The three-dimensional X-ray structure of cytochrome c3 from a sulfate reducing bacterium, Desulfovibrio desulfuricans ATCC 27774 (107 residues, 4 heme groups), has been determined by the method of molecular replacement [Frazao et al. (1994) Acta Crystallogr. D50, 233-236] and refined at 1.75 A to an R-factor of 17.8%. When compared with the homologous proteins isolated from Desulfovibrio gigas, Desulfovibrio vulgaris Hildenborough, Desulfovibrio vulgaris Miyazaki F, and Desulfomicrobium baculatus, the general outlines of the structure are essentialy kept [heme-heme distances, heme-heme angles, His-His (axial heme ligands) dihedral angles, and the geometry of the conserved aromatic residues]. The three-dimensional structure of D. desulfuricans ATCC 27774 cytochrome c3Dd was modeled on the basis of the crystal structures available and amino acid sequence comparisons within this homologous family of multiheme cytochromes [Palma et al. (1994) Biochemistry 33, 6394-6407]. This model is compared with the refined crystal structure now reported, in order to discuss the validity of structure prediction methods and critically evaluate the steps used to predict protein structures by homology modeling. The four heme midpoint redox potentials were determined by using deconvoluted electron paramagnetic resonance (EPR) redox titrations. Structural criteria (electrostatic potentials, heme ligand orientation, EPR g values, heme exposure, data from protein-protein interaction studies) are invoked to assign the redox potentials corresponding to each specific heme in the three-dimensional structure.

Resonance Raman spectra of the molybdenum containing aldehyde oxidoreductase from Desulfovibrio gigas were recorded at liquid nitrogen temperature with various excitation wavelengths. The spectra indicate that all the iron atoms are organised in [2Fe-2S] type centers consistent with cysteine ligations. No vibrational modes involving molybdenum could be clearly identified. The features between 280 and 420 cm-1 are similar but different from those of typical plant ferredoxin-like [2Fe-2S] cluster. The data are consistent with the presence of a plant ferredoxin-like cluster (center I) and a unique [2Fe-2S] cluster (center II), as suggested by other spectroscopic studies. The Raman features of center II are different from those of other [2Fe-2S] clusters in proteins. In addition, a strong peak at ca. 683 cm-1, which is not present in other [2Fe-2S] clusters in proteins, was observed with purple excitation (406.7-413.1 nm). The peak is assigned to enhanced cysteinyl C-S stretching in center II, suggesting a novel geometry for this center.

Mossbauer 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. Mossbauer 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 Mossbauer 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.

All organisms utilize ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) to catalyze the terminal step of the heme biosynthetic pathway, which involves the insertion of ferrous ion into protoporphyrin IX. Kinetic methods and Mossbauer spectroscopy have been used in an effort to characterize the ferrous ion-binding active site of recombinant murine ferrochelatase. The kinetic studies indicate that dithiothreitol, a reducing agent commonly used in ferrochelatase activity assays, interferes with the enzymatic production of heme. Ferrochelatase specific activity values determined under strictly anaerobic conditions are much greater than those obtained for the same enzyme under aerobic conditions and in the presence of dithiothreitol. Mossbauer spectroscopy conclusively demonstrates that, under the commonly used assay conditions, dithiothreitol chelates ferrous ion and hence competes with the enzyme for binding the ferrous substrate. Mossbauer spectroscopy of ferrous ion incubated with ferrochelatase in the absence of dithiothreitol shows a somewhat broad quadrupole doublet. Spectral analysis indicates that when 0.1 mM Fe(II) is added to 1.75 mM ferrochelatase, the overwhelming majority of the added ferrous ion is bound to the protein. The spectroscopic parameters for this bound species are delta = 1.36 +/- 0.03 mm/s and delta EQ = 3.04 +/- 0.06 mm/s, distinct from the larger delta EQ of a control sample of Fe(II) in buffer only. The parameters for the bound species are consistent with an active site composed of nitrogenous/oxygenous ligands and inconsistent with the presence of sulfur ligands. This finding is in accord with the absence of conserved cysteines among the known ferrochelatase sequences. The implications these results have with regard to the mechanism of ferrochelatase activity are discussed.

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.

Desulforedoxin is a protein purified from cellular extracts of Desulfovibrio gigas. It is a small (7.9 kDa) dimeric protein that contains a distorted rubredoxin like center (one single iron coordinated by four cysteinyl residues). Due to the simplicity of the polypeptide chain and of the iron center, an attempt was made to chemically produce this protein. A 36 amino acid polypeptide chain was synthesized based on the known sequence of native Desulforedoxin. The iron center was then reconstituted and the biochemical and spectroscopic characteristics of this synthetic protein were investigated. The final product has an equal sequence to the protein purified from D. gigas. The synthetic and natural Dx are very similar, in terms redox potential and spectroscopic properties (UV-Visible, EPR, Mossbauer).

Quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni (QH-EDH) contains two cofactors, 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinoline-4,5-dione (PQQ) and heme c. Since previous studies on the kinetics of this enzyme suggested that both participate in electron transfer, spectroscopic investigations were performed of the oxidized and reduced holo- and apoenzyme (without PQQ but with heme c) to reveal the nature of the interaction between the two redox centers. From this it appears that the properties of the heme in the enzyme are affected by the presence of PQQ, as judged from the shift of the maxima in the ultraviolet/visible absorption spectra of the heme moiety in both reduced and oxidized QH-EDH and the 60-mV increase of the heme midpoint redox potential caused by PQQ addition. Also 1H-NMR spectroscopy was indicative for interaction since binding of PQQ induced shifts in the resonances of the methyl groups of the porphyrin ring in the oxidized form of the apoenzyme and a shift in the methionine heme ligand resonance of the reduced form of the apoenzyme. On the other hand, resonance Raman spectra of the heme in the different enzyme forms were nearly similar. These results suggest that a major effect of PQQ binding to apo-QH-EDH is a rotation of the methionine ligand of heme c. Since no intermediate 1H-NMR spectra were observed upon titration of apoenzyme with PQQ, apparently no exchange occurs of PQQ between (oxidized) holo- and apoenzyme at the NMR time scale and at that of the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

After up to 21 days without food, adult male quails (Coturnix coturnix japonica) lost about 45% of the initial body weight (100-150 g). As in naturally fast-adapted and larger birds, three phases were identified during prolonged fasting in quails. Phase I lasted 2-3 days and was characterized by a rapid decrease in the rate of body weight loss and high fat mobilization. Phase II was longer and characterized by a slow and steady decline in the rates of body weight loss and of nitrogen excretion. The third (critical) period was marked by an abrupt increase in the rates of body weight loss and of nitrogen excretion. Despite their small size, the duration of phase II in quails was relatively long, a clear advantage for the study of the relationships between the several metabolic events that occur during this crucial adaptative period. Also, the beginning of phase III could be precisely determined. Changes in blood glucose, plasma FFA and triacylglycerols levels, as well as in liver and carcass lipid content were similar to those found in other species of birds. Therefore, quails seem to be a suitable model to investigate the biochemical mechanisms involved in the metabolic adjustments to prolonged food deprivation in non fasting-adapted birds.

Desulfovibrio desulfuricans ATCC 27774 is a sulfate reducer that can adapt to nitrate respiration, inducing the enzymes required to utilize this alternative metabolic pathway. Nitrite reductase from this organism has been previously isolated and characterized, but no information was available on the enzyme involved in the reduction of nitrate. This is the first report of purification to homogeneity of a nitrate reductase from a sulfate reducing organism, thus completing the enzymatic system required to convert nitrate (through nitrite) to ammonia. D. desulfuricans nitrate reductase is a monomeric (circa 70 kDa) periplasmic enzyme with a specific activity of 5.4 K(m) for nitrate was estimated to be 20 microM. EPR signals due to one [4Fe-4S] cluster and Mo(V) were identified in dithionite reduced samples and in the presence of nitrate.

The binding of Ca2+ to the dihaem cytochrome-c peroxidase from Paracoccus denitrificans was analysed by following perturbations in the visible and 1H-NMR spectra of both haem groups. The enzyme contains at least two types of Ca(2+)-binding site. Site I is occupied in the isolated enzyme, binds Ca2+ with a redox-state-independent Kd of 1.2 microM and accommodates neither Mg2+ nor Mn2+. Site II is unoccupied in dilute solutions of the isolated oxidised enzyme and binds Ca2+ cooperatively with a Kd of 0.52 mM. In the mixed valence form, the binding affinity increases to resemble that of site I. The cooperativity was shown by -Ca2+ binding to site II, the titration of haem methyl 1H-NMR resonances, and a half-of-sites effect observed for modification of an essential histidine with diethylpyrocarbonate. These are all consistent with site II being situated at the interface between two monomers of a dimeric enzyme. Thus the equilibrium of binding to site II is a reflection of the equilibrium for dimerisation and conditions which shift that equilibrium towards the dimer, such as increased ionic strength or high protein concentration, also increase Ca2+ affinity. Binding of Ca2+ to site II is required for formation of the active high spin state at the peroxidatic haem.

Nitrite reductase (cytochrome cd1) was purified to electrophoretic homogeneity from the soluble extract of the marine denitrifying bacterium Pseudomonas nautica strain 617. Cells were anaerobically grown with 10 mM nitrate as final electron acceptor. The soluble fraction was purified by four successive chromatographic steps and the purest cytochrome cd1 exhibited an A280 nm(oxidized)/A410nm(oxidized) coefficient of 0.90. In the course of purification, cytochrome cd1 specific activity presented a maximum value of 0.048 units/mg of protein. This periplasmic enzyme is a homodimer and each 60 kDa subunit contains one heme c and one heme d1 as prosthetic moieties, both in a low spin state. Redox potentials of hemes c and d1 were determined at three different pH values (6.6, 7.6 and 8.6) and did not show any pH dependence. The first 20 amino acids of the NH2-terminal region of the protein were identified and the sequence showed 45% identity with the corresponding region of Pseudomonas aeruginosa nitrite reductase but no homology to Pseudomonas stutzeri and Paracoccus denitrificans enzymes. Spectroscopic properties of Pseudomonas nautica 617 cytochrome cd1 in the ultraviolet-visible range and in electron paramagnetic resonance are described. The formation of a heme d1 -nitric-oxide complex as an intermediate of nitrite reduction was demonstrated by electron paramagnetic resonance experiments.

A c-type hexaheme nitrite reductase (NiR) isolated from nitrate-grown cells of Desulfovibrio desulfuricans (Dd) ATCC 27774 catalyses the six-electron reduction of nitrite to ammonia. Previous electrochemical studies demonstrated that a simple electrocatalytic mechanism can be applied to this system (Moreno, C., Costa, C., Moura, I., LeGall, J., Liu, M. Y., Payne, W. J., Van Dijk, C. and Moura, J. J. G. (1992) Eur.J.Biochem. 212, 79-86). Its substrate specificity, availability and stability under ambient conditions makes this enzymatic system a promising candidate for use in a biosensor device. An electrochemical study of gel-immobilized Dd NiR on a glassy carbon electrode revealed both enzymatic activity and amperometric response to nitrite. In this study it was observed that the catalytic current density is a function of the nitrite concentration in solution and follows a characteristic Michaelis-Menten-type substrate dependence. Such a biosensor device (NiR-electrode) bears the option to be used for analytical determination of nitrite in complex media.

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.

The thermal aquation rate constant and the photoaquation quantum yield of [Cr(CN)(6)](3-) are reduced by a factor of 40 and 3, respectively, when the complex forms a 1:1 adduct with the protonated form of the polyaza macrocycle [32]ane-N-8. On the other hand, the same macrocycle has practically no effect on the photoaquation of [Cr(CN)(5)(H2O)](2-) and a very small effect on the thermal reaction of this complex. These results are discussed in relation to the thermal and photochemical reaction mechanisms and to the steric configuration of the adducts between complexes and macrocycle.

The steady-state fluorescence emission spectra of the azacyclophanes 2,5,8,11-tetraaza[12] paracyclophane (L(1)), 2,6,9,13-tetraaza[14]paracyclophane (L(2)), 14,15,17,18-tetramethyl-2,5,8,11-tetraaza-[12]paracyclophane (L(3)) and 16,17,19,20-tetramethyl-2,6,9,13-tetraaza[14]paracyclophane (L(4)) as a function of pH have been measured. The fully protonated species of each cyclophane gives the highest fluorescence-emission quantum yield. The shapes of the titration curves have been explained by the existence of an electron-transfer quenching effect from a non-protonated amine to the benzene chromophore. This effect is greater for macrocycles in which the first deprotonated amine group is closer to the benzene. The association constants for the interaction of the four fully protonated macrocycles with K-3[Co(CN)(6)] have been measured either by potentiometry or from fluorescence-emission measurements, and increase in the order L(3) approximate to L(4) < L(1) approximate to L(2). The photoaquation quantum yields of K-3[Co(CN)(6)] have been measured in the presence of the macrocycles L(1) and L(2). and indicate that three of the CN nitrogens of the complex are involved in adduct formation with the fully protonated macrocycles, as supported by molecular modelling.

When biacetyl is imprisoned into Cram's hemicarcerand 1, its absorption, fluorescence, and phosphorescence maxima are red shifted compared to the values obtained for solutions of free biacetyl in any solvent. Furthermore, the lifetime of the T-1 excited state of imprisoned biacetyl is unaffected by solvent nature and presence of dioxygen. These results show that inclusion into the hemicarcerand (i) shields biacetyl from interaction with the solvent molecules and (ii) prevents deactivation of its long-lived T-1 excited state by energy transfer to dioxygen. The perturbation provided by the cavity on the spectroscopic properties of biacetyl is much smaller than that provided by even the most ''innocent'' solvent. The consequent picture is that of a biacetyl molecule which is contained in a not-too-tight cavity where no specific host-guest interaction takes place. The peculiar spectroscopic and excited-state behavior of biacetyl imprisoned in hemicarcerand 1 supports Cram's view that the inner phase of carcerands and hemicarcerands is to be considered as a new phase of matter.

Three-quarters of the carbon-13 resonances of nuclei attached to the four haems of Desulfovibrio vulgaris ferricytochrome c3 are assigned. Preliminary analysis of their Fermi contact interactions shows that the shifts are directly related to the orientation of both of the axial histidine ligands in each case and the approach can therefore be used to obtain structural information in other cytochromes with bis-histidinyl coordination. The implications for the control of redox potential in cytochromes are discussed.

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The crystal structure of desulforedoxin from Desulfovibrio gigas, a new homo-dimeric (2x36 amino acids) non-heme iron protein, has been solved by the SIRAS method using the indium-substituted protein as the single derivative. The structure was refined to a crystallographic X-factor of 16.9% at 1.8 Angstrom resolution. Native desulforedoxin crystals were grown from either PEG 4K or lithium sulfate, with cell constants a = b = 42.18 Angstrom, = 72.22 Angstrom (for crystals grown from PEG 4K), and they belong to space group P3(2)21. The indium-substituted protein crystallized isomorphously under the same conditions. The 2-fold symmetric dimer is firmly hydrogen bonded and folds as an incomplete beta-barrel with the two iron centers placed on opposite poles of the molecule. Each iron atom is coordinated to four cysteinyl residues in a distorted tetrahedral arrangement. Both iron atoms are 16 Angstrom apart but connected across the 2-fold axis by 14 covalent bonds along the polypeptide chain plus two hydrogen bonds. Desulforedoxin and rubredoxin share some structural features but show significant differences in terms of metal environment and water structure, which account for the known spectroscopic differences between rubredoxin and desulforedoxin. (C) 1995 Academic Press Limited