Permanent cardiac pacing in elderly patients with recurrent falls, dizziness and syncope, and a hypersensitive cardioinhibitory reflex.

The study was designed to assess the outcome of treatment with permanent dual-chamber pacing of elderly patients with falls, dizziness and syncope associated with the demonstration of a hypersensitive cardioinhibitory reflex. Questionnaires were sent to patients (and their general practitioners) who had been referred to a regional pacing centre with recurrent falls, dizziness or syncope diagnosed as likely to be secondary to cardioinhibitory carotid sinus syndrome or predominantly cardioinhibitory vasovagal syndrome. After pacemaker insertion, 84% of patients had no further syncope over a mean follow-up period of 10 (range 1.5 to 30) months. Minor symptoms persisted in only 40% of all patients. Symptoms were unchanged in 22%. It was concluded that permanent dual-chamber pacing is an effective treatment for elderly patients with recurrent falls, dizziness and syncope in whom a hypersensitive cardioinhibitory reflex is found. Good results were obtained in this group with a simple diagnostic work-up.

Structure of ferric soybean leghemoglobin a nicotinate at 2.3 A resolution.

Soybean leghemoglobin a is a small (16 kDa) protein facilitating the transport of O(2) to respiring N(2)-fixing bacteria at low free-O(2) tension. The crystal structure of soybean ferric leghemoglobin a nicotinate has been refined at 2.3 A resolution. The final R factor is 15.8% for 6877 reflections between 6.0 and 2.3 A. The structure of soybean leghemoglobin a (143 residues) is closely similar to that of lupin leghemoglobin II (153 residues), the proteins having 82 identical residues when the sequences are aligned. The new structure provides support for the conclusion that the unique properties of leghemoglobin arise principally from a heme pocket considerably larger and more flexible than that of myoglobin, a strongly ruffled heme group, and a proximal histidine orientation more favourable to ligand binding.

A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum.

It has been a long-standing hypothesis that the endosymbiotic rhizobia (bacteroids) cope with a concentration of 10 to 20 nM free O2 in legume root nodules by the use of a specialized respiratory electron transport chain terminating with an oxidase that ought to have a high affinity for O2. Previously, we suggested that the microaerobically and anaerobically induced fixNOQP operon of Bradyrhizobium japonicum might code for such a special oxidase. Here we report the biochemical characteristics of this terminal oxidase after a 27-fold enrichment from membranes of anaerobically grown B. japonicum wild-type cells. The purified oxidase has TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity as well as cytochrome c oxidase activity. N-terminal amino acid sequencing of its major constituent subunits confirmed that presence of the fixN,fixO, and fixP gene products. FixN is a highly hydrophobic, heme B-binding protein. FixO and FixP are membrane-anchored c-type cytochromes (apparent Mrs of 29,000 and 31,000, respectively), as shown by their peroxidase activities in sodium dodecyl sulfate-polyacrylamide gels. All oxidase properties are diagnostic for it to be a member of the cbb3-type subfamily of heme-copper oxidases. The FixP protein was immunologically detectable in membranes isolated from root nodule bacteroids, and 85% of the total cytochrome c oxidase activity in bacteroid membranes was contributed by the cbb3-type oxidase. The Km values for O2 of the purified enzyme and of membranes from different B. japonicum wild-type and mutant strains were determined by a spectrophotometric method with oxygenated soybean leghemoglobin as the sole O2 delivery system. The derived Km value for O2 of the cbb3-type oxidase in membranes was 7 nM, which is six- to eightfold lower than that determined for the aerobic aa3-type cytochrome c oxidase. We conclude that the cbb3-type oxidase supports microaerobic respiration in endosymbiotic bacteroids.

Bradyrhizobium japonicum cytochrome c550 is required for nitrate respiration but not for symbiotic nitrogen fixation.

Bradyrhizobium japonicum possesses three soluble c-type cytochromes, c550, c552, and c555. The genes for cytochromes c552 (cycB) and c555 (cycC) were characterized previously. Here we report the cloning, sequencing, and mutational analysis of the cytochrome c550 gene (cycA). A B. japonicum mutant with an insertion in cycA failed to synthesize a 12-kDa c-type cytochrome. This protein was detectable in the cycA mutant complemented with cloned cycA, which proves that it is the cycA gene product. The cycA mutant, a cycB-cycC double mutant, and a cycA-cycB-cycC triple mutant elicited N2-fixing root nodules on soybean (Nod+ Fix+ phenotype); hence, none of these three cytochromes c is essential for respiration supporting symbiotic N2 fixation. However, cytochrome c550, in contrast to cytochromes c552 and c555, was shown to be essential for anaerobic growth of B. japonicum, using nitrate as the terminal electron acceptor.

Characterization of three soluble c-type cytochromes isolated from soybean root nodule bacteroids of Bradyrhizobium japonicum strain CC705.

Three soluble, low molecular mass cytochromes c (Mr 8000-15,000) were isolated and purified from soybean root nodule bacteroids of Bradyrhizobium japonicum strain CC705. On the basis of their alpha: absorbance peaks in the reduced forms, they were named cytochromes c550, c552 and c555. Cytochrome c552 reacted very fast, c555 very slowly and c550 not at all with carbon monoxide. The complete amino acid sequence (73 residues) of cytochrome c552 was established which identifies it as a monoheme, class I cytochrome c with some remote similarity to the cytochrome c6 family.

Cloning, sequencing and mutational analysis of the cytochrome c552 gene (cycB) from Bradyrhizobium japonicum strain 110.

We report the cloning and nucleotide sequence analysis of the cytochrome c552 gene (cycB) of Bradyrhizobium japonicum strain 110. The gene was identified with help of an oligonucleotide that was designed on the basis of the amino acid sequence determined for purified cytochrome c552 of B. japonicum strain CC705. The cycB gene product has an N-terminal 23-amino acid signal peptide that is missing in the mature cytochrome c552 protein. A B. japonicum cycB insertion mutant was constructed which had no observable phenotypic defects in denitrification and symbiotic nitrogen fixation. Thus, the function of c552 remains unknown.

Effect of pO(2) on the Formation and Status of Leghemoglobin in Nodules of Cowpea and Soybean.

Nodulated cowpea (Vigna unguiculata [L.] Walp. cv Vita 3: Bradyrhizobium strain CB756) and soybean (Glycine max [L.] Merr. cv White Eye: Bradyrhizobium strain CB1809) were grown with their root systems maintained in a flowing gas stream containing a range of pO(2) (1-80%, v/v) in N(2) for up to 28 days after planting. At the extremes of sub- and supra-ambient pO(2), the levels of leghemoglobin (Lb) in nodules were reduced. However, neither the proportional composition of Lb component proteins (eight in soybean, three in cowpea) nor their oxidation state was affected by pO(2). Short-term changes in pO(2) (transferring plants grown with sub- or supra-ambient pO(2) in the rhizosphere to air or vice versa) caused a significant decline in Lb content and, in cowpea but not soybean, where pO(2) was increased, a higher percentage of oxidation of Lb. Combining data on changes in Lb level of cowpea nodules grown in sub-ambient pO(2) with those for their structural adaptation to an under supply of O(2) indicated that, despite the nodules having a lower level of Lb, the amount per infected cell was increased by up to twofold and per bacteroid up to fivefold (in those from 1% O(2)) compared to those grown in air. Progressive decline in pO(2) resulted in a progressive increase on this basis, indicating a close relationship between Lb content and the adaptation of nodule functioning to external O(2) level.

Characterization of a soluble catalase-peroxidase hemoprotein b-590, previously identified as 'cytochrome alpha 1' from Bradyrhizobium japonicum bacteroids.

The cytochrome "a1" or P-428, previously proposed to be a high affinity terminal oxidase in nitrogen-fixing bacteroids of Bradyrhizobium japonicum has been purified. The water-soluble native hemoprotein has an Mr of 136,000, lacks heme a and is a high-spin ferric protohemoprotein: It is slowly reduced with dithionite to give a species with an optical spectrum like that of hemoprotein b-590 (Escherichia coli; peak at 555 nm, shoulder at 590 nm), and which reacts slowly with CO. It has catalase and peroxidase activities, again resembling the E. coli b-590. Neither hemoprotein forms a stable oxy complex under conditions in which dithionite-reduced horseradish peroxidase reacts with oxygen to form such a complex. The hemoprotein, which we name hemoprotein b-590 (Bradyrhizobium japonicum), may play a role in removal of peroxides generated during respiration in the bacteroids of several Rhizobium and Bradyrhizobium species. The high-affinity terminal oxidase under nitrogen-fixing conditions remains to be identified.

Nicotinate, nicotinamide, and the reactivity of leghemoglobin in soybean root nodules.

Nicotinate has been postulated to interfere with the binding of O(2) to ferrous leghemoglobin in soybean (Glycine max) root nodules. For such a function, the levels of nicotinate in nodules must be sufficiently high to bind a significant amount of leghemoglobin. We have measured levels of nicotinate, nicotinamide, and leghemoglobin in soybean nodules from plants 34 to 73 days after planting in a glasshouse. On a per gram nodule fresh weight basis, levels between 10.4 and 21 nanomoles for nicotinate, 19.2 and 37.8 nanomoles for nicotinamide, and 170 to 280 nanomoles for leghemoglobin were measured. Even if all the nicotinate were bound to ferrous leghemoglobin, only 11% or less of the total leghemoglobin would be unavailable for binding O(2). Using the measured levels of nicotinate and a pH of 6.8 in the cytosol of presenescent soybean nodules, we estimate that the proportion of ferrous leghemoglobin bound to nicotinate in such nodules would be less than 1%. These levels of nicotinate are too low to interfere with the reaction between ferrous leghemoglobin and O(2) in soybean root nodules.

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].

The kinetics of ligand binding to plant hemoglobins. Structural implications.

The rates of reaction of oxygen, carbon monoxide, and nitric oxide with 14 plant hemoglobins have been determined by relaxation and stopped-flow methods. The combination rates for oxygen lie between 0.12 and 0.26 x 10(9)/M.s, for carbon monoxide between 0.01 and 0.07 x 10(9)/M.s, and for nitric oxide between 0.12 and 0.25 x 10(9)/M.s. The dissociation velocities for oxygen range from 5 to 25/s, and for CO from 0.005 to 0.011 s. The oxygen dissociation constants range only from 36 to 78 nM. Nanosecond relaxation experiments show large differences between the proteins. Five have known primary structures which correlate closely with the nanosecond relaxations and less immediately with the millisecond reactions. The relevant amino acid substitutions are concentrated in the C-E interhelical region.

NMR studies of the conformations of leghemoglobins from soybean and lupin.

Phase-sensitive two-dimensional NMR methods have been used to obtain extensive proton resonance assignments for the carbon monoxide complexes of lupin leghemoglobins I and II and soybean leghemoglobin a. The assigned resonances provide information on the solution conformations of the proteins, particularly in the vicinity of the heme. The structure of the CO complex of lupin leghemoglobin II in solution is compared with the X-ray crystal structure of the cyanide complex by comparison of observed and calculated ring current shifts. The structures are generally very similar but significant differences are observed for the ligand contact residues, Phe30, His63 and Val67, and for the proximal His97 ligand. Certain residues are disordered and adopt two interconverting conformations in lupin leghemoglobin II in solution. The proximal heme pocket structure is closely conserved in the lupin leghemoglobins I and II but small differences in conformation in the distal heme pocket are apparent. Larger conformational differences are observed when comparisons are made with the CO complex of soybean leghemoglobin. Altered protein-heme packing is indicated on the proximal side of the heme and some conformational differences are evident in the distal heme pocket. The small conformational differences between the three leghemoglobins probably contribute to the known differences in their O2 and CO association and dissociation kinetics. The heme pocket conformations of the three leghemoglobins are more closely related to each other than to sperm whale myoglobin. The most notable differences between the leghemoglobins and myoglobin are: (a) reduced steric crowding of the ligand binding site in the leghemoglobins, (b) different orientations of the distal histidine, and (c) small but significant differences in proximal histidine coordination geometry. These changes probably contribute to the large differences in ligand binding kinetics between the leghemoglobins and myoglobin.

Amino acid sequences of hemoglobins I and II from root nodules of the non-leguminous Parasponia rigida-rhizobium symbiosis, and a correction of the sequence of hemoglobin I from Parasponia andersonii.

The amino acid sequence of hemoglobins I (pI 6.15 as oxyhemoglobin) and II (pI 5.64 as oxyhemoglobin) from the nitrogen-fixing root nodules of Parasponia rigida have been determined by protein sequencing. The sequence of hemoglobin I (pI 6.16, as oxyhemoglobin) from Parasponia andersonii was re-examined and the corrected primary structure, now in agreement with that predicted from the DNA sequence, is reported. The three Parasponia hemoglobins contain 161 amino acid residues (Mr approximately equal to 18,700 including the heme) with a single cysteine residue and five methionine residues. The N-terminal serine is blocked by an acetyl group. The primary structure of the Parasponia hemoglobins is highly conserved. Hemoglobins I from the two species of Parasponia are identical; both show microheterogeneity at position 30 (Asp/Glu substitution) and hemoglobin I from P. rigida shows microheterogeneity at position 150 (Ala/Val) while hemoglobin I from P. andersonii has only an Ala at 150. P. rigida hemoglobin II shows no microheterogeneity at these positions, having Asp and Val residues respectively, and it contains a single amino acid change of a Gln for an Arg at position 85, which accounts for the 0.5 unit difference in isoelectric point observed between hemoglobins I and II. The sequence data are consistent with allelic heterogeneity at a single locus rather than different genes.

Functioning haemoglobin genes in non-nodulating plants.

Haemoglobin has previously been recorded in plants only in the nitrogen-fixing nodules formed by symbiotic association between Rhizobium or Frankia and legume or non-legume hosts. Structural similarities amongst these and animal haemoglobins at the protein and gene level suggested a common evolutionary origin. This suggests that haemoglobin genes, inherited from an ancestor common to plants and animals, might be present in all plants. We report here the isolation of a haemoglobin gene from Trema tomentosa, a non-nodulating relative of Parasponia (Ulmaceae). The gene has three introns located at positions identical to those in the haemoglobin genes of nodulating plant species, strengthening the case for a common origin of all plant haemoglobin genes. The data argue strongly against horizontal haemoglobin gene transfer from animals to plants. The Trema gene has a tissue-specific pattern of transcription and translation, producing monomeric haemoglobin in Trema roots. We have also found that the Parasponia haemoglobin gene is transcribed in roots of non-nodulated plants. These results suggest that haemoglobin has a role in the respiratory metabolism of root cells of all plant species. We propose that its special role in nitrogen-fixing nodules has required adaptation of the haemoglobin-gene regulation pathway, to give high expression in the specialized environment of the nodule.

Sesbania rostrata root and stem nodule leghemoglobins: purification, and relationships among the seven major components.

By anion-exchange chromatography, the nitrogen fixing photosynthetic stem nodules and nonphotosynthetic root nodules of Sesbania rostrata are shown to contain the same seven major components of leghemoglobin (Lb), numbered LbI-LbVII in order of elution, although in different proportions. No novel component was found in photosynthetic nodules. All components of Sesbania Lb are monomeric, with molecular weights varying between 15,000 and 17,000, and at least six of them are separate gene products. It is suspected that variable conjugation with nonprotein moieties might be partially responsible for the molecular weight differences and anomalous behavior observed between isoelectric focusing and anion-exchange chromatography.

The kinetics of the reactions of Parasponia andersonii hemoglobin with oxygen, carbon monoxide, and nitric oxide.

Hemoglobin I was isolated from nodules formed on the roots of Parasponia andersonii inoculated with Rhizobium strain CP 283. The rate of oxygen dissociation from Parasponia hemoglobin increases about 12-fold between pH 4 and 7, with apparent pK 6.4, to reach a limiting value of 14.8s-1. The optical spectrum of oxyhemoglobin in the visible region is also dependent on pH with pK near 6.4. The rate constant for oxygen combination with Parasponia hemoglobin increases about 7-8-fold between pH 4 and 7, with apparent pK 5.37, to reach a value of 1.67 X 10(8) M-1 s-1 at pH 7. The optical spectrum of deoxyhemoglobin in the visible region and the rate constant for carbon monoxide combination are also dependent on pH with apparent pK 5.65 and 5.75, respectively. The rate constant for carbon monoxide dissociation is independent of pH. The oxygen affinity of Parasponia hemoglobin, P50 = 0.049 torr at 20 degrees C, calculated from the kinetic constants at pH 7, is very great. At alkaline pH there is a prominent geminate reaction with oxygen and nitric oxide, with both subnanosecond and tens of nanosecond components. These reactions disappear at acid pH, with pK 6.4, and the effective quantum yield is reduced. In general, the reactions of Parasponia hemoglobin with oxygen and carbon monoxide resemble those of soybean leghemoglobin. In each, great oxygen affinity is achieved by unusually rapid oxygen combination together with a moderate rate of oxygen dissociation. We suggest that protonation of a heme-linked group with pK near 6.4 controls many properties of Parasponia oxyhemoglobin, and protonation of a group with pK near 5.5 controls many properties of Parasponia deoxyhemoglobin.

Haemoprotein b-590 (Escherichia coli), a reducible catalase and peroxidase: evidence for its close relationship to hydroperoxidase I and a 'cytochrome a1b' preparation.

A reducible hydroperoxidase, haemoprotein b-590, has been purified 16-fold from a soluble fraction of Escherichia coli K12, grown anaerobically with glycerol and fumarate. The Mr of the native protein, determined by gel filtration, was 331,000 although a minor, smaller species with a Mr of 188,000 was also detected; both had catalase activities. Based on the subunit Mr, determined from SDS gel electrophoresis to be 75,000, the above species are tentatively identified as tetramers and dimers, respectively. The isoelectric point of both species was 4.4. The absorption spectrum of the isolated haemoprotein is typical of ferric, high-spin haem. The A405/A280 ratio never exceeded 0.27, a value half of that obtained for E. coli hydroperoxidase I. On reduction with dithionite, the gamma, beta, and alpha bands were at 441, 559 and 590 nm respectively, the alpha-band being unusually distinct. Treatment of the reduced form with CO gave a sharp prominent gamma-band at 426 nm and caused significant shifts of the alpha and beta bands to shorter (574 and 545 nm) wavelengths. The pyridine haemochrome spectra showed the haem to be protohaem IX; the spectra were featureless between 580 and 630 nm, thus excluding the presence of haem a. However, some features of the difference spectra of the haemoprotein were reminiscent of cytochrome a1, notably the maxima in reduced minus oxidized spectra at 444 and 593 nm and the peaks and troughs in CO difference spectra at 426 and 446 nm respectively. The haemoprotein had high catalase activity: Vmax was 2.3 X 10(6) mol H2O2 (mol haem)-1 min-1 and the Km was 11 mM. At 10 mM-H2O2 the first order rate constant was 0.3 X 10(7) M-1 s-1. The haemoprotein was also a peroxidase with o-dianisidine or 2,3',6-trichloroindophenol as substrates; for the latter substrate, the Km was 0.18 mM. It is concluded that haemoprotein b-590 strongly resembles the hydroperoxidase I purified by Claiborne & Fridovich (Journal of Biological Chemistry 254, 4245-4252, 1979) and that a similar haemoprotein was mistaken for a cytochrome a1 b complex by Barrett & Sinclair (Abstracts of the 7th International Congress of Biochemistry, Tokyo, H-107, p. 907, 1967).

NMR studies of oxyleghemoglobin: assignment of distal histidine proton resonances and evidence for pH-dependent changes in conformation.

The resonance of the C-2 proton of the distal histidine has been assigned in the 400 MHz 1H-NMR spectrum of soybean ozyleghemoglobin a. This resonance is subject to a very large ring current shift from the heme and occurs to high field of the residual HO2H peak. The pH dependence was measured from a series of nuclear Overhauser effect difference spectra over a range of pH values. The resonance moves to high field with decreasing pH and reflects titration of a one proton-dissociable group with pK 5.5. Resonances of the heme substituents and distal amino acid side-chains are also sensitive to this titration. Changes in ring-current shifts and nuclear Overhauser effects indicate that a conformational change occurs in the heme pocket upon titration of the pK 5.5 group. We propose that protonation of the distal histidine with pK 5.5 is accompanied by movement of the imidazole ring towards the heme normal. This movement would allow interaction between the ligated oxygen molecule and the protonated distal histidine at acid pH.

Hemoglobin in a nonleguminous plant, parasponia: possible genetic origin and function in nitrogen fixation.

A dimeric hemoglobin was purified from nitrogen-fixing root nodules formed by association of Rhizobium with a nonleguminous plant, Parasponia. The oxygen dissociation rate constant is probably sufficiently high to allow Parasponia hemoglobin to function in a fashion similar to that of leghemoglobin, by oxygen buffering and transport during symbiotic nitrogen fixation. The identification of hemoglobin in a nonlegume raises important questions about the evolution of plant hemoglobin genes.

Leghemoglobin. Kinetic, nuclear magnetic resonance, and optical studies of pH dependence of oxygen and carbon monoxide binding.

The rate of dissociation of oxygen from soybean oxyleghemoglobin a increases about 5-fold between pH 4 and pH 7, with apparent pK = 5.46 and n = 1. The rate of dissociation of carbon monoxide from carbon monoxyleghemoglobin a and the rates of combination of oxygen and carbon monoxide with ferrous leghemoglobin a are all invariant in this range of pH. The optical spectrum of oxyleghemoglobin in the visible region and the resonances of the four heme meso protons (protons of the bridge carbon atoms) in the NMR spectrum of oxyleghemoglobin are also dependent on pH with pK near 5.5. We suggest that protonation of the imidazole of the distal histidine residue (His 61) leads to formation of a hydrogen bond to the bound oxygen molecule which affects the electronic configuration or conformation of the heme and decreases the rate of oxygen dissociation.