Systems utilizing oxygenated leghemoglobin and myoglobin as sources of free dissolved O2 at low concentrations for experiments with bacteria

Bergersen, F. J.; Turner, G. L.

doi:10.1016/0003-2697(79)90569-4

Cited by 51 publications

(62 citation statements)

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“…It has been previously shown that 50% inhibition of activity and synthesis of nitrogenase occurred at about 100nM-0, which was close to the K, of the dominant terminal oxidase ; this was estimated from 02-consumption measurements with whole bacteria using the same system for 0, determination as used in the present work for measurement of the K, value for the purified oxidase (Bergersen & Turner, 1979 ;Bergersen et al, 1982 ;Hill et al, 1984). The estimated value using whole bacteria was about 80 nM.…”

Section: Requirement For Activity Of the Oxidase For Detergent Or Neusupporting

confidence: 73%

“…This is presumably due to provision of ATP for nitrogenase activity by way of 0,-dependent respiration. Higher O2 concentrations inhibit both synthesis and activity of nitrogenase, with 50% inhibition occurring at about 1 0 0 n~-O , which is close to the estimated K, of the dominant terminal oxidase (about 80 nM, estimated from 02-consumption measurements with whole bacteria) (Bergersen & Turner, 1979;Bergersen et al, 1982;Hill et al, 1984). This raises the important question of the nature of the oxidase(s) responsible for respiration at the very low O2 (microaerobic) concentrations that support more efficient nitrogen fixation, and which is involved in creating these conditions suitable for derepression of ni trogenase synthesis.…”

Section: Introductionsupporting

confidence: 73%

“…This was measured by following the deoxygenation of leghaemoglobin. Ferric azide leghaemoglobin was a kind gift of F. J. Bergersen (CSIRO, Canberra, Australia); it was converted for use to the ferrous oxygenated form by the method of Bergersen & Turner (1979) and oxygen affinity measured as described by Appleby & Bergersen (1980). A glass cuvette contained 50 pM-oxyleghaemoglobin, 0.05 % Tween 20, 55 pM-ubiquinol-1 and 8 mM-dithiothreitol in 50 mM-sodium phosphate buffer (pH 7.4), to give a total volume of 1.9 ml completely filling the cuvette which was sealed with a Suba-seal so as to exclude all air.…”

Section: Methodsmentioning

confidence: 99%

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The purification, characterization and role of the d-type cytochrome oxidase of Klebsiella pneumoniae during nitrogen fixation

Smith

Hill

Anthony

1990

Journal of General Microbiology

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Klebsiella pneumoniae synthesized only 6-type and d-type cytochromes under the wide range of growth conditions tested, and reaction with CO revealed two potential oxidases. The 0-type oxidase was produced only in the presence of O2 and appeared to be repressed by glucose. The d-type oxidase was, by contrast, produced only in the absence of measurable O2 (< 1 p~), and was the only oxidase expressed in nitrogen-fixing conditions. It was extracted from the membrane, purified and shown to be similar to that from E. coli in being a heterodimer (subunits of M, 52000 and 35000), in containing two distinguishable b haems and haem d (one or two molecules per molecule of oxidase), and in being able to react with O2 to give a stable oxygenated intermediate. The purified d-type cytochrome oxidase had a very high affinity for O2 (K, 20 nM; measured by the spectral properties of leghaemoglobin). It is proposed that this provides a role for this oxidase in lowering the O2 concentration to allow nitrogenase synthesis and function, and to provide a terminal oxidase to permit electron-transport-coupled ATP synthesis which supports the increase in efficiency of nitrogen fixation observed under microaerobic conditions.

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Section: Requirement For Activity Of the Oxidase For Detergent Or Neusupporting

confidence: 73%

Section: Introductionsupporting

confidence: 73%

Section: Methodsmentioning

confidence: 99%

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The purification, characterization and role of the d-type cytochrome oxidase of Klebsiella pneumoniae during nitrogen fixation

Smith

Hill

Anthony

1990

Journal of General Microbiology

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“…The H2 oxidation reaction proceeded perceptibly at concentrations of 02 below 400 nm (Fig. 1 (8). The results of one such experiment is shown in Figure 2.…”

Section: Resultsmentioning

confidence: 93%

“…While preparing this manuscript, a paper by Bergersen and Turner (8) was published in which they described a method of supplying low concentrations of free dissolved O2 to bacteroids by the deoxygenation of oxyleghemoglobin. Their method is essentially the same as ours although an entirely different apparatus was utilized.…”

Section: Methodsmentioning

confidence: 99%

Oxyleghemoglobin-mediated Hydrogen Oxidation by Rhizobium japonicum USDA 122 DES Bacteroids

Emerich

Albrecht²,

Russell³

et al. 1980

Plant Physiol.

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Oxyleghemoglobin was used to supply low concentrations of 02 to H2-oxidizing bacteroids from Rhizobium japoncum USDA 122 DES. The H2 oxidation system of these bacteroids was capable of effectively utilizing 02 at the low concentrations of 02 expected to be found in soybean nodules.Apparent Km values of approximately 10 nanomolar 02 have been calculated for the oxyhydrogen reaction. These values include the Km values for both H2 oxidation and endogenous substrate oxidation. Even in the presence of oxyleghemoglobin, H2 additions stimulated C2H2 reduction, reduced the rate of endogenous respiration and maintained the ATP contents of bacteroids. In our reconstituted oxyleghemoglobin and bacteroid system, we estimate that the H2 oxidation system is capable of recycling all of the H2 evolved during the N2 fixation process.are correct, there may be insufficient O2 at the bacteroid surface to accommodate an appreciable rate of H2 oxidation. Soybean nodules formed by inoculation with H2 uptake positive (Hup+) strains of R japonicum rarely evolve H2, whereas nodules formed by H2 uptake negative (Hup-) strains of R japonicum evolve H2 at rates amounting up to 40%o of the energy supplied to the N2-fixing process (12,14). Thus, the H2 oxidation system efficiently utilizes physiological concentrations of 02 to recycle H2 evolved by nitrogenase.Here, we report that bacteroids possessing the H2 oxidation system are capable of effectively utilizing low levels of 02 for H2 oxidation. We independently developed a method whereby oxyleghemoglobin is used to establish low levels of 02 in bacteroid suspensions. The method is similar to that recently described by Bergersen and Turner (8). Our results show that oxyleghemoglobin provides O2 for utilization by the H2 oxidation system in bacteroids, but we do not imply that the interaction of the H2 oxidation system with oxyleghemoglobin is direct.Certain strains of Rhizobium japonicum (10) possess a H2 oxidation system that is capable of recycling the H2 that is evolved during the N2-fixing process (1,(11)(12)(13)(14)(15). H2 evolution during N2 reduction by nitrogenase represents a considerable proportion of the energy supplied for the fixation process (12)(13)(14). A part of the energy lost through H2 evolution may be conserved through H2-dependent ATP formation (11).Oxygen is the physiological terminal electron acceptor of the H2 oxidation system in R japonicum bacteroids (14,15). The apparent Km value for O2 measured amperometrically in suspensions of washed bacteroids was reported to be 1 iUM (12, 15). The free-dissolved O2 concentration at the bacteroid surface, however, has been estimated to be approximately 10 nM (2,22,24 MATERIALS AND METHODS Suspensions of washed bacteroids were prepared and amperometric assays for H2 and 02 uptake were conducted as described previously (11). A specifically constructed chamber was used for the simultaneous measurement of the rate of H2 oxidation and the rate of deoxygenation ofoxyleghemoglobin (see Fig. 2, inset). This chamber consisted ...

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Leghämoglobin aus Wurzelknöllchenextrakten von Luzerne bei genotypischer Variation der Symbiose

Hoffmann

Kaemmer

Miksch

et al. 1990

J. Basic Microbiol.

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The host plant genotype significantly influenced the nodule Lb content in all of the combinations examined between 5 alfalfa (M<,dicago satizu) cultivars and 5 Rhimbium nieliloti strains inoculated. on the contrary not the bacterial genotype. The inoculation with the strains exhibiting an increased activity of uptake hydrogenase caused an elevation of the nodule Lb content. By IEF 4 major Lb componcnts were found. Their proportions were changed throughout the nodule development.Leghamoglobin, das in den Wurzel-sowie Stammknollchen der Leguminosen vorkommt, beeinflu& wesentlich die Effektivitat der N,-Fixierung (APPLEBY 1974(APPLEBY , 1984. Lb') ist fur die Sauerstoffversorgung der Bakteroide in den Knollchenzellen verantwortlich (BERGEK-SEN et a/. 1973, WITTENBEKC cif ni. 174, APPLEBY-1984). Es ist auch in nichtinfizicrten Zellen der Knollchen gefunden worden, allerdings in geringerer Konzentration als in infizierten Zellen (VAN DEN BOSCH und NEWCOMB i988).Mit Hilfe von IEF und HPLC wurden in Wurzelknollchenextrakten von Sojabohnenpflanzen verschiedene Lb-Formen getrennt und isoliert. Die Nomenklatur der mittels IEP getrennten Lb-Komponenten ist an absteigenden IEP-Werten orientiert. Vier Lb-Hauptkomponenten, Lba, Lbc,, Lbc, und Lbc, ,wurden identifiziert und mit Lbb, Lbd,, Lbd, und Lbd, die gering vorkommenden, jeweils am N-Terminus acetylierten Lb-Formen bezeichnet (FUCHSMAN und APPLEBY 1979, WHITTAKER et al. 1981, PUPPO und RIGAUII 1987. In Ahhangigkeit vom Kniillchenalter sind zwei proteolytisch modifizierte Lb-Formen nachsewiesen worden, Lba' und Lbc' (SAKATFI et ul. 1986). Wiihrend der Entwicklung der SojabohnenKnollchen andern sich die Anteile der einzelnen Lb-Isoproteine, weshalb eine entwicklungskontrollierte Expression angenommen wird. Die funktionale Bedeutung der Lb-Heterogenitiit ist unklar (POWELL und GANNON 1988

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Systems utilizing oxygenated leghemoglobin and myoglobin as sources of free dissolved O2 at low concentrations for experiments with bacteria

Cited by 51 publications

References 10 publications

The purification, characterization and role of the d-type cytochrome oxidase of Klebsiella pneumoniae during nitrogen fixation

The purification, characterization and role of the d-type cytochrome oxidase of Klebsiella pneumoniae during nitrogen fixation

Oxyleghemoglobin-mediated Hydrogen Oxidation by Rhizobium japonicum USDA 122 DES Bacteroids

Leghämoglobin aus Wurzelknöllchenextrakten von Luzerne bei genotypischer Variation der Symbiose

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