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PubMed1991

Lipid peroxidation in peribacteroid membranes from French-bean nodules.

Enriched peribacteroid membranes were prepared from Phaseolus vulgaris nodules and, in the presence of metleghemoglobin and H(2)O(2), membranal lipid peroxidation was observed. The initial rate of the reaction was low and increased with time. Ferrous leghemoglobin was unable to induce this peroxidation with H(2)O(2). Thus, it appears that leghemoglobin (IV) is not the activated species involved in this process. Heme plays a role in this peroxidation and the hydroxyl radical is not an intermediate of the reaction. Lipid peroxidation in peribacteroid membranes was also observed in the presence of iron ions. A mixture of iron (III) and iron (II) produced a maximal peroxidation. Senescing nodule extracts were able to provoke membranal lipid peroxidation; they contained nonprotein-bound iron. Peribacteroid membranes were more sensitive than microsomes to peroxidation, as measured by malonaldehyde formation.

Journal Article↗
PubMed1990

Nonlegume hemoglobin genes retain organ-specific expression in heterologous transgenic plants.

Hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa have been isolated [Landsmann et al. (1986). Nature 324, 166-168; Bogusz et al. (1988). Nature 331, 178-180]. The promoters of these genes have been linked to a beta-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus. Both promoters directed root-specific expression in transgenic tobacco. When transgenic Lotus plants were nodulated by Rhizobium loti, both promoter constructs showed a high level of nodule-specific expression confined to the central bacteroid-containing portion of the nodule corresponding to the expression seen for the endogenous Lotus leghemoglobin gene. The T. tomentosa promoter was also expressed at a low level in the vascular tissue of the Lotus roots. The hemoglobin promoters from both nonlegumes, including the non-nodulating species, must contain conserved cis-acting DNA signals that are responsible for nodule-specific expression in legumes. We have identified sequence motifs postulated previously as the nodule-specific regulatory elements of the soybean leghemoglobin genes [Stougaard et al. (1987). EMBO J. 6, 3565-3569].

Base Sequence↗
PubMed1987

Tn5-induced cytochrome mutants of Bradyrhizobium japonicum: effects of the mutations on cells grown symbiotically and in culture.

Two Bradyrhizobium japonicum cytochrome mutants were obtained by Tn5 mutagenesis of strain LO and were characterized in free-living cultures and in symbiosis in soybean root nodules. One mutant strain, LO501, expressed no cytochrome aa3 in culture; it had wild-type levels of succinate oxidase activity but could not oxidize NADH or N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The cytochrome content of LO501 root nodule bacteroids was nearly identical to that of the wild type, but the mutant expressed over fourfold more bacteroid cytochrome c oxidase activity than was found in strain LO. The Tn5 insertion of the second mutant, LO505, had a pleiotropic effect; this strain was missing cytochromes c and aa3 in culture and had a diminished amount of cytochrome b as well. The oxidations of TMPD, NADH, and succinate by cultured LO505 cells were very similar to those by the cytochrome aa3 mutant LO501, supporting the conclusion that cytochromes c and aa3 are part of the same branch of the electron transport system. Nodules formed from the symbiosis of strain LO505 with soybean contained no detectable amount of leghemoglobin and had no N2 fixation activity. LO505 bacteroids were cytochrome deficient but contained nearly wild-type levels of bacteroid cytochrome c oxidase activity. The absence of leghemoglobin and the diminished bacterial cytochrome content in nodules from strain LO505 suggest that this mutant may be deficient in some aspect of heme biosynthesis.

Cytochromes↗
PubMed2002

Molecular cloning, functional characterization, and subcellular localization of soybean nodule dihydrolipoamide reductase.

Nodule ferric leghemoglobin reductase (FLbR) and leaf dihydrolipoamide reductase (DLDH) belong to the same family of pyridine nucleotide-disulfide oxidoreductases. We report here the cloning, expression, and characterization of a second protein with FLbR activity, FLbR-2, from soybean (Glycine max) nodules. The cDNA is 1,779 bp in length and codes for a precursor protein comprising a 30-residue mitochondrial transit peptide and a 470-residue mature protein of 50 kD. The derived protein has considerable homology with soybean nodule FLbR-1 (93% identity) and pea (Pisum sativum) leaf mitochondria DLDH (89% identity). The cDNA encoding the mature protein was overexpressed in Escherichia coli. The recombinant enzyme showed Km and kcat values for ferric leghemoglobin that were very similar to those of DLDH. The transcripts of FLbR-2 were more abundant in stems and roots than in nodules and leaves. Immunoblots of nodule fractions revealed that an antibody raised against pea leaf DLDH cross-reacted with recombinant FLbR-2, native FLbR-2 of soybean nodule mitochondria, DLDH from bacteroids, and an unknown protein of approximately 70 kD localized in the nodule cytosol. Immunogold labeling was also observed in the mitochondria, cytosol, and bacteroids of soybean nodules. The similar biochemical, kinetic, and immunological properties, as well as the high amino acid sequence identity and mitochondrial localization, draw us to conclude that FLbR-2 is soybean DLDH.

Amino Acid Sequence↗
PubMed1978

Acid and alkaline forms of the higher oxidation state of kangaroo, horse, and sperm whale myoglobin.

Myoglobin(IV), the derivative of myoglobin at the formal oxidation state IV, prepared from kangaroo (Megaleia rufa), horse, or sperm whale myoglobin, when cooled to liquid nitrogen temperature, assumes acid and alkaline forms with different optical spectra. The essential features of the optical spectra of the acid forms are the same as those of leghemoglobin(IV) and are very similar to those of optical spectra of the red higher oxidation states of catalases and peroxidases. This shows that the configuration of the heme iron is the same throughout these compounds. That configuration is believed to be Fe(IV) in a porphyrin environment. The optical spectra of alkaline mammalian myoglobin(IV), like that of alkaline leghemoglobin(IV), resemble those of the alkaline low spin ferric proteins. Kangaroo myoglobin(IV) may be prepared by reaction of ferrous myoglobin with hydrogen peroxide. The acid forms of myoglobin(IV) are conveniently prepared by cooling solutions in borate buffers, initially pH 8.3, to liquid nitrogen temperature. At this temperature borate buffers become acidic.

Animals↗
PubMedDate not supplied

[Contribution of protein conformation to stereochemistry and reactivity of the active center of heme proteins and enzymes. The existence of horseradish peroxidase conformations and their possible role in the catalysis mechanism].

In the spectral region 350-800 nm at 4.2 K we measured magnetic circular dichroism (MCD) spectra of the pentacoordinated complex of protcheme with 2-methylimidazole, deoxyleghemoglobin, neutral and alkaline forms of reduced horseradish peroxidase in the equilibrium states, as well as in non-equilibrium states produced by low-temperature photolysis of their carbon monoxide derivatives. Earlier the corresponding results have been obtained for myoglobin, hemoglobin and cytochromes P-450 and P-420. The energies of Fe-N (proximal His) and Fe-N(pyrroles) bonds and their changes upon ligand binding in heme proteins and enzymes were compared with those in the model heme complex thus providing conformational contribution into stereochemistry of the active site. The examples of weak and strong conformational "pressure" on stereochemistry were analysed and observed. If conformational energy contribution into stereochemistry prevails the electronic one the heme stereochemistry remains unchanged on ligand binding as it was observed for leghemoglobin and alkaline horseradish peroxidase. The change of bond energies in myoglobin and hemoglobin on ligand binding are comparable with those in protein free pentacoordinated protoheme, giving an example of weak conformational contribution to heme stereochemistry. The role of protein conformation energy in the modulation of ligand binding properties of heme in leghemoglobin relative to those in myoglobins is discussed. The most striking result were obtained in the study of reduced horseradish peroxidase in the pH region of 6.0-10.2. It was found that such different perturbations as ligand binding and heme-linked ionization of the distal amino acid residue induce identical changes in heme stereochemistry. Neither heme-linked ionization in the carbon monoxide complex nor the geometry of Fe-Co bond affect the heme local structure of photoproducts. These and other findings suggest a very low conformation mobility of horseradish peroxidase whose protein constraints appear to allow only two preferable geometries of specific amino acid residues that form the heme pocket. The role of the two tertiary structure constraints on the heme in the mechanism of horseradish peroxidase function is discussed. It is supposed that one conformation produces a heme environment suitable for two-electron oxidation of the native enzyme to compound I by hydrogen peroxide while another conformation changes the heme stereochemistry in the direction favourable for back reduction of compound I by the substrate to the resting enzyme through two one-electron steps. The switch from one tertiary structure to another is expected to be induced by substrate bind

Binding Sites↗
PubMed1989

The oxygen dependency of the redox state of heme and copper in cytochrome oxidase in vitro.

The oxidation-reduction state of cytochrome oxidase in isolated mitochondria at low oxygen concentrations was measured by the use of leghemoglobin as an oxygen indicator. P50 a + a3 varied with energy state as well as the respiratory rate. In contrast to heme a + a3, copper was slower to reduce than heme a + a3. The P50Cu of 8 x 10(-8)M in State 4 and 7.4 x 10(-8)M in State 3 was independent of both the energy state and the respiratory rate.

Aerobiosis↗
PubMed1994

A dioxygenase gene (Ids2) expressed under iron deficiency conditions in the roots of Hordeum vulgare.

A lambda zapII cDNA library was constructed from mRNA isolated from Fe-deficient barley roots and screened with cDNA probes made from mRNA of Fe-deficient and Fe-sufficient (control) barley roots. Seven clones were selected. Among them a clone having the putative full-length mRNA of dioxygenase as judged by northern hybridization was selected and named Ids2 (iron deficiency-specific clone 2). Using a cDNA fragment as probe, two clones from the genomic library (lambda EMBL-III) were isolated and one was sequenced. The predicted amino acid sequence of Ids2 resembled that of 2-oxoglutarate-dependent dioxygenase. Ids2 is expressed in the Fe-deficient barley roots but is not in the leaves. The expression is repressed by the availability of Fe. Ids2 was also strongly expressed under Mn deficiency and weakly under Zn deficiency or excess NaCl (0.5%). The upstream 5'-flanking region of Ids2 has a root-specific cis element of the CaMV 35S promoter and a nodule-specific element of leghemoglobin, a metal regulatory element (MRE) and several Cu regulatory elements (UAS) of yeast metallothionein (CUP1).

Amino Acid Sequence↗
PubMed1995

Expression of nodulin genes in plant-determined ineffective nodules of pea.

The mutant E135 (sym 13) of pea (Pisum sativum L.) forms a normal number of small white nodules that contain bacteroids, but these bacteroids lack nitrogenase activity. To evaluate the effects of the sym 13 gene on the expression of nodulin genes, cDNA clones for nodulins were isolated from pea nodules and the expression of nodulin genes in ineffective E135 nodules was compared with that in nitrogen-fixing nodules on the wild-type parent, cv. Sparkle. Nineteen cDNA clones for nodulins, including ENOD2 and cDNAs for two distinct leghemoglobins (Lbs), were isolated from Sparkle nodules by a subtractive hybridization procedure. All the nodulin genes examined were expressed in nodules on both E135 and Sparkle plants. However, the level of expression of seven genes, one of which was an Lb gene that corresponded to PsN5, was significantly lower in E135 nodules. The levels of Lb apo-proteins, with the exception of Lb-III and Lb-IV, in E135 nodules resembled those in Sparkle nodules, but the level of heme in E135 nodules was lower than that in Sparkle nodules. Although the expression of the two Lb genes that corresponded to PsN5 and PsN120 in E135 nodules was slightly depressed by exogenous ammonia, the level of the PsN5 transcript was still lower than the control level in Sparkle nodules. Our results indicate that the plant gene sym 13 does not influence the induction of nodulin genes but does influence the level of the expression of some genes, one of which is a gene for Lb, as well as the level of heme.

Ammonia↗
PubMed1990

Characterization of a novel nodulin gene in soybean that shares sequence similarity to the gene for nodulin-24.

A gene encoding for nodulin-16 (N-16) was isolated from a soybean genomic library. Nucleotide sequence analysis of the cDNA and the genomic clone of N-16 indicated that the coding region of this gene is 330 bp long and is interrupted by a single intron of 494 bp. The coding region of the N-16 gene shows a high degree of localized sequence similarity with the coding sequence of soybean nodulin-24 (N-24). Sequence similarity between the two genes is limited to the coding region of 90 bp in the first exon and the first 54 bp in the second exon of the N-16 gene which is repeated as the 2nd, 3rd, and 4th exons in the N-24 gene. The N-24 gene has been postulated to be a result of repeated duplication of an insertion element consisting of the 54 bp exon and the flanking intron sequences. In the absence of sequence similarity in the regions flanking the 54 bp sequence between the N-16 and N-24 genes, the N-16 gene does not appear to be the ancestral gene. Both N-16 and N-24 have a similar hydrophobic amino terminal end suggesting that N-16 like N-24 is targeted to the peribacteroid membrane. Southern analysis of soybean genomic DNA shows the presence of other related sequences to the N-16 gene, one of which is found to be closely linked to it. Analysis of the temporal accumulation of the N-16 transcripts during nodule development in effective and ineffective nodules suggests that N-16 and related genes might differ from leghemoglobin and some other late nodulin genes in their mechanism of regulation.

Amino Acid Sequence↗
PubMed2003

The Lotus japonicus Sen1 gene controls rhizobial differentiation into nitrogen-fixing bacteroids in nodules.

A Lotus japonicus mutant, Ljsym75, which forms ineffective symbiotic nodules and defines a new locus involved in the process of nitrogen fixation, was characterized in detail in order to identify the stage of developmental arrest of the nodules. No nitrogen-fixing activity was detectable in Ljsym75 nodules at any stage during plant development, and plant growth was markedly retarded. Ljsym75 plants formed twice as many nodules as the wild-type Gifu, and this phenotype was not influenced by the application of low concentrations of nitrate. Although the ineffective nodules formed on Ljsym75 were anatomically similar to effective Gifu nodules, Ljsym75 nodules senesced prematurely. Microscopic examination revealed that bacteria endocytosed into Ljsym75 nodules failed to differentiate into bacteroids. Moreover, the bacteria contained no nitrogenase proteins, whereas leghemoglobin was detected in the cytosol of the nodules. These results indicate that Ljsym75 is required for bacterial differentiation into nitrogen-fixing bacteroids in nodules, and thus the Ljsym75 gene was renamed sen1 (for stationary endosymbiont nodule). Linkage analysis using DNA markers showed that Sen1 is located on chromosome 4.

Chromosome Mapping↗
PubMed2005

Kinetics and mechanistic studies of the reactions of metleghemoglobin, ferrylleghemoglobin, and nitrosylleghemoglobin with reactive nitrogen species.

It is now established that nitrogen monoxide is produced not only in animals but also in plants. However, much less is known about the pathways of generation and the functions of NO. in planta. One of the possible targets of NO. is leghemoglobin (Lb), the hemoprotein found in high concentrations in the root nodules of legumes that establish a symbiosis with nitrogen-fixing bacteria. In analogy to hemoglobin and myoglobin, we have shown that different forms of Lb react not only with NO. but also with so-called reactive nitrogen species derived from it, among others peroxynitrite and nitrite. Because of the wider active-site pocket, the rate constants measured in this work for NO. and for nitrite binding to metLb are 1 order of magnitude larger than the corresponding values for binding of these species to metmyoglobin and methemoglobin. Moreover, we showed that reactive nitrogen species are able to react with two forms of Lb that are produced in vivo but that cannot bind oxygen: ferrylLb is reduced by NO. and nitrite, and nitrosylLb is oxidized by peroxynitrite. The second-order rate constants of these reactions are on the order of 10(2), 10(6), and 10(5) M-1 s-1, respectively. In all cases, the final reaction product is metLb, a further Lb form that has been detected in vivo. Since a specific reductase is active in nodules, which reduces metLb, reactive nitrogen species could contribute to the recycling of these inactive forms to regenerate deoxyLb, the oxygen-binding form of Lb.

Carbon Dioxide↗
PubMed1979

Development of the nitrogen-fixing and protein-synthesizing apparatus of bacteroids in pea root nodules.

Some aspects of root nodule development of Pisum sativum inoculated with Rhizobium leguminosarum were examined. 1. Nitrogenase activity (measured as acetylene reduction) appears to be preceded by leghemoglobin synthesis (measured immunologically). 2. Syntheses of component I and component II of nitrogenase are not strictly coordinated. Synthesis of component I starts before component II. 3. Plant and bacteroid protein synthesis (measured by [35S]sulfate labeling) in root nodules declines rapidly during nodule development. Corresponding with this decline is a decrease in quantity and quality of rRNA.

Fabaceae↗
PubMed1987

1H resonances of proximal histidine in CO complexes of hemoglobins provide a sensitive probe of coordination geometry.

A straightforward strategy for assignment of the C epsilon H, C delta H and N delta H proton resonances of the proximal histidine ligand in diamagnetic complexes of monomeric hemoglobins and myoglobins is reported. These resonances are subject to large ring current shifts and are highly sensitive to coordination geometry. There are no significant differences between the CO complexes of myoglobin, leghemoglobin or hemoglobin alpha-subunits in proximal His coordination geometry or hydrogen bonding to the backbone at Leu F4. Ring current calculations show that the His F8 coordination geometry in the CO complexes of myoglobin and hemoglobin alpha-subunits is very similar in crystal and solution.

Carbon Monoxide↗
PubMedDate not supplied

Parasitic origins of nitrogen-mixing Rhizobium-legume symbioses. A review of the evidence.

This paper is divided into two sections. The first part (I) reviews the literature on the legume-Rhizobium association with emphasis on the processes leading to the establishment of the association. In the second part (II) it is proposed that the legume-Rhizobium association was originally necrotrophic , beginning when the free-living, nitrogen-fixing, saprotrophic Rhizobium developed the ability to infect the plant. The pre-infection events, infection processes and nodulation in the colonization of the legumes by the Rhizobium are similar to those of other parasitic associations. Likewise, the host responses to the Rhizobium entry, infection thread synthesis and bacteroid formation are comparable to those of other plants when they encounter phytopathogens . Evolutionary processes acted in the selection of biotrophy , the fine control and regulation of the extracellular enzymes of the necrotrophic Rhizobium converted the association into biotrophy . The nutritional dependence of the Rhizobium on the legume, the requirement of the plant for combined nitrogen and the Rhizobium potential to meet this requirement drove the biotrophic association into mutualism . This became possible when regulation of the nitrogen-fixing system of the Rhizobium was modified and the oxygen carrying protein leghemoglobin was acquired or evolved by the legume to enhance nitrogen fixation.

Biological Evolution↗
PubMed1986

Heme protein fluorescence versus pressure.

Fluorescence spectra of several ferric heme proteins have been measured vs. pressure to 6,000 bars. Sperm whale myoglobin (SW Mb), Aplysia myoglobin, leghemoglobin (Lb), and cytochrome P450 all show excitation and emission spectra characteristic of tryptophan in proteins with peak emission at 330-340 nm. At one bar, the fluorescence is weak due to energy transfer to the heme group, which makes the yield a sensitive probe of protein unfolding at high pressure. After an initial decrease of a few percent per kbar, the protein shows a large increase in fluorescence at high pressure. The increase is pH dependent and the results indicate that several high pressure states occur. For SW Mb at 15 degrees C an increase of a factor of 20 occurs with midpoint at 2,000 bars at pH 5 and is only partially reversible, while the increase at pH 7 occurs at 4,000 bars and is only half as large and is completely reversible. Aplysia Mb and Lb show a similar effect, but unfold at a higher pressure than SW Mb. P450 also shows a transition to a state of higher fluorescence, but the transition in this case is irreversible as a stable form, P420, is formed. The fluorescence intensity measurements permit an estimation of the increase in the TRY-heme distance in the high pressure state.

Animals↗
PubMed1997

An application of NMR microimaging to investigate nitrogen fixing root nodules.

Various techniques to obtain high-resolution NMR images (voxel size down to 39 x 39 x 250 microns) of nitrogen-fixing root nodules from soybean [Glycine max (Merr.)] and peanut (Arachis hypogaea L.) are compared. We describe the artefacts arising from changes in the magnetic susceptibility throughout the sample and how these can be minimised. A series of T1 (TR = 220 to 3020 ms) and T2 (TE = 9.3 to 33.6 ms) weighted images are presented. From these it has been possible to locate the oxygen diffusion barrier. A possible interpretation in terms of nodule biochemistry and physiology are given. The data and parameters presented are shown to serve as a basis for more extensive investigations of root nodules (e.g., the oxygen diffusion barrier or the mechanisms driving the regulation of the oxygen concentration in the infected zone by leghemoglobin) by NMR microimaging.

Arachis↗
PubMed2003

Plants, humans and hemoglobins.

New developments have forced a re-evaluation of our understanding of the structure and function of hemoglobins. Leghemoglobins regulate oxygen affinity through a mechanism different from that of myoglobin using a novel combination of heme pocket amino acids that lower the oxygen affinity. The hexacoordinate hemoglobins are characterized by intramolecular coordination of the ligand binding site at the heme iron, and were first identified in plants as the 'non-symbiotic plant hemoglobins'. They are now known to be present in animals and bacteria. Many of these proteins are upregulated in both plants and animals during hypoxia or similar stresses. Therefore, there might be a common physiological function for hexacoordinate hemoglobins in plants and animals.

Animals↗