Proline inhibition of pyrroline-5-carboxylate reductase: differences in enzymes obtained from animal and tissue culture sources

The complete amino acid sequence for the 148 amino acid flavodoxin from $\text{Desulfovibrio}\text{vulgaris}$ is presented. This is the first flavoenzyme for which both the complete amino acid sequence and a 2.5 Å resolution x-ray diffraction structure are now known. The position of some important residues in the binding of FMN are given. The D. vulgaris sequence is compared with other published flavodoxin sequences.     

Purification and characterization of cytochrome C3 (Mr 26,000) isolated from Desulfovibriodesulfuricans Norway strain

An acidic cytochrome c (P_i = 4.8) has been purified from $\text{Desulfovibrio}\text{desulfuricans}$ Norway. Its molecular weight was estimated to be 26,000 but a monomeric form of 13,500 molecular weight has been obtained. The comparison of its amino acid composition and N terminal sequence has characterized this cytochrome as a new cytochrome, different from cytochrome c₃ (Mr 13,000) and cytochrome c_553(550) studied in the same organism. Its optical spectrum was similar to cytochrome c₃ (Mr 13,000) accordingly it has 4 haems per subunit. The absence of absorption at 695 nm indicates that two histidine residues are implicated as fifth and sixth ligand for haem iron. This new cytochrome is homologous to the cytochrome C₃ (Mr 26,000) previously described for $\text{Desulfovibrio}\text{gigas}$ and $\text{Desulfovibrio}\text{vulgaris}$.     

Electron transport components from methanogenic bacteria: The ferredoxin from Methanosarcinabarkeri (strain Fusaro)

A ferredoxin has been isolated from the methanogenic organism $\text{Methanosarcina}\text{barkeri}$ (strain Fusaro). The protein appears to be constituted by two identical subunits of molecular weight approx. 6000 daltons. The UV-visible spectrum of the protein is characterized by two broad absorption peaks centered at 410 and 300 nm and an absorbance ratio $\text{A}{}_{410}\text{A}{}_{300}\text{= 0.8}$. The molar extinction coefficients at 410 and 300 nm are 36,500 and 45,625 M^?1 cm^?1, respectively. The amino acid compsition of $\text{M.}\text{barkeri}$ ferredoxin shows a preponderance of acidic residues and lacks five amino acids. The protein contains 8 cysteine residues and approx. 7 iron atoms and 7–8 acid-labile sulfide groups per molecule which are indicative of the presence of two iron-sulfur clusters in the molecule. The N-terminal sequence shows a high degree of homology with the sequences of ferredoxins from $\text{Clostridium}\text{pasteurianum}\text{,}\text{Desulfovibrio}\text{gigas}$ and $\text{Desulfovibrio}\text{africanus}\text{.}\text{M.}\text{barkeri}$ ferredoxin functions as an electron carrier in the pyruvate dehydrogenase system. Its possible role in a variety of electron transfer reactions is discussed.     

Amino acid sequence of Desulfovibrio gigas ferredoxin: revisions

Reexamination of the amino acid sequence of $\text{Desulfovibrio}$$\text{gigas}$ ferredoxin revealed that the sequence published in 1971 should be revised. This sequence was determined using automatic protein sequencer in liquid phase and in solid phase. Peptides derived from tryptic hydrolysis, $\text{Staphylococcus}$$\text{aureus}$ protease hydrolysis, cyanogen bromide cleavage were used to construct the total sequence. This ferredoxin contains 6 cysteines per minimum molecular weight of 6,400. 4 cysteines are linked to a (4 Fe-4 S) cluster and the two others possibly participate in a disulfide bridge.     

Cytochrome C553 from Desulfovibrio,vulgaris: Potentiometric characterization by optical and EPR studies

Cytochrome c₅₅₃ is a monohaemic c type cytochrome isolated from the sulfate reducing bacteria $\text{Desulfovibrio}$,$\text{vulgaris}$. Its midpoint potential value, determined by optical, EPR and polarographic studies is significantly lower than the midpoint potentials reported for other monohaemic cytochromes c (+ 10 mV instead of + 290 mV). In an attempt to study correlations between amino acid sequence, haem iron coordination and haem exposure in cytochromes c, cytochrome c₅₅₃ is compared with mitochondrial and bacterial c type cytochromes.     

Phospholipid Composition of Desulfovibrio Species

The phospholipids of Desulfovibrio desulfuricans, Norway strain, D. vulgaris, and D. gigas were examined in relationship to their qualitative and quantitative composition. D. desulfuricans and D. vulgaris exhibited an essentially identical phospholipid composition consisting of phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and lysophosphatidylserine. Phosphatidylserine (10.9%) was present in D. desulfuricans but was not detected in D. vulgaris. D. gigas was found to contain only two phospholipids, phosphatidylethanolamine (30%) and phosphatidylglycerol (70%). An ornithine-containing lipid was detected in D. gigas which was not present in the other two Desulfovibrio species.     

Redox reactions of the hydrogenase-cytochrome C3 system from Desulfovibrio gigas with the synthetic analogue of ferredoxin active sites [Fe4S4 (SCH2CH2OH)4]2−

The synthetic cluster [Fe₄S₄ (SCH₂CH₂OH)₄]^2? and ferredoxin I from $\text{Desulfovibrio}\text{gigas}$ have been comparatively reacted in the presence of hydrogen with two enzyme components isolated from the $\text{D.}\text{gigas}$ electron carrier system: hydrogenase and cytochrome c₃. The reactions have been followed by spectrophotometric and manometric methods. No reduction is detected unless both enzymes are simultaneously present. The easy reduction in the presence of hydrogenase-cytochrome c₃ combination takes place in two stages corresponding each to one equivalent of reduction. The first stage is reversible while the second is not and yields a super reduced species deriving from the cluster complex.     

Whole-cell antigens of members of the sulfate-reducing genus Desulfovibrio

Antisera have been developed against the wholecell antigens of Desulfovibrio africanus Benghazi and Walvis Bay, D. vulgaris Hildenborough, D. salexigens British Guiana, D. gigas, and D. desulfuricans Essex 6. An enzymelinked immunoadsorption assay (ELISA) was developed to measure the reaction of these antisera with the homologous and heterologous antigens. The ELISA method demonstrated a reaction between pre-immune sera and cells of D. africanus, D. gigas and D. desulfuricans, suggesting the presence of a lectin-like substance on these cell surfaces. Extensive cross-reactions were seen between the antisera and heterologous cells, suggesting the sharing of a number of surface antigens amongst the Desulfovibrio. However, the pattern of these cross-reactions was different from that observed for an ELISA reaction developed for the cytochrome c₃ from various Desulfovibrio.Abbreviation ELISA enzyme-linked immunoadsorption assay     

The amino acid sequence of desulforedoxin, a new type of non heme iron protein from Desulfovibrio gigas

A new type of non heme iron protein called desulforedoxin has been isolated from the sulfate reducing bacterium, $\text{Desulfovibrio gigas}$. The complete amino acid sequence has been established. The 36 amino acid residues of the sequence are aligned with the aid of peptides obtained by cyanogen bromide cleavage and by hydrolysis with a peptidase isolated from $\text{Staphylococcus aureus}$. Desulforedoxin has been described as a non heme iron protein of molecular weight 7,600 with 2 iron atoms linked to eight cysteine residues. In fact, sequence elucidation shows that it consists of a dimer of a peptide containing 36 aminoacids. We do not know whether if each monomer contains 1 iron atom linked to 4 cysteine residues or whether the two iron cross link the two monomers. Additional studies on the elucidation of the structure of this new cluster are presently under study.     

Purification and some properties of cytochrome C553(550) isolated from Desulfovibrio desulfuricans Norway.

A new c-type cytochrome containing a single heme group, cytochrome c_553(550) has been purified from $\text{Desulfovibrio desulfuricans}$ (Norway strain) and some of its properties have been investigated. It has an isoelectric point of 6.6 and a higher redox potential than cytochrome c₃ isolated from the same bacteria. Its molecular weight was estimated to be 9,200 by gel filtration. The main absorption peaks are at 553, 522.5 and 417 nm in the reduced form and at 690, 529, 411, 357 and 280 nm in the oxidized form. The asymmetric α band of the reduced state is similar to the one reported for socalled “split α” cytochromes c. The cytochrome contains 86 amino acid residues with 5 methionine, two cysteine and two histidine residues. The N terminal sequence of $\text{D. desulfuricans}$ Norway cytochrome c_553(550) presents no evident homology with that of $\text{Desulfovibrio vulgaris}$ Hildenborough cytochrome c₅₅₃.     

Isolation and characterization of a molybdenum iron-sulfur protein from Desulfovibrio africanus.

A molybdenum-containing iron-sulfur protein has been isolated from the sulfate reducer $\text{Desulfovibrio}\text{africanus}$. The protein appears to be a complex protein of high molecular weight (112,000) composed of 10 subunits (mol. wt. 11,500) and containing a high amount of molybdenum (5–6 atoms/mole) with approx. 20 atoms each of iron and labile sulfide. The spectrum shows peaks at around 615, 410 and 325 nm with a protein peak at 280 nm. Its millimolar extinction coefficients at 615, 410 and 280 nm are 48.4, 64.4 and 141 respectively. The protein contains 106 amino-acid residues per subunit of mol. wt. 11,262 and the number of cysteine residues is 2 per subunit. The N-terminal sequence which has been determined up to 26 residues is characterized by its high degree of hydrophobicity.     

The presence of redox-sensitive nickel in the periplasmic hydrogenase from Desulfovibrio gigas

A new and improved method for the purification of the periplasmic hydrogenase from $\text{Desulfovibrio}\text{gigas}$is described. This preparation of hydrogenase was found to contain 0.64 g atom of nickel per mole of protein. In the oxidized state, the hydrogenase exhibited an isotropic signal at g = 2.02 and a characteristic Ni(III) signal with g-values at 2.31, 2.20 and ～2.0. The EPR spectrum of the reduced enzyme consisted of multiple species. One set of g-values are determined as 2.17, 2.08 and 2.04. The other minor species exhibited a resonance at g = 2.28. On partial reoxidation of the hydrogenase, the initial Ni(III) signals reappeared along with additional signals attributed to multiple Ni(III) species. It is proposed that Ni is an important functional unit in this hydrogenase.     

Time-resolved fluorescence studies of flavodoxin. Fluorescence decay and fluorescence anisotropy decay of tryptophan in Desulfovibrio flavodoxins

The time-resolved fluorescence characteristics of tryptophan in flavodoxin isolated from the sulfate-reducing bacteria Desulfovibrio vulgaris and Desulfovibrio gigas have been examined. By comparing the results of protein preparations of normal and FMN-depleted flavodoxin, radiationless energy transfer from tryptophan to FMN has been demonstrated. Since the crystal structure of the D. vulgaris flavodoxin is known, transfer rate constants from the two excited states ¹ L _a and ¹ L _b can be calculated for both tryptophan residues (Trp 60 and Trp 140). Residue Trp 60, which is very close to the flavin, transfers energy very rapidly to FMN, whereas the rate of energy transfer from the remote Trp 140 to FMN is much smaller. Both tryptophan residues have the indole rings oriented in such a way that transfer will preferentially take place from the ¹ L _a excited state. The fluorescence decay of all protein preparations turned out to be complex, the parameter values being dependent on the emission wavelength. Several decay curves were analyzed globally using a model in which tryptophan is involved in some nanosecond relaxation process. A relaxation time of about 2 ns was found for both D. gigas apo- and holoflavodoxin. The fluorescence anisotropy decay of both Desulfovibrio FMN-depleted flavodoxins is exponential, whereas that of the two holoproteins is clearly non-exponential. The anisotropy decay was analyzed using the same model as that applied for fluorescence decay. The tryptophan residues turned out to be immobilized in the protein. A time constant of a few nanoseconds results from energy transfer from tryptophan to flavin, at least for D. gigas flavodoxin. The single tryptophan residue in D. gigas flavodoxin occupies a position in the polypeptide chain remote from the flavin prosthetic group. Because of the close resemblance of steady-state and time-resolved fluorescence properties of tryptophan in both flavodoxins, the center to center distance between tryptophan and FMN in D. gigas flavodoxin is probably very similar to the distance between Trp 140 and FMN in D. vulgaris flavodoxin (i.e. 20 Å). Offprint requests to: A.J.W.G. Visser     

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 absorbance ratio of $\text{A}{}_{385}\text{A}{}_{283}\text{= 0.74}$. Storage results in a bleaching of the chromophore; the denatured ferredoxin is reconstitutable with iron and sulfide. The instability temperature is 52°C.The rubredoxin does not differ markedly from rubredoxins from other anaerobic bacteria.     

Hybrid Cluster Proteins and Flavodiiron Proteins Afford Protection to Desulfovibrio vulgaris upon Macrophage Infection

Desulfovibrio species are Gram-negative anaerobic sulfate-reducing bacteria that colonize the human gut. Recently, Desulfovibrio spp. have been implicated in gastrointestinal diseases and shown to stimulate the epithelial immune response, leading to increased production of inflammatory cytokines by macrophages. Activated macrophages are key cells of the immune system that impose nitrosative stress during phagocytosis. Hence, we have analyzed the in vitro and in vivo responses of Desulfovibrio vulgaris Hildenborough to nitric oxide (NO) and the role of the hybrid cluster proteins (HCP1 and HCP2) and rubredoxin oxygen oxidoreductases (ROO1 and ROO2) in NO protection. Among the four genes, hcp2 was the gene most highly induced by NO, and the hcp2 transposon mutant exhibited the lowest viability under conditions of NO stress. Studies in murine macrophages revealed that D. vulgaris survives incubation with these phagocytes and triggers NO production at levels similar to those stimulated by the cytokine gamma interferon (IFN-γ). Furthermore, D. vulgaris hcp and roo mutants exhibited reduced viability when incubated with macrophages, revealing that these gene products contribute to the survival of D. vulgaris during macrophage infection.     

The detection of a bound ferredoxin in the photosynthetic lamellae of blue-green algae and other oxygen evolving photosynthetic organisms

The presence of an electron transport component with an EPR spectrum similar to that of a ferredoxin has been demonstrated in the blue-green alga $\text{Anabaena}$$\text{cylindrica}$, the green alga $\text{Euglena}$$\text{gracilis}$, and in chloroplasts from sorghum ($\text{Sorghum}$$\text{bicolour}$) and beans ($\text{Phaseolus}$$\text{vulgaris}$). The component is photoreduced at 77°K and is very similar to that previously reported in spinach. It seems likely that this component is a primary electron acceptor in photosynthesis in all of these organisms.     

Detection of triplet states in algae by zero-field resonance.

Triplet states have been detected and characterized by zero-field splitting parameters in $\text{Anacystis nidulans}$, $\text{Euglena gracilis}$, $\text{Porphyridium cruentum}$, and $\text{Chlorella vulgaris}$, using fluorescence-detected magnetic resonance in zero-field at 4.2 K. Monitoring the 720 nm photosystem I emission, transitions between triplet spin levels have been assigned to antenna chlorophyll of one of both photosystems; photochemical reactions of chlorophyll are observed in the presence of an inhibitor and strong light, probably resulting in photoreduction and pheophytinization.     

Analysis of the primary structure of collagen for the origins of molecular packing

The amino acid sequence in the triplet region of the α1 chain of collagen was analyzed for complementary relationships that would explain the stagger of multiples of 670 Å between the rod-like molecules in the fibril. The analysis was done by moving the sequence of 1011 amino acids past itself and scoring for complementarity between opposing amino acids allowing a range of ±2 to 3 residues. It was found that interactions between amino acids of opposite charge and between large hydrophobic amino acids in the overlapping region between two chains are maximal when the chains are staggered by 0D, 1D, 2D, 3D and 4D, where D = 234 ± 1 residues. The residue repeat derived from this value is 2.86 ± 0.02 Å. The existence of a D separation between interacting residues was shown to be reflected in the actual distribution of large hydrophobic amino acids. Surprisingly, the distribution approximates the pattern ($\text{2D}\text{11}$)₅($\text{D}\text{11}$) repeated over 4.4D intervals. The regularity may arise from structural constraints imposed by super-coiling. The distribution of charged residues is less regular and does not show a well-defined periodicity. However, positively-charged residues tend to be near negatively-charged residues, allowing intramolecular charge neutralization as well as strong intermolecular charge interactions at 0D.     

Desulforedoxin: Preliminary X-ray diffraction study of a new iron-containing protein

Desulforedoxin, a two-iron, two-chain protein from the bacterium Desulfovibrio gigas, has been crystallized. X-ray diffraction photos show symmetry consistent with space group P3₁21 or the enantiomorph P3₂21. Cell parameters are $\text{a = b = 42.28}\text{A}\text{?}\text{, c = 72.46}\text{A}\text{?}$.Crystals develop sufficiently well for an X-ray diffraction analysis of at least 2.0 Å resolution to be performed.