When purified, a high-potential c-type monohaem cytochrome from the nitrate-respiring organism, Wollinella 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 (Ka = 5 x 10(5) M-1) 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 1H-NMR spectroscopy. In the oxidized state, the pH dependence of the haem 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 pKa 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 degrees C) was extremely temperature-dependent [delta(323 K) - delta(273 K) = 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 10(6) M-1 s-1 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 11 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.

Extracted human teeth are frequently used for research or educational purposes. Therefore, it is necessary to store them in disinfectant solutions that do not alter dental structures. Thus, this study evaluated the influence of storage solution on enamel demineralization. For that purpose, sixty samples were divided into the following groups: enamel stored in formaldehyde (F1), stored in thymol (T1), stored in formaldehyde and submitted to pH cycling (F2), stored in thymol and submitted to pH cycling (T2). All samples were evaluated by cross-sectional microhardness analysis and had their percentage of mineral volume versus micrometer (integrated area) determined. Differences between groups were found up to 30-microm depth from the enamel surface (p < 0.05), where samples from group T2 were more demineralized. It was concluded that the storage solution influenced the reaction of a dental substrate to a cariogenic challenge, suggesting that formaldehyde may increase enamel resistance to demineralization, when compared to demineralization occurring in enamel stored in thymol solution.

Thirteen cows maintained on natural bracken fern (Pteridium aquilinum) were analyzed cytogenetically. The frequency of structural chromosome aberrations detected in peripheral blood cells was significantly higher when compared to that detected in animals raised on pasture containing no bracken fern. We discuss the clastogenic action of fern and its synergistic action with infection by type 2 and 4 papilloma virus in the same animals.

The reduction of nitrate to nitrogen gas via nitrite, nitric oxide and nitrous oxide is the metabolic pathway usually known as denitrification, a key step in the nitrogen cycle. As observed for other elemental cycles, a battery of enzymes are utilized, namely the reductases for nitrate, nitrite, nitric oxide and nitrous oxide, as well as multiple electron donors that interact with these enzymes, in order to carry out the stepwise reactions that involve key intermediates. Because of the importance of this pathway (of parallel importance to the nitrogen-fixation pathway), efforts are underway to understand the structures of the participating enzymes and to uncover mechanistic aspects. Three-dimensional structures have been solved for the majority of these enzymes in the past few years, revealing the architecture of the active metal sites as well as global structural aspects, and possible mechanistic aspects. In addition, the recognition of specific electron-transfer partners raises important questions regarding specific electron-transfer pathways, partner recognition and control of metabolism.

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. Mossbauer 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 Mossbauer 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.

Desulfovibrio gigas ferredoxin II (FdII) contains a single 3Fe cluster [Huynh, B.H., Moura, J.J.G., Moura, I., Kent, T.A., LeGall, J., Xavier, A.V., and Munck, E. (1980) J. Biol. Chem. 255, 3242-3244]. In the oxidized state the protein exhibits an intense electron paramagnetic resonance (EPR) signal at g = 2.02. Upon one-electron reduction the center becomes EPR silent. In the presence of D. gigas crude cell extracts, devoid of acidic electron carriers and supplemented with pyruvate and FdII, an EPR signal typical of reduced [4Fe-4S] centers is obtained. The appearance of this signal correlates with the beginning of stimulation of the phosphoroclastic reaction as judged by the production of H2. These results, supported by the occurrence of easy chemical conversion of the 3Fe cluster of D. gigas ferredoxin into 4Fe structures [Moura, J.J.G., Moura, I., Kent, T.A., Lipscomb, J.D., Huynh, B.H., LeGall, J., Xavier, A.V., and Munch, E. (1982) J. Biol. Chem. 257, 6259-6267], suggest that cluster conversion takes place in conditions close to the situation in vivo. This cluster interconversion is discussed in the context of some of the relevant metabolic pathways of Desulfovibrio spp.

Potentiometric titration followed by e.p.r. measurements were used to determine the midpoint reduction potentials of the redox centres of a molybdenum-containing iron-sulphur protein previously isolated from Desulfovibrio gigas, a sulphate-reducing bacterium (Moura, Xavier, Bruschi, Le Gall, Hall & Cammack (1976) Biochem. Biophys. Res. Commun. 728 782-789; Moura, Xavier, Bruschi, Le Gall & Cabral (1977) J. Less Common Metals 54, 555-562). The iron-sulphur centres could readily be distinguished into three types by means of g values, temperature effect, oxidation-reduction potential values and reduction rates. The type-I Fe-S centres are observed at 77 K. They show mid-point potential values of -260mV (Fe-S type IA) and -440 mV (Fe-S type IB). Centres of types IA and IB appear to have similar spectra at 77 K and 24 K. The Fe-S type-II centres are only observed below 65 K and have a midpoint potential of -28mV. Long equilibration times (30 min) with dye mediators under reducing conditions were necessary to observe the very slow equilibrating molybdenum signals. The potential values associated with this signal were estimated to be approx. -415 mV for Mo(VI)/Mo(V) and-530mV for Mo(V)/Mo(IV).

Two new low molecular weight proteins with sulfite reductase activity, isolated from Methanosarcina barkeri (DSM 800) and Desulfuromonas acetoxidans (strain 5071), were studied by EPR and optical spectroscopic techniques. Both proteins have visible spectra similar to that of the low-spin sulfite reductase of Desulfovibrio vulgaris strain Hildenborough and no band at 715 nm, characteristic of high-spin Fe3+ complexes in isobacteriochlorins is observed. EPR shows that as isolated the siroheme is in a low-spin ferric state (S = 1/2) with g-values at 2.40, 2.30 and 1.88 for the Methanosarcina barkeri enzyme and g-values at 2.44, 2.33 and 1.81 for the Desulfuromonas acetoxidans enzyme. Chemical analysis shows that both proteins contain one siroheme and one [Fe4S4] center per polypeptidic chain. These results suggest that the low molecular weight, low-spin non-heme iron siroheme proteins represent a new homologous class of sulfite reductases common to anaerobic microorganisms.

The most thoroughly characterized non-heme iron center in biology is Rubredoxin, the simplest member of the iron-sulfur: class of metalloproteins. Rubredoxin contains a high-spin iron atom with tetrahedral coordination by four cysteinyl sulfur atoms. A structural variant of this center is found in Desulforedoxin, the smallest known Rubredoxin type protein. The 3D structure of both Rd and Dr has been determined at high resolution. These two proteins can therefore be used as case studies in which structural control by the polypeptide chain over the metal site can be discussed in detail.