroxide, the peak-like signal of cytochrome c-551 is converted into a sigmoidal wave form characteristic of an E(r)C'(i) catalytic mechanism. An intermolecular electron transfer rate constant of (4 ± 1) × 10(5) M(-1) s(-1) was estimated for both forms of the enzyme, as well as a similar Michaelis-Menten constant. These results show that neither the intermolecular electron transfer nor the catalytic activity is kinetically controlled by the activation mechanism of CCP in the case of the P. stutzeri enzyme. Direct enzyme catalysis using protein film voltammetry was unsuccessful for the analysis of the activation mechanism, since P. stutzeri CCP undergoes an undesirable interaction with the pyrolytic graphite surface. This interaction, previously reported for the Paracoccus pantotrophus CCP, induces the formation of a non-native conformation state of the electron-transferring haem, which has a redox potential 200 mV lower than that of the native state and maintains peroxidatic activity.

The effect of the sixth sulfur ligand in the catalytic mechanism of periplasmic nitrate reductase.

The catalytic mechanism of nitrate reduction by periplasmic nitrate reductases has been investigated using theoretical and computational means. We have found that the nitrate molecule binds to the active site with the Mo ion in the +6 oxidation state. Electron transfer to the active site occurs only in the proton-electron transfer stage, where the Mo(V) species plays an important role in catalysis. The presence of the sulfur atom in the molybdenum coordination sphere creates a pseudo-dithiolene ligand that protects it from any direct attack from the solvent. Upon the nitrate binding there is a conformational rearrangement of this ring that allows the direct contact of the nitrate with Mo(VI) ion. This rearrangement is stabilized by the conserved methionines Met141 and Met308. The reduction of nitrate into nitrite occurs in the second step of the mechanism where the two dimethyl-dithiolene ligands have a key role in spreading the excess of negative charge near the Mo atom to make it available for the chemical reaction. The reaction involves the oxidation of the sulfur atoms and not of the molybdenum as previously suggested. The mechanism involves a molybdenum and sulfur-based redox chemistry instead of the currently accepted redox chemistry based only on the Mo ion. The second part of the mechanism involves two protonation steps that are promoted by the presence of Mo(V) species. Mo(VI) intermediates might also be present in this stage depending on the availability of protons and electrons. Once the water molecule is generated only the Mo(VI) species allow water molecule dissociation, and, the concomitant enzymatic turnover.

Crystal structure of the xanthine oxidase-related aldehyde oxido-reductase from D. gigas.

The crystal structure of the aldehyde oxido-reductase (Mop) from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas has been determined at 2.25 A resolution by multiple isomorphous replacement and refined. The protein, a homodimer of 907 amino acid residues subunits, is a member of the xanthine oxidase family. The protein contains a molybdopterin cofactor (Mo-co) and two different [2Fe-2S] centers. It is folded into four domains of which the first two bind the iron sulfur centers and the last two are involved in Mo-co binding. Mo-co is a molybdenum molybdopterin cytosine dinucleotide. Molybdopterin forms a tricyclic system with the pterin bicycle annealed to a pyran ring. The molybdopterin dinucleotide is deeply buried in the protein. The cis-dithiolene group of the pyran ring binds the molybdenum, which is coordinated by three more (oxygen) ligands.

Role of vitamin B12 in methyl transfer for methane biosynthesis by Methanosarcina barkeri.

When Methanosarcina barkeri is grown on methanol as the sole carbon source, a B12-containing protein is synthesized by this organism. This B12 protein contains bound aquocobalamin, and when this cofactor is reduced and methylated with [14C]methyl iodide, the resultant [14C]methyl B12 protein is extremely active in the biosynthesis of 14C-labeled methane. These findings indicate that a B12-dependent system is operative in the biological formation of methane in addition to other systems that are B12-independent.

Cobalt-, zinc- and iron-bound forms of adenylate kinase (AK) from the sulfate-reducing bacterium Desulfovibrio gigas: purification, crystallization and preliminary X-ray diffraction analysis.

Adenylate kinase (AK; ATP:AMP phosphotransferase; EC 2.7.4.3) is involved in the reversible transfer of the terminal phosphate group from ATP to AMP. AKs contribute to the maintenance of a constant level of cellular adenine nucleotides, which is necessary for the energetic metabolism of the cell. Three metal ions, cobalt, zinc and iron(II), have been reported to be present in AKs from some Gram-negative bacteria. Native zinc-containing AK from Desulfovibrio gigas was purified to homogeneity and crystallized. The crystals diffracted to beyond 1.8 A resolution. Furthermore, cobalt- and iron-containing crystal forms of recombinant AK were also obtained and diffracted to 2.0 and 3.0 A resolution, respectively. Zn(2+)-AK and Fe(2+)-AK crystallized in space group I222 with similar unit-cell parameters, whereas Co(2+)-AK crystallized in space group C2; a monomer was present in the asymmetric unit for both the Zn(2+)-AK and Fe(2+)-AK forms and a dimer was present for the Co(2+)-AK form. The structures of the three metal-bound forms of AK will provide new insights into the role and selectivity of the metal in these enzymes.

Sarcoplasmic reticulum calcium ATPase is inhibited by organic vanadium coordination compounds: pyridine-2,6-dicarboxylatodioxovanadium(V), BMOV, and an amavadine analogue.

The general affinity of the sarcoplasmic reticulum (SR) Ca (2+)-ATPase was examined for three different classes of vanadium coordination complexes including a vanadium(V) compound, pyridine-2,6-dicarboxylatodioxovanadium(V) (PDC-V(V)), and two vanadium(IV) compounds, bis(maltolato)oxovanadium(IV) (BMOV), and an analogue of amavadine, bis( N-hydroxylamidoiminodiacetato)vanadium(IV) (HAIDA-V(IV)). The ability of vanadate to act either as a phosphate analogue or as a transition-state analogue with enzymes' catalysis phosphoryl group transfer suggests that vanadium coordination compounds may reveal mechanistic preferences in these classes of enzymes. Two of these compounds investigated, PDC-V(V) and BMOV, were hydrolytically and oxidatively reactive at neutral pH, and one, HAIDA-V(IV), does not hydrolyze, oxidize, or otherwise decompose to a measurable extent during the enzyme assay. The SR Ca (2+)-ATPase was inhibited by all three of these complexes. The relative order of inhibition was PDC-V(V) > BMOV > vanadate > HAIDA-V(IV), and the IC 50 values were 25, 40, 80, and 325 microM, respectively. Because the observed inhibition is more potent for PDC-V(V) and BMOV than that of oxovanadates, the inhibition cannot be explained by oxovanadate formation during enzyme assays. Furthermore, the hydrolytically and redox stable amavadine analogue HAIDA-V(IV) inhibited the Ca (2+)-ATPase less than oxovanadates. To gauge the importance of the lipid environment, studies of oxidized BMOV in microemulsions were performed and showed that this system remained in the aqueous pool even though PDC-V(V) is able to penetrate lipid interfaces. These findings suggest that the hydrolytic properties of these complexes may be important in the inhibition of the calcium pump. Our results show that two simple coordination complexes with known insulin enhancing effects can invoke a response in calcium homeostasis and the regulation of muscle contraction through the SR Ca (2+)-ATPase.

Vanadium distribution, lipid peroxidation and oxidative stress markers upon decavanadate in vivo administration.

The contribution of decameric vanadate species to vanadate toxic effects in cardiac muscle was studied following an intravenous administration of a decavanadate solution (1mM total vanadium) in Sparus aurata. Although decameric vanadate is unstable in the assay medium, it decomposes with a half-life time of 16 allowing studying its effects not only in vitro but also in vivo. After 1, 6 and 12h upon decavanadate administration the increase of vanadium in blood plasma, red blood cells and in cardiac mitochondria and cytosol is not affected in comparison to the administration of a metavanadate solution containing labile oxovanadates. Cardiac tissue lipid peroxidation increases up to 20%, 1, 6 and 12h after metavanadate administration, whilst for decavanadate no effects were observed except 1h after treatment (+20%). Metavanadate administration clearly differs from decavanadate by enhancing, 12h after exposure, mitochondrial superoxide dismutase (SOD) activity (+115%) and not affecting catalase (CAT) activity whereas decavanadate increases SOD activity by 20% and decreases (-55%) mitochondrial CAT activity. At early times of exposure, 1 and 6h, the only effect observed upon decavanadate administration was the increase by 20% of SOD activity. In conclusion, decavanadate has a different response pattern of lipid peroxidation and oxidative stress markers, in spite of the same vanadium distribution in cardiac cells observed after decavanadate and metavanadate administration. It is suggested that once formed decameric vanadate species has a different reactivity than vanadate, thus, pointing out that the differential contribution of vanadium oligomers should be taken into account to rationalize in vivo vanadate toxicity.

The diseases we cause: Iatrogenic illness in a department of internal medicine.

BACKGROUND: The aim of this study was to estimate the incidence, main causes, and risk factors of iatrogenic disease occurring in a department of internal medicine. METHODS: Over a 1-year period, physicians systematically filled out a 2-page questionnaire for all patients admitted to the ward. A database was created and the data were statistically analyzed. Patients undergoing immunosuppressive, chemo-, or radiation therapy were excluded. Missing data were completed by reviewing the patients' charts. The patients were then divided into two groups: those with and those without iatrogenic disease. The groups were compared using several parameters including gender, age, social features, days of hospitalization, associated illness, functional status, medical impression, prognosis, associated renal or liver function impairment, drugs taken daily, and outcome. In the group with iatrogenic disease, the type, severity, and predictability were also analyzed. RESULTS: Of the 879 patients admitted to the ward, 445 completed questionnaires and were included in the study. A total of 102 patients (22.9%) developed 121 iatrogenic events. Forty-four patients (43.1%) were admitted for iatrogenic illness, 10 (9.8%) developed life-threatening events, and in 3 (6.8%) it was the cause of death. Fifty-eight patients (56.8%) registered 77 episodes of iatrogenic disease during their hospital stay, 20 (19.6%) developed life-threatening events, and 9 (11.7%) died, 4 (5.2%) of an iatrogenic cause (nosocomial infections). Significant differences were found in 20 out of 26 parameters studied (p<0.005 for all cases; 95% confidence interval). Eighteen percent of all iatrogenic disease was severe, 61.9% predictable, 54.5% avoidable, and 59% drug-related, 80% of which was due to side effects or adverse reactions. Infection and metabolic and electrolyte disorders were the most frequent effects. CONCLUSIONS: It is possible to identify risk factors for iatrogenic events. Chronically ill elderly inpatients are the main target of iatrogenic events.

Metalloenzymes of the denitrification pathway.

Denitrification, or dissimilative nitrate reduction, is an anaerobic process used by some bacteria for energy generation. This process is important in many aspects, but its environmental implications have been given particular relevance. Nitrate accumulation and release of nitrous oxide in the atmosphere due to excess use of fertilizers in agriculture are examples of two environmental problems where denitrification plays a central role. The reduction of nitrate to nitrogen gas is accomplished by four different types of metalloenzymes in four simple steps: nitrate is reduced to nitrite, then to nitric oxide, followed by the reduction to nitrous oxide and by a final reduction to dinitrogen. In this manuscript we present a concise updated review of the bioinorganic aspects of denitrification.

Bacterial nitrate reductases: Molecular and biological aspects of nitrate reduction.

Nitrogen is a vital component in living organisms as it participates in the making of essential biomolecules such as proteins, nucleic acids, etc. In the biosphere, nitrogen cycles between the oxidation states +V and -III producing many species that constitute the biogeochemical cycle of nitrogen. All reductive branches of this cycle involve the conversion of nitrate to nitrite, which is catalyzed by the enzyme nitrate reductase. The characterization of nitrate reductases from prokaryotic organisms has allowed us to gain considerable information on the molecular basis of nitrate reduction. Prokaryotic nitrate reductases are mononuclear Mo-containing enzymes sub-grouped as respiratory nitrate reductases, periplasmic nitrate reductases and assimilatory nitrate reductases. We review here the biological and molecular properties of these three enzymes along with their gene organization and expression, which are necessary to understand the biological processes involved in nitrate reduction.

Redox chemistry of low-pH forms of tetrahemic cytochrome c3.

Desulfovibrio vulgaris Hildenborough cytochrome c(3) contains four hemes in a low-spin state with bis-histidinyl coordination. High-spin forms of cytochrome c(3) can be generated by protonation of the axial ligands in order to probe spin equilibrium (low-spin/high-spin). The spin alterations occurring at acid pH, the associated changes in redox potentials, as well as the reactivity towards external ligands were followed by the conjunction of square wave voltammetry and UV-visible, CD, NMR and EPR spectroscopies. These processes may be used for modelling the action of enzymes that use spin equilibrium to promote enzyme activity and reactivity towards small molecules.

Crystal structure of the first dissimilatory nitrate reductase at 1.9 A solved by MAD methods.

BACKGROUND: The periplasmic nitrate reductase (NAP) from the sulphate reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is induced by growth on nitrate and catalyses the reduction of nitrate to nitrite for respiration. NAP is a molybdenum-containing enzyme with one bis-molybdopterin guanine dinucleotide (MGD) cofactor and one [4Fe-4S] cluster in a single polypeptide chain of 723 amino acid residues. To date, there is no crystal structure of a nitrate reductase. RESULTS: The first crystal structure of a dissimilatory (respiratory) nitrate reductase was determined at 1.9 A resolution by multiwavelength anomalous diffraction (MAD) methods. The structure is folded into four domains with an alpha/beta-type topology and all four domains are involved in cofactor binding. The [4Fe-4S] centre is located near the periphery of the molecule, whereas the MGD cofactor extends across the interior of the molecule interacting with residues from all four domains. The molybdenum atom is located at the bottom of a 15 A deep crevice, and is positioned 12 A from the [4Fe-4S] cluster. The structure of NAP reveals the details of the catalytic molybdenum site, which is coordinated to two MGD cofactors, Cys140, and a water/hydroxo ligand. A facile electron-transfer pathway through bonds connects the molybdenum and the [4Fe-4S] cluster. CONCLUSIONS: The polypeptide fold of NAP and the arrangement of the cofactors is related to that of Escherichia coli formate dehydrogenase (FDH) and distantly resembles dimethylsulphoxide reductase. The close structural homology of NAP and FDH shows how small changes in the vicinity of the molybdenum catalytic site are sufficient for the substrate specificity.

Metabolic syndrome in dyslipidemia consultations.

AIMS: To evaluate the prevalence of the metabolic syndrome (MS) in dyslipidemia consultations, according to the criteria established by the National Cholesterol Education Program Adult Treatment Panel III (ATP III); to classify patients with MS according to ATP III risk categories and prevention type (primary versus secondary); and to evaluate evolution to type 2 diabetes in these patients. METHODS: A retrospective study was conducted based on analysis of the clinical records of 470 patients followed in dyslipidemia consultations at Internal Medicine II of Coimbra University Hospitals. MS was defined as the presence of three or more of the following abnormalities: waist circumference > 102/88 cm (male/female), triglyceride levels > or =150 mg/dl, HDL-cholesterol level < 40/50 mg/dl (male/female), blood pressure > or = 130/85 mmHg and fasting glucose > or = 110 mg/dl. The patients were classified into three risk categories according to the major risk factors defined by ATP III, regardless of LDL-cholesterol level. RESULTS: MS was diagnosed in 31.3% of the patients; 53.7% were male and mean age was 52.7+/-11.9 years. The most prevalent anomaly was hypertriglyceridemia (95.9%), followed by waist circumference (76.1%), HDL cholesterol (73.5%), blood pressure (69.4%) and fasting glycemia (36.7%). Based on the risk categories identified by ATP III, 30.6% of the patients had no or only one risk factor, 66.7% had multiple (2 or more) risk factors and 2.7% had coronary disease or an equivalent risk profile. 97.3% of the patients with MS were in primary prevention. 18.4% developed diabetes, the majority within 1-3 years of the first consultation. CONCLUSIONS: Our study identified a high MS prevalence in patients followed in dyslipidemia consultations, particularly in older age-groups. More than half of the MS patients had an intermediate risk profile and most of them were in primary prevention. We also found that a fifth of non-diabetic patients developed diabetes during follow-up.

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

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)

Iron-sulphur cluster composition and redox properties of two ferredoxins from Desulfovibrio desulfuricans Norway strain.

Two ferredoxins from Desulfovibrio desulfuricans, Norway Strain, were investigated by EPR spectroscopy. Ferredoxin I appears to be a conventional [4Fe-4S]2+;1+ ferredoxin, with a midpoint reduction potential of -374 mV at pH 8. Ferredoxin II when reduced, at first showed a more complex spectrum, indicating an interaction between two [4Fe-4S] clusters, and probably, has two clusters per protein subunit. Upon reductive titration ferredoxin II changed to give a spectrum in which no intercluster interaction was seen. The midpoint potentials of the native and modified ferredoxin at pH 8 were estimated to be -500 and -440 mV, respectively.

Flavodoxin and rubredoxin from Desulphovibrio salexigens.

A flavodoxin and a rubredoxin have been isolated from the sulfate-reducing bacterium Desulphovibrio salexigens (strain British Guiana, NICB 8403). Their amino acid composition and spectral characteristics did not differ markedly from the homologous proteins presented in other Desulphovibrio spp. Flavodoxin was shown to be active in the electron transport of the sulfite reductase system.

Structural and functional approach toward a classification of the complex cytochrome c system found in sulfate-reducing bacteria.

Following the discovery of the tetraheme cytochrome c3 in the strict anaerobic sulfate-reducing bacteria (Postgate, J.R. (1954) Biochem. J. 59, xi; Ishimoto et al. (1954) Bull. Chem. Soc. Japan 27, 564-565), a variety of c-type cytochromes (and others) have been reported, indicating that the array of heme proteins in these bacteria is complex. We are proposing here a tentative classification of sulfate- (and sulfur-) reducing bacteria cytochromes c based on: number of hemes per monomer, heme axial ligation, heme spin state and primary structures (whole or fragmentary). Different and complementary spectroscopic tools have been used to reveal the structural features of the heme sites.

NMR characterization of three forms of ferredoxin from Desulphovibrio gigas, a sulphate reducer.

A NMR and magnetic susceptibility study of the oxidized and reduced states of three different oligomers (forms) of a [4Fe-4S] ferrodoxin protein from Desulphovibrio gigas, FdI, FdI', and FdII was carried out. FdI and FdI' are different trimers and FdII a tetramer of the same basic subunit. A probable assignment of the contact shifted resonances is indicated. Since the temperature dependences of the contact shifted responances associated with each [4Fe-4S] are not all similar a delocalized model for the spin densities on the 4Fe does not apply. The exchange rate between oxidized and reduced states is slow on the NMR time scale. The three oligomers are not magnetically equivalent. Using the "three state hypothesis" terminology it is shown that FdIox is predominantly in the C2- state and changes upon reduction into the C3- state, while FdIIox is in the C- state and changes into the C2- state. FdI' does not easily fit into this classification. This study shows a similarity of magnetic behaviour between FdI and bacterial ferredoxins (e.g. Bacillus polymyxa) and between FdII and HiPIP from Chromatium sp. The influence of the quaternary structure on the stabilization of the different oxidation states of ferredoxins as well as on their redox potentials is discussed.

Isolation and characterization of a rubredoxin and an (8Fe-8S) ferredoxin from Desulfuromonas acetoxidans.

A two cluster (4Fe-4S) ferredoxin and a rubredoxin have been isolated from the sulfur-reducing bacterium Desulfuromonas acetoxidans. Their amino acid compositions are reported and compared to those of other iron-sulfur proteins. The ferredoxin contains 8 cysteine residues, 8 atoms of iron and 8 atoms of labile sulfur per molecule; its minimum molecular weight is 6163. The protein exhibits an abosrbance ratio of A385/A283 = 0.74. Storage results in a bleaching of the chromophore; the denatured ferredoxin is reconstitutable with iron and sulfide. The instability temperature is 52 degrees C. The rubredoxin does not differ markedly from rubredoxins from other anaerobic bacteria.