Characterization and cloning of an extremely thermostable, Pyrococcus furiosus-type 4Fe ferredoxin from Thermococcus profundus.

An extremely thermostable [4Fe-4S] ferredoxin was isolated under anaerobic conditions from a hyperthermophilic archaeon Thermococcus profundus, and the ferredoxin gene was cloned and sequenced. The nucleotide sequence of the ferredoxin gene shows the ferredoxin to comprise 62 amino acid residues with a sequence similar to those of many bacterial and archaeal 4Fe (3Fe) ferredoxins. The unusual Fe-S cluster type, which was identified in the resonance Raman and EPR spectra, has three cysteines and one aspartate as the cluster ligands, as in the Pyrococcus furiosus 4Fe ferredoxin. Under aerobic conditions, a ferredoxin was purified that contains a [3Fe-4S] cluster as the major Fe-S cluster and a small amount of the [4Fe-4S] cluster. Its N-terminal amino acid sequence is the same as that of the anaerobically-purified ferredoxin up to the 26th residue. These results indicate that the 4Fe ferredoxin was degraded to 3Fe ferredoxin during aerobic purification. The aerobically-purified ferredoxin was reversibly converted back to the [4Fe-4S] ferredoxin by the addition of ferrous ions under reducing conditions. The anaerobically-purified [4Fe-4S] ferredoxin is quite stable; little degradtion was observed over 20 h at 100 degrees C, while the half-life of the aerobically-purified ferredoxin is 10 h at 100 degrees C. Both the anaerobically- and aerobically-purified ferredoxins were found to function as electron acceptors for the pyruvate-ferredoxin oxidoreductase purified from the same archaeon.

Preoviposition activation of cathepsin-like proteinases in degenerating ovarian follicles of the mosquito Culex pipiens pallens.

Within developing ovaries of many insects, some developing follicles or oocytes usually degenerate (follicular atresia or oosorption), while the others may continue to grow to maturity, thus maintaining the balance between the number of eggs and reproductive circumstances such as available nutrients. To help clarify the phenomenon of follicular atresia during ovarian development, we examined cysteine proteinases stored in mosquito Culex pipiens pallens ovaries. First, analysis using synthesized substrates showed that cathepsin B- and L-like proteinases gradually accumulated in the developing ovaries after a blood meal, which required more than 10 min of preincubation under acidic conditions to reach their maximum activities. However, homogenates of degenerating follicles 3 days after feeding showed proteolytic activities without acid treatment, suggesting that the proteinases had already been activated, while the extract of normally developing follicles collected from the same ovaries required more than 10 min of acid preincubation to reach the optimum activities, suggesting that the enzymes remained as inactive forms. Chemical and immunohistochemical analyses showed that more proteinases are located in the cytoplasm, rather than being associated with yolk granules. Ovarian proteinases, which are believed to become activated at the onset of embryogenesis, should also be activated during oogenesis, presumably to enhance oosorption.

Induction of oogenesis in mosquitoes (Diptera: Culicidae) by infusion of the hemocoel with amino acids.

As done previously with adult females of Culex pipiens pallens Coquillett, a mixture of 17 amino acids was infused into the hemocoel of females of seven anautogenous and one autogenous mosquito species belonging to three genera. In Culex. p. quinquefasciatus Say, Cx. tritaeniorhynchus Giles, Cx. kyotoensis Yamaguti & LaCasse, Aedes albopictus (Skuse), Armigeres subalbatus (Coquillett), and Cx. p. molestus Forskal, which previously had laid autogenously matured first batch of eggs, ovarian development was stimulated and frequently continued to maturity. In most mosquitoes, the number of mature follicles nearly doubled when the period of infusion was extended from 24 to 48 h. Therefore, the two previously indicated roles of amino acids, one to initiate ovarian development and the other to regulate the number of maturing oocytes, were confirmed in these species. In Cx. halifaxii Theobald and Ae. japonicus (Theobald), however, the frequency of activation and maturation of ovaries was low compared with the other species, indicating that those species may require some factors other than an increase in amino acids for normal ovarian development after a blood meal.

Spectroscopic investigation of selective cluster conversion of archaeal zinc-containing ferredoxin from Sulfolobus sp. strain 7.

Archaeal zinc-containing ferredoxin from Sulfolobus sp. strain 7 contains one [3Fe-4S] cluster (cluster I), one [4Fe-4S] cluster (cluster II), and one isolated zinc center. Oxidative degradation of this ferredoxin led to the formation of a stable intermediate with 1 zinc and approximately 6 iron atoms. The metal centers of this intermediate were analyzed by electron paramagnetic resonance (EPR), low temperature resonance Raman, x-ray absorption, and (1)H NMR spectroscopies. The spectroscopic data suggest that (i) cluster II was selectively converted to a cubane [3Fe-4S](1+) cluster in the intermediate, without forming a stable radical species, and that (ii) the local metric environments of cluster I and the isolated zinc site did not change significantly in the intermediate. It is concluded that the initial step of oxidative degradation of the archaeal zinc-containing ferredoxin is selective conversion of cluster II, generating a novel intermediate containing two [3Fe-4S] clusters and an isolated zinc center. At this stage, significant structural rearrangement of the protein does not occur. We propose a new scheme for oxidative degradation of dicluster ferredoxins in which each cluster converts in a stepwise manner, prior to apoprotein formation, and discuss its structural and evolutionary implications.

Novel zinc-containing ferredoxin family in thermoacidophilic archaea.

The dicluster-type ferredoxins from the thermoacidophilic archaea such as Thermoplasma acidophilum and Sulfolobus sp. are known to contain an unusually long extension of unknown function in the N-terminal region. Recent x-ray structural analysis of the Sulfolobus ferredoxin has revealed the presence of a novel zinc center, which is coordinated by three histidine ligand residues in the N-terminal region and one aspartate in the ferredoxin core domain. We report here the quantitative metal analyses together with electron paramagnetic resonance and resonance Raman spectra of T. acidophilum ferredoxin, demonstrating the presence of a novel zinc center in addition to one [3Fe-4S] and one [4Fe-4S] cluster (Fe/Zn = 6.8 mol/mol). A phylogenetic tree constructed for several archaeal monocluster and dicluster type ferredoxins suggests that the zinc-containing ferredoxins of T. acidophilum and Sulfolobus sp. form an independent subgroup, which is more distantly related to the ferredoxins from the hyperthermophiles than those from the methanogenic archaea, indicating the existence of a novel group of ferredoxins, namely, a "zinc-containing ferredoxin family" in the thermoacidophilic archaea. Inspection of the N-terminal extension regions of the archaeal zinc-containing ferredoxins suggested strict conservation of three histidine and one aspartate residues as possible ligands to the novel zinc center.

A novel 6Fe (2 x [3Fe-4S]) ferredoxin from Mycobacterium smegmatis.

A novel ferredoxin was purified from Mycobacterium smegmatis by a series of hydrophobic chromatographies in the presence of high concentrations of ammonium sulfate and sodium chloride. The ferredoxin exhibited the same peptide map and N-terminal amino acid sequence as the known 7Fe ferredoxin from the same bacterium. On the other hand, this ferredoxin was found to contain approximately 6 Fe/mol ferredoxin and was also shown to contain only [3Fe-4S] clusters by resonance Raman spectroscopy, indicating that it is a novel 6Fe ferredoxin which contains two [3Fe-4S] clusters.

pH-dependent activity change of superoxide dismutase from Mycobacterium smegmatis.

Superoxide dismutase (SOD), purified from Mycobacterium smegmatis, was found to contain both manganese and iron. Since the Fe and Mn-reconstituted enzymes had specific activities of 190 and 2810 units/mg protein/g atom of metal/mol of subunit, respectively, the Mycobacterial SOD can be classified with SODs showing activity with either iron or manganese as the active-site metal (a cambialistic SOD). Mn-reconstituted enzyme showed an enzymatic reaction rate constant of 1.4 x 10(8) M-1 s-1 at pH 7.8. This rate only slightly increased with decreasing pH. Fe-reconstituted enzyme showed a rate constant of 2.7 x 10(7) M-1 s-1 at pH 7.8, but this rate increased with decreasing pH to become 1.7 x 10(8) M-1 s-1 at pH 5.7 with two pK values of 6.6 and 9.0. These results show that the metal specificity of the enzymatic activity of M. smegmatis superoxide dismutase shows manganese predominance at pH 7.8, but changes to be equal for either metal at acidic pH.

[Serum superoxide dismutase (SOD) activity in patients with renal disease by a spintrap method using electron spin resonance (ESR)].

Superoxide dismutase (SOD) activity in serum samples of patients with chronic glomerulonephritis (CGN) and chronic renal failure (CRF) was measured by a spin trap method using electron spin resonance (ESR). Twenty-three patients with CGN, 10 patients with CRF and 10 healthy adults were examined. Among 23 patients with CGN, there were 12 patients with IgA nephropathy and one patient with membranous nephropathy diagnosed by immunofluorescence of renal biopsy specimens. Other CGN patients were diagnosed by its clinical criteria. The serum activity of SOD in patients with CGN or CRF was significantly higher than those in healthy adults (p < 0.05). The serum SOD activity in patients with CRF was also higher than those in patients with CGN (p < 0.05). Marked high levels of serum SOD activity were observed histologically in the advanced stage of IgA nephropathy. These results suggest an increase in serum SOD activity may reflect renal injuries in patients with CGN and CRF.

Changes in free amino acid concentration in the hemolymph of the female Culex pipiens pallens (Diptera: Culicidae), after a blood meal.

The analysis of the free amino acids in the hemolymph of female Culex pipiens pallens L. indicated that asparagine, glutamine, glycine, leucine, serine, threonine, tyrosine, and valine noticeably increased in concentration during a blood meal digestion at 22 degrees C. The concentrations started to rise at about 4 h, reaching maximal level at 12-24 h, and then gradually returned to the prefeeding level by the fifth day after the blood meal. Before the sharp increase, the concentrations of asparagine, glutamine, glycine, serine, threonine, and tyrosine declined slightly during the first 1-2 h, probably because of hemolymph dilution by water absorbed from the blood meal. Histidine and lysine also showed a minor decrease soon after blood feeding, followed by a moderate but noticeable increase, delaying maximal concentrations until 48 h after the blood meal. Proline and alanine both exhibited a high hemolymph content and changed greatly, but with a large variation between the two samples analyzed. The changing pattern of individual hemolymph amino acids depended to a large extent upon the content of each amino acid in the blood meal given to the mosquitoes. Other free amino acids detected in the hemolymph were at very small concentrations and except for phenylalanine did not show any changes after a blood meal. The total free amino acid concentration was 50 nmol (or 6 micrograms) per microliters hemolymph before a blood meal, and increased to a maximal concentration of 88 nmol (or 11 micrograms) per microliters hemolymph at 18 h after the blood meal.

Purification and some properties of a 2Fe ferredoxin in Pseudomonas ovalis.

A [2Fe-2S] ferredoxin was found in Pseudomonas ovalis which was grown in a medium supplemented with glucose and ammonium sulfate. The molecular weight of the 2Fe ferredoxin was estimated to be 13,000. It contained 2.2 gramatoms of non-heme iron and 2.3 gramatoms of acid-labile sulfur per mole protein. The absorption and circular dichroism spectra were characteristic of those of [2Fe-2S] type ferredoxins, especially adrenodoxin and putidaredoxin. The electron paramagnetic resonance spectrum of the reduced protein showed an axial symmetry (g = 2.020, g = 1.939). The amino acid composition was determined.

Difference between amino acid residues in the metal-ligand environments of iron- and manganese-superoxide dismutases.

Alignment of the amino acid sequences of the Pseudomonas ovalis and Photobacterium leiognathi iron-superoxide dismutases (Fe-SODs) with the known sequences of the manganese-superoxide dismutases (Mn-SODs) shows that both types of SOD are highly homologous (33-53% identity) and share residues for the metal coordination. The amino acid residues that form the environment of the metal ions appear to be also conserved between the Fe- and Mn-SODs, except that the Phe-84 and Gln-154 in the Mn-SODs are replaced by Tyr and Ala, respectively, in the Fe-enzymes. Since this latter residue contributes to formation of the hydrophobic metal-ligand environment through hydrogen bonding with Trp-133 and Tyr-34 in the Mn-SODs, its substitution by Ala should cause different micro environments between the metal centers of the Fe- and Mn-SODs. This difference may account for the metal specificity of both types of SODs demonstrated by previous reconstitution experiments.

Amino acid sequence of iron-superoxide dismutase from Pseudomonas ovalis.

The amino acid sequence of iron-superoxide dismutase from Pseudomonas ovalis was deduced by the analyses of peptides derived from limited hydrolysis of the aminoethylated or pyridylethylated apoprotein with trypsin, Staphylococcus aureus V8 protease, and dilute acid hydrolysis. The polypeptide chain contains 195 amino acid residues and has a calculated Mr of 21,421. The sequence is highly homologous (65% identity) to the recently published sequence of the iron-superoxide dismutase from Photobacterium leiognathi. It is also homologous to the known sequences of the manganese-superoxide dismutase by sharing 33-53% identical residues. Alignment of the superoxide dismutase sequences and the available structural information from X-ray crystallography suggest that the ligands to the iron in the P. ovalis superoxide dismutase are His-26, His-74, Asp-156 and His-160, which align with the ligands to the manganese in the Thermus thermophilus manganese-superoxide dismutase. The sequence information of the P. ovalis dismutase will facilitate refinement of the X-ray crystallographic data that are now available at 2.9 A resolution.

Proton magnetic resonance studies of 7Fe ferredoxins. Three redox states of the [4Fe-4S] cluster in a Pseudomonas ovalis ferredoxin.

The oxidizability of a redox couple, [4Fe-4S], in a 7Fe ferredoxin extracted from Pseudomonas ovalis was monitored by 1H-NMR. The iron-sulfur cluster in the ferredoxin was not only reducible (Nagayama et al., 1983) but also oxidizable in its native form. This result provided the first verification of 3 redox states for a redox center in ferredoxin, 4Fe, in the native form of the protein.

Localization, isolation and characterization of Mn-superoxide dismutase in bovine adrenocortical cells.

Cyanide-insensitive superoxide dismutase activity was present in both cytosol (26%) and mitochondrial (64%) fractions of bovine adrenal cells. The cyanide-insensitive superoxide dismutase was isolated from the mitochondrial fraction. It contained 2.2 g atoms of manganese per mol of enzyme. The enzyme had a molecular weight of 82,000 and a subunit molecular weight of 22,000. The isoelectric point, amino acid composition, and spectra of visible and ultraviolet regions were similar to those of the Mn-superoxide dismutase from the other mitochondria.

Structure of iron superoxide dismutase from Pseudomonas ovalis at 2.9-A resolution.

The three-dimensional structure of the iron-containing superoxide dismutase (EC 1.15.1.1) from Pseudomonas ovalis has been determined at 2.9-A resolution by the method of multiple isomorphous replacement. The molecule is a dimer of two identical subunits with the iron atom per monomer. The conformation of the enzyme is completely different from that of the eukaryotic copper-zinc superoxide dismutase. Each subunit consists of about 50% alpha-helix plus three strands of antiparallel pleated sheet. The iron atoms are coordinated by four protein ligands, one of which is the side-chain of histidine-26. Crystals of complexes with the inhibitors azide or fluoride are considerably more resistant to irradiation than those of the free enzyme. The structure of the apoprotein is identical to that of the iron-containing molecule.

The iron content of iron superoxide dismutase: determination by anomalous scattering.

The number of iron atoms in the dimeric iron-containing superoxide dismutase from Pseudomonas ovalis and their atomic positions have been determined directly from anomalous scattering measurements on crystals of the native enzyme. To resolve the long-standing question of the total amount of iron per molecule for this class of dismutase, the occupancy of each site was refined against the measured Bijvoet differences. The enzyme is a symmetrical dimer with one iron site in each subunit. The iron position is 9 A from the intersubunit interface. The total iron content of the dimer is 1.2 +/- 0.2 moles per mole of protein. This is divided between the subunits in the ratio 0.65:0.55; the difference between them is probably not significant. Since each subunit contains, on average, slightly more than half an iron atom we conclude that the normal state of this enzyme is two iron atoms per dimer but that some of the metal is lost during purification of the protein. Although the crystals are obviously a mixture of holo- and apo-enzymes, the 2.9 A electron density map is uniformly clean, even at the iron site. We conclude that the three-dimensional structures of the iron-bound enzyme and the apo-enzyme are identical.