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.

A conductometric biosensor for nitrite detection was developed using cytochrome c nitrite reductase (ccNiR) extracted from Desulfovibrio desulfuricans ATCC 27774 cells immobilized on a planar interdigitated electrode by cross-linking with saturated glutaraldehyde (GA) vapour in the presence of bovine serum albumin, methyl viologen (MV), Nafion, and glycerol. The configuration parameters for this biosensor, including the enzyme concentration, ccNiR/BSA ratio, MV concentration, and Nafion concentration, were optimized. Various experimental parameters, such as sodium dithionite added, working buffer solution, and temperature, were investigated with regard to their effect on the conductance response of the biosensor to nitrite. Under the optimum conditions at room temperature (about 25 degrees C), the conductometric biosensor showed a fast response to nitrite (about 10s) with a linear range of 0.2-120 microM, a sensitivity of 0.194 microS/microM [NO(2)(-)], and a detection limit of 0.05 microM. The biosensor also showed satisfactory reproducibility (relative standard deviation of 6%, n=5). The apparent Michaelis-Menten constant (K(M,app)) was 338 microM. When stored in potassium phosphate buffer (100mM, pH 7.6) at 4 degrees C, the biosensor showed good stability over 1 month. No obvious interference from other ionic species familiar in natural waters was detected. The application experiments show that the biosensor is suitable for use in real water samples.

Nitrous oxide (N20) is a greenhouse gas, the third most significant contributor to global warming. As a key process for N20 elimination from the biosphere, N20 reductases catalyze the two-electron reduction of N20 to N2. 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 N20 reductase from Pseudomonas nautica by multiwavelength anomalous dispersion, to a resolution of 2.4 A. 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. N20 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.

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).

A soluble [NiFe] hydrogenase has been partially purified from the obligate thermophilic sulfate-reducing bacterium Thermodesulfobacterium mobile. A 17% purification yield was obtained after four chromatographic steps and the hydrogenase presents a purity index (A398 nm/A277 nm) equal to 0.21. This protein appears to be 75% pure on SDS-gel electrophoresis showing two major bands of molecular mass around 55 and 15 kDa. This hydrogenase contains 0.6-0.7 nickel atom and 7-8 iron atoms per mole of enzyme and has a specific activity of 783 in the hydrogen uptake reaction, of 231 in the hydrogen production assay and of 84 in the deuterium-proton exchange reaction. The H2/HD ratio is lower than one in the D2-H+ exchange reaction. The enzyme is very sensitive to NO, relatively little inhibited by CO but unaffected by NO2-. The EPR spectrum of the native hydrogenase shows the presence of a [3Fe-4S] oxidized cluster and of a Ni(III) species.

Nitrate reductase from Desulfovibrio desulfuricans ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harboring a bis(molybdopterin guanine dinucleotide) active site and a [4Fe-4S] cluster. Previous electron paramagnetic resonance (EPR) studies in both catalytic and inhibiting conditions showed that the molybdenum center has high coordination flexibility when reacted with reducing agents, substrates or inhibitors. As-prepared DdNapA samples, as well as those reacted with substrates and inhibitors, were crystallized and the corresponding structures were solved at resolutions ranging from 1.99 to 2.45 A. The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on that basis, the sixth molybdenum ligand, originally proposed to be an OH/OH(2) ligand, was assigned as a sulfur atom after refinement and analysis of the B factors of all the structures. This unexpected result was confirmed by a single-wavelength anomalous diffraction experiment below the iron edge (lambda = 1.77 A) of the as-purified enzyme. Furthermore, for six of the seven datasets, the S-S distance between the sulfur ligand and the Sgamma atom of the molybdenum ligand Cys(A140) was substantially shorter than the van der Waals contact distance and varies between 2.2 and 2.85 A, indicating a partial disulfide bond. Preliminary EPR studies under catalytic conditions showed an EPR signal designated as a turnover signal (g values 1.999, 1.990, 1.982) showing hyperfine structure originating from a nucleus of unknown nature. Spectropotentiometric studies show that reduced methyl viologen, the electron donor used in the catalytic reaction, does not interact directly with the redox cofactors. The turnover signal can be obtained only in the presence of the reaction substrates. With use of the optimized conditions determined by spectropotentiometric titration, the turnover signal was developed with (15)N-labeled nitrate and in D(2)O-exchanged DdNapA samples. These studies indicate that this signal is not associated with a Mo(V)-nitrate adduct and that the hyperfine structure originates from two equivalent solvent-exchangeable protons. The new coordination sphere of molybdenum proposed on the basis of our studies led us to revise the currently accepted reaction mechanism for periplasmic nitrate reductases. Proposals for a new mechanism are discussed taking into account a molybdenum and ligand-based redox chemistry, rather than the currently accepted redox chemistry based solely on the molybdenum atom.

Increased levels of unconjugated bilirubin, the end product of heme catabolism, impair crucial aspects of nerve cell function. In previous studies, we demonstrated that bilirubin toxicity may be due to cell death by apoptosis. To characterize the sequence of events leading to neurotoxicity, we exposed developing rat brain astrocytes and neurons to unconjugated bilirubin and investigated whether changes in membrane dynamic properties can mediate apoptosis. Bilirubin induced a rapid, dose-dependent increase in apoptosis, which was nevertheless preceded by impaired mitochondrial metabolism. Using spin labels and electron paramagnetic resonance spectroscopy analysis of whole cell and isolated mitochondrial membranes exposed to bilirubin, we detected major membrane perturbation. By physically interacting with cell membranes, bilirubin induced an almost immediate increase in lipid polarity sensed at a superficial level. The enhanced membrane permeability coincided with an increase in lipid fluidity and protein mobility and was associated with significant oxidative injury to membrane lipids. In conclusion, apoptosis of nerve cells induced by bilirubin is mediated by its primary effect at physically perturbing the cell membrane. Bilirubin directly interacts with membranes influencing lipid polarity and fluidity, protein order, and redox status. These data suggest that nerve cell membranes are primary targets of bilirubin toxicity.

On photolysis of solutions of azulene and uranyl nitrate in alcohols, a dark, amorphous precipitate is formed. Various analytical techniques show that this is a mixture of a uranium salt and an organic component, suggested to be polyazulene. The effects of various parameters on the yield of the product have been studied and it is found that oxygen facilitates the reaction. Electron spin resonance studies show that the product is paramagnetic, in agreement with the established ease of oxidation of polyazulene, and suggest that it is formed via electron transfer from azulene to excited uranyl ion, followed by successive dimerizations and deprotonations of radical cation intermediates.

A c-type monohemic ferricytochrome c552 (11 kDa) was isolated from the soluble extract of a marine denitrifier, Pseudomonas nautica strain 617, grown under anaerobic conditions with nitrate as final electron acceptor. The NH2-terminal sequence and the amino acid composition of the cytochrome were determined. The heme iron of the cytochrome c552 has histidine-methionine as axial ligands, and a pH-dependent mid-point redox potential, equal to 250 mV at pH 7.6. The presence of methionine was demonstrated by visible, EPR and NMR spectroscopies. The assignment of most of the hemic protons was performed applying two-dimensional NOE spectroscopy (NOESY), and the aromatic region was assigned through two-dimensional correlated spectroscopy (COSY) experiments. The EPR spectrum of the oxidised form of the cytochrome c552 is typical of a low-spin ferric heme.

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 (57)Fe 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.

Two proteins were purified and preliminarily characterized from the soluble extract of cells (310 g, wet weight) of the aminolytic and peptidolytic sulfate-reducing bacterium Desulfovibrio (D.) aminophilus DSM 12254. The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5'-phosphosulfate, APS) reductase, a key enzyme in the microbial dissimilatory sulfate reduction, has been purified in three chromatographic steps (DEAE-Biogel A, Source 15, and Superdex 200 columns). It contains two different subunits with molecular masses of 75 and 18 kDa. The fraction after the last purification step had a purity index (A(278nm) / A(388nm)) of 5.34, which was used for further EPR spectroscopic studies. The D. aminophilus APS reductase is very similar to the homologous enzymes isolated from D. gigas and D. desulfuricans ATCC 27774. A tetraheme cytochrome c(3) (His-heme iron-His) has been purified in three chromatographic steps (DEAE- Biogel A, Source 15, and Biogel-HTP columns) and preliminarily characterized. It has a purity index ([A(553nm) - A(570nm)](red) / A(280nm)) of 2.9 and a molecular mass of around 15 kDa, and its spectroscopic characterization (NMR and EPR) has been carried out. This hemoprotein presents similarities with the tetraheme cytochrome c(3) from Desulfomicrobium (Des.) norvegicum (NMR spectra, and N-terminal amino acid sequence).

Three forms of ferredoxin FdI, FdI', and FdII have been isolated from Desulfovibrio gigas, a sulfate reducer. They are separated by a combination of DEAE-cellulose and gel filtration chromatographic procedures. FdI and FdI' present a slight difference in isoelectric point which enables the separation of the two forms over DEAE-cellulose, while FdII is easily separated from the two other forms by gel filtration. The three forms have the same amino acid composition and are isolated in different aggregation states. Molecular weight determinations by gel filtration gave values of 18 000 for FdI and FdI' and 24 000 for FdII, whereas a value of 6000 is determined when dissociation is accomplished with sodium dodecyl sulfate. The electronic spectra are different and their ultraviolet-visible absorbance rations are 0.77, 0.87 and 0.68 respectively for FdI, FdI' and FdII. Despite these differences, the physiological activities of the three forms are similar as far as the reduction of sulfite by molecular hydrogen is concerned.

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 Cu(A) and Cu(Z). Cu(Z) 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 approximately 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 mT, g(z) = 2.178, A(z)= 4 mT) and absorption bands at 480, 540, and approximately 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the Cu(A) center. In form A, Cu(A) is predominantly oxidized (S = (1)/(2), Cu(1.5+)-Cu(1.5+)), while in form B it is mostly in the one-electron reduced state (S = 0, Cu(1+)-Cu(1+)). In both forms, Cu(Z) remains reduced (S = 1/2). Complete crystallographic data at 2.4 A 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 al. (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.

Native zinc/cobalt-containing ATP sulfurylase (ATPS; EC 2.7.7.4; MgATP:sulfate adenylyltransferase) from Desulfovibrio desulfuricans ATCC 27774 was purified to homogeneity and crystallized. The orthorhombic crystals diffracted to beyond 2.5 A resolution and the X-ray data collected should allow the determination of the structure of the zinc-bound form of this ATPS. Although previous biochemical studies of this protein indicated the presence of a homotrimer in solution, a dimer was found in the asymmetric unit. Elucidation of this structure will permit a better understanding of the role of the metal in the activity and stability of this family of enzymes.

A soluble hydrogenase from the halophilic sulfate reducing bacterium Desulfovibrio salexigens, strain British Guiana (NCIB 8403) has been purified to apparent homogeneity with a final specific activity of 760 mumoles H2 evolved/min/mg (an overall 180-fold purification with 20% recovery yield). The enzyme is composed of two non-identical subunits of molecular masses 62 and 36 kDa, respectively, and contains approximately 1 Ni, 12-15 Fe and 1 Se atoms/mole. The hydrogenase shows a visible absorption spectrum typical of an iron-sulfur containing protein (A400/A280 = 0.275) and a molar absorbance of 54 mM-1cm-1 at 400 nm. In the native state (as isolated, under aerobic conditions), the enzyme is almost EPR silent at 100 K and below. However, upon reduction under H2 atmosphere a rhombic EPR signal develops at g-values 2.22, 2.16 and around 2.0, which is optimally detected at 40 K. This EPR signal is reminiscent of the nickel signal C (g-values 2.19, 2.16 and 2.02) observed in intermediate redox states of the well characterized D. gigas nickel containing hydrogenase and assigned to nickel by 61 Ni isotopic substitution (J.J.G. Moura, M. Teixeira, I. Moura, A.V. Xavier and J. Le Gall (1984), J. Mol. Cat., 23, 305-314). Upon longer incubation with H2 the "2.22" EPR signal decreases. During the course of a redox titration under H2, this EPR signal attains a maximal intensity around--380 mV. At redox states where this "2.22" signal develops (or at lower redox potentials), low temperature studies (below 10 K) reveals the presence of other EPR species with g-values at 2.23, 2.21, 2.14 with broad components at higher fields. This new signal (fast relaxing) exhibits a different microwave power dependence from that of the "2.22" signal, which readily saturates with microwave power (slow relaxing). Also at low temperature (8 K) typical reduced iron-sulfur EPR signals are concomitantly observed with gmed approximately 1.94. The catalytic properties of the enzyme were also followed by substrate isotopic exchange D2/H+ and H2 production measurements.

The enthalpy and entropy changes associated with protein reduction (deltaHdegrees,(rc), deltaSdegrees,(rc)) were determined for a number of low-potential iron-sulfur proteins through variable temperature direct electrochemical experiments. These data add to previous estimates making available, overall, the reduction thermodynamics for twenty species from various sources containing all the different types of metal centers. These parameters are discussed with reference to structural data and calculated electrostatic metal-environment interaction energies, and redox properties of model complexes. This work, which is the first systematic investigation on the reduction thermodynamics of Fe-S proteins, contributes to the comprehension of the determinants of the differences in reduction potential among different protein families within a novel perspective. Moreover, comparison with analogous data obtained previously for electron transport (ET) metalloproteins with positive reduction potentials, i.e., cytochromes c, blue copper proteins, and HiPIPs, helps our understanding of the factors controlling the reduction potential in ET species containing different metal cofactors. The main results of this work can be summarized as follows.

Dissimilatory nitrite reduction, carried out by hexaheme proteins, gives ammonia as the final product. Representatives of this enzyme group from 3 bacterial species can also reduce NO to either ammonia or N2O. The redox regulation of the nitrite/nitric oxide activities is discussed in the context of the denitrifying pathway.