A bacterioferritin from the strict anaerobe Desulfovibrio desulfuricans ATCC 27774.

A bacterioferritin was isolated from the anaerobic bacterium Desulfovibrio desulfuricans ATCC 27774, grown with nitrate as the terminal electron acceptor, which is the first example of a bacterioferritin from a strict anaerobic organism. This new bacterioferritin was isolated mainly as a 24-mer of 20 kDa identical subunits, containing 0.5 noncovalently bound heme and 2 iron atoms per monomer. Although its N-terminal sequence is significantly homologous with ferritins from other microorganisms and the ligands to the di-iron ferroxidase center are conserved, it is one of the most divergent bacterioferritins so far characterized. Also, in contrast to all other known bacterioferritins, its heme is not of the B type; its chromatographic behavior is identical to that of iron uroporphyrin. Thus, D. desulfuricans bacterioferritin appears to be the second example of a protein unexpectedly containing this heme cofactor, or a closely related porphyrin, after its finding in Desulfovibrio gigas rubredoxin:oxygen oxidoreductase ¿Timkovich, R., Burkhalter, R. S., Xavier, A. V., Chen, L., and Le Gall, J. (1994) Bioorg. Chem. 22, 284-293. The oxidized form of the protein has a visible spectrum characteristic of low-spin ferric hemes, exhibiting a weak absorption band at 715 nm, indicative of bis-methionine heme axial coordination; upon reduction, the alpha-band appears at 550 nm and a splitting of the Soret band occurs, with two maxima at 410 and 425 nm. The heme center has a reduction potential of 140 +/- 10 mV (pH 7.6), a value unusually high compared to that of other bacterioferritins (ca. -200 mV).

Effect of the ionic environment on the molecular structure of bacteriophage SPP1 portal protein.

Bacteriophage SPP1 portal protein is a large cyclical homo-oligomer composed of 13 subunits. The solution structure and assembly behavior of this protein with high-point rotational symmetry was characterized. The purified protein was present as a monodisperse population of 13-mers, named gp6H, at univalent salt concentrations in the hundred millimolar range (>/= 250 mM NaCl) or in the presence of bivalent cations in the millimolar range (>/= 5 mM MgCl2). Gp6H had a slightly higher sedimentation coefficient, a smaller shape-dependent frictional ratio, and a higher rate of intersubunit cross-linking in the presence of magnesium than in its absence. In the absence of bivalent cations and at univalent salt concentrations below 250 mM, the 13-mer molecules dissociated partially into stable monomers, named gp6L. The monomer had a somewhat different shape from the subunit present in the 13-mer, but maintained a defined tertiary structure. The association-dissociation equilibrium was mainly between the monomer and the 13-mer with a minor population of intermediate oligomers. Their interconversion was strongly influenced by the ionic environment. Under physiological conditions, the concentration of Mg2+ found in the Bacillus subtilis cytoplasm (10-50 mM) probably promotes complete association of gp6 into 13-mer rings with a compact conformation.

Characterisation of a new rubredoxin isolated from Desulfovibrio desulfuricans 27774: definition of a new family of rubredoxins.

A new rubredoxin from the sulphate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774, grown with nitrate as terminal electron acceptor, was isolated and characterised. The protein is an 8.5 kDa monomer containing one iron atom per molecule, with a reduction potential of 25 +/- 5 mV at pH 7.6. Like the recombinant Rdl protein from D. vulgaris, expressed in Escherichia coli [Lumpio, H.L., Shenvi, N.V., Garg, R.P., Summers, A.O. and Kurtz, D.M., J. Bacteriol. 179 (1997) 4607-4615], it contains an unusual spacing of four amino acids between the first two of the iron coordinating cysteinyl residues. This difference is reflected in the structure of the iron centre, as observed by visible and EPR spectroscopies. All together, these features make these proteins the first members of a new family of rubredoxins.

Primary sequence, oxidation-reduction potentials and tertiary-structure prediction of Desulfovibrio desulfuricans ATCC 27774 flavodoxin.

Flavodoxin was isolated and purified from Desulfovibrio desulfuricans ATCC 27774, a sulfate-reducing organism that can also utilize nitrate as an alternative electron acceptor. Mid-point oxidation-reduction potentials of this flavodoxin were determined by ultraviolet/visible and EPR methods coupled to potentiometric measurements and their pH dependence studied in detail. The redox potential E2, for the couple oxidized/semiquinone forms at pH 6.7 and 25 degrees C is -40 mV, while the value for the semiquinone/hydroquinone forms (E1), at the same pH, -387 mV. E2 varies linearly with pH, while E1 is independent of pH at high values. However, at low pH (< 7.0), this value is less negative, compatible with a redox-linked protonation of the flavodoxin hydroquinone. A comparative study is presented for Desulfovibrio salexigens NCIB 8403 flavodoxin [Moura, I., Moura, J.J.G., Bruschi, M. & LeGall, J. (1980) Biochim. Biophys. Acta 591, 1-8]. The complete primary amino acid sequence was obtained by automated Edman degradation from peptides obtained by chemical and enzymic procedures. The amino acid sequence was confirmed by FAB/MS. Using the previously determined tridimensional structure of Desulfovibrio vulgaris flavodoxin as a model [similarity, 48.6%; Watenpaugh, K.D., Sieker, L.C., Jensen, L.H., LeGall, J. & Dubourdieu M. (1972) Proc. Natl Acad. Sci. USA 69, 3185-3188], the tridimensional structure of D. desulfuricans ATCC 27774 flavodoxin was predicted using AMBER force-field calculations.

Cytochrome c6 from Monoraphidium braunii. A cytochrome with an unusual heme axial coordination.

A soluble monoheme c-type cytochrome (cytochrome c6) has been isolated from the green alga Monoraphidium braunii. It has a molecular mass of 9.3 kDa, an isoelectric point of 3.6 and a reduction potential of 358 mV at pH 7. The determined amino acid sequence allows its classification as a class-I c-type cytochrome. The ferric and ferrous cytochrome forms and their pH equilibria have been studied using 1H-NMR, ultraviolet/visible, EPR and Mössbauer spectroscopies. The pH equilibria are complex, several pKa values and pH-dependent forms being observed. The amino acid sequence, the reduction-potential value and the visible and NMR spectroscopies data in the pH range 4-9 indicate that the heme iron has a methionine-histidine axial coordination. However, the EPR and Mössbauer data obtained for the ferricytochrome show that in this pH range two distinct forms are present: form I, gz = 3.27, gy = 2.05 and gx = 1.05; form II, gz = 2.95, gy = 2.29 and gx = 1.43. While form I has crystal-field parameters typical of a methionine-histidine coordination, those associated with form II would suggest a histidine-histidine axial ligation. This possibility was extensively analyzed by spectroscopic methods and by chemical modification of a histidine residue. It was concluded that form II actually corresponds to an unusual type of methionine-histidine axial coordination. Straightforward examples of this type of coordination have recently been found in other c-type hemeproteins [Teixeira, M., Campos, A. P., Aguiar, A. P., Costa, H. S., Santos, H., Turner, D. L. & Xavier, A. V. (1993) FEBS Lett. 317, 233-236], corroborating our proposal. Since both forms, with very distinct crystal-field parameters, are shown to have the same reduction potential, it may be concluded that the axial and rhombic distortions of the heme-iron ligand field cannot be directly correlated with the heme-reduction potential. The pH-dependence studies have also shown that the form I and form II are interconvertible, with pKa approximately 5. To establish a possible physiological significance for this process, in particular for the interaction of the cytochrome with the membrane-bound electron-transfer complexes b6f and photosystem I, the effect of surfactants on the spectroscopic characteristics of cytochrome c6 has been studied.