Membrane energization in relation with nitrogen fixation in Azotobacter vinelandii and Rhizobium leguminosarum bacteroids

Veeger, C.; Laane, C.; Scherings, G.; Wolbers, Louise van Zeeland

doi:10.1016/s0300-9084(78)80820-7

Cited by 19 publications

(14 citation statements)

References 9 publications

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“…Cordewener, unpublished). Another experiment that can be explained differently now is the reduction of flavodoxin to the hydroquinone form by NADPH with purified NAD(P)H-flavodoxin oxidoreductase [42]. The hydroquinone that is formed originates most likely from Fld I (El) = -320 mV), while Fld I1 is not reduced beyond the semiquinone state (El = -500 mV, E2 = -250 mV).…”

Section: Discussionmentioning

confidence: 99%

Characterization of three different flavodoxins from Azotobacter vinelandii

Klugkist¹,

Voorberg²,

Haaker³

et al. 1986

European Journal of Biochemistry

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The flavodoxins from Azotobacter vinelandii cells grown N2-fixing and from cells grown on NH40Ac have been purified and characterized. The purified flavodoxins from these cells are a mixture of three different flavodoxins (Fld I,11, 111) with different primary structures. The three proteins were separated by fast protein liquid chromatography; Fld I eluted at 0.38 M KC1, Fld I1 at 0.43 M KC1 and Fld I11 at 0.45 M KC1. The most striking difference between the three flavodoxins was the midpoint potential (pH 7.0, 25 "C) of the semiquinone/ hydroquinone couple, which was -320 mV for Fld I and -500 mV for the other two flavodoxins (Fld 11 and Fld 111).All three flavodoxins were present in cells grown on NH40Ac. In cells grown on N2 as N source only Fld 1. and Fld I1 were found. The concentration of Fld I1 was 10-fold higher in N2-fixing cells than in cells grown on NH40Ac. Evidence has been obtained that Fld I1 is involved in electron transport to nitrogenase.As will be discussed, our observation that preparations of Azotobacter flavodoxin are heterogeneous, has consequences for the published data.Flavodoxin from Azotobacter vinelandii was first isolated by Shethna et al. in 1964 [l, The protein consists of a single polypeptide chain with 179 amino acid residues, it contains one FMN and has a relative molecular mass of 19990 [S]. There is one single cysteine residue present which can cause dimerization of two flavodoxin molecules, a process which results in the loss of biological activity [9]. In addition to the 5'-phosphate ester on the FMN, flavodoxin contains 2 mol tightly bound phosphate/mol [lo]. One phosphate group is covalently bound to the protein in a phosphodiester linkage between serine and threonine residues. It has been suggested that the other is an acid-labile phosphate in an acyl phosphate linkage with a protein COOH group [ll]. At pH 8.0 and 25°C the redox potential of the quinone/semiquinone couple (E2) of flavodoxin is -250 mV [12-141. However an anomalous value of + 50 mV was also reported for E2 [15]. The redox potential of the semiquinone/hydroquinone couple ( E l ) is -500 mV The primary function of the flavodoxin in Azotobacter species was suggested to be electron transport to nitrogenase. [12-161.In 1969, Benemann et al. [4, 171 showed that flavodoxin was one of the four factors native to A. vinelandii cells needed for electron transport from NADPH to nitrogenase; however, the reported rate was just a fraction of the activity obtained with dithionite as electron donor. It appeared that the endogenous enzyme system was not capable of reducing flavodoxin effectively beyond the semiquinone state, whereas the hydroquinone form is necessary for nitrogenase activity [18, 191. In fact, completely reduced flavodoxin was found to be a good electron donor for nitrogenase, activities being 50% higher than with dithionite [I9 -211. Furthermore flavodoxin from Azotobacter chroococcurn was shown to be nif specific [=I.What argues against flavodoxin being the unique physiological electron donor for ni...

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Section: Discussionmentioning

confidence: 99%

Characterization of three different flavodoxins from Azotobacter vinelandii

Klugkist¹,

Voorberg²,

Haaker³

et al. 1986

European Journal of Biochemistry

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“…P/O ratios were determined with the assay system previously described [16,17]. With this system it is possible to control the respiration rate of a membrane suspension from zero to the maximum respiration rate and to measure the free 0 2 concentration continously.…”

Section: Methodsmentioning

confidence: 99%

“…We reported recently [17] that a difference can be observed between vesicles prepared from bacteroids in the presence of high salt concentrations (NaCl') or in the presence of sucrose (NaCl-). The rate of succinate oxidation is the same in both vesicles preparations, but NaCl' vesicles can be energized by succinate oxidation, while NaC1-vesicles cannot or only to a small extent.…”

Section: Effect Of 0 2 On the Coupling Between Oxidation And Phosphormentioning

confidence: 99%

On the Efficiency of Oxidative Phosphorylation in Membrane Vesicles of Azotobacter vinelandii and of Rhizobium leguminosarum Bacteroids

Laane¹,

Haaker²,

Veeger³

1979

European Journal of Biochemistry

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1. The effect of 0 2 on the rate of ATP synthesis and the P/O ratio has been studied in membrane vesicles of Azotobacter vinelandii and Rhizobium leguminosarum bacteroids (strain PRE). It was observed that the P/O ratio rises to a maximum value when the O2 input is increased, but declines when 0 2 becomes detectable in the medium.2. Evidence is presented, that in A . vinelundii HZ donates its electrons at a fourth proton-translocating site. The presence and the efficiency of this site is independent of the growth conditions and it appeared that no flavoproteins are directly involved in the oxidation of Hz. The unidirectional hydrogenase in membrane vesicles of A. vinelandii is not inhibited by acetylene. The function of hydrogenase in the process of nitrogen fixation by A . vinelandii is discussed.3. Two types of membrane vesicles of R. leguminosarum bacteroids were prepared. Membranes isolated in a low salt medium lose the ability to couple succinate oxidation to oxidative phosphorylation; upon isolation in a high salt medium this ability is preserved. In contrast the efficiency of oxidative phosphorylation with NADH is not affected by the isolation procedure. The efficiency of energy coupling in membranes vesicles of R. leguminosarum bacteroids can be enhanced by the addition of fatty-acid-free bovine serum albumin. No active membrane-bound hydrogenase could be detected in membrane vesicles of R. leguminosarum bacteroids.Several reports have appeared in the literature on attempts to assess the efficiency of oxidative phosphorylation in intact cells and isolated respiratory membranes of Azotobacter vinelandii [l -51. The reported P/O ratios of respiratory membrane vesicles are usually low (< 0.5 with NADH) [1,5-71, although Ackrell and Jones [3] obtained values as high as 1.1. Much higher P/O ratios were calculated during studies on whole cells of this organism and the authors suggest that there are at least two and more likely three coupling sites [2,4]. One site at the level of NADH dehydrogenase, one in the central ubiquinone-cytochrome b region and a third coupling site at the minor branch of the terminal cytochrome system; the major cytochrome branch appears to be uncoupled.No data are present in the literature about the efficiency of energy coupling in membrane vesicles of Rhizobium leguminosarum bacteroids. In general P/O ratios are obtained from measurements at air-satuAbbreviation. Tes, 2-{ [tris(hydroxymethyl)methyl]amino)-ethanesulfonic acid. rated 0 2 concentrations, which are non-physiological for aerobic Nz-fixing organisms. We, therefore devised a technique which enabled us to maintain different rates of respiration at non-saturating 0 2 concentrations and to study the effect of 0 2 on the P/O ratio in membrane vesicles of both A . vinelundii and R. leguminosarum bacteroids.It has been known for several years that a number of nitrogen fixing organisms contain a hydrogenase, which enables these organisms to recover some of the energy lost during the wasteful reduction of protons by nitrogenase [8 ...

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“…At the highest flavodoxin concentration tested (366 pM), with a midpoint potential of -495 mV at pH 7.0, it can be calculated that at -480 mV the flavodoxin hydroquinone concentration is 132 pM. However under continuous illumination flavodoxin concentrations around 10 pM give maximum activity [7,23,24]. We can conclude that A .…”

Section: Figurementioning

confidence: 75%

The Effect of the Redox Potential on the Activity of the Nitrogenase and on the Fe-Protein of Azotobacter vinelandi

et al. 1982

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The redox potential dependence on the nitrogenase reaction catalyzed by different nitrogenase cornplexcs from Azotohacter vinelundii has been studied by two methods. The redox potential was set with the redox couplc SO;p/SO:-and the effect on the nitrogenase activity was determined. The oxidation of photochemically reduced low-potential electron carriers by nitrogenase was followed spectrophotometrically. Simultaneously the nitrogenase activity was estimated by the production of hydrogen. It was found that when component I1 of the nitrogenase is in redox equilibrium with the electron donor, the nitrogenase activity is maximum and independent of the redox potential up to -440 mV. At higher potentials the nitrogenase activity declines and no significant activity was detectable at potentials above -350 mV.The effect of the redox potential on the oxidation reduction state of the 4Fe-4S cluster of Azotohacter nitrogenase component I1 was studied by electron paramagnetic resonance spectroscopy. Without adenine nucleotides present, component I1 of the nitrogenase shows a midpoint potential of -393 mV and the oxidation reduction reaction is characterized by the transfer of two electrons per redox step. In the presence of MgATP the midpoint potential of component I1 of the nitrogenase undergoes a negative shift of 42 mV. The curve fitting to the experimental points is also characteristic of a two-electron redox step. In contrast, it is found that in the presence of MgADP, the curve fitting to the experimental points is characterized by a one-electron step with a midpoint potential of -473 mV. It is demonstrated that the nitrogenase activity correlates with the oxidation reduction state of component I1 of the nitrogenase without adenine nucleotides bound. The implications of this observation, indicating a sequential reaction of the reduced Fe protein with the Mo-Fe protein, for thc present kinetic models of nitrogenase will be discussed.

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Membrane energization in relation with nitrogen fixation in Azotobacter vinelandii and Rhizobium leguminosarum bacteroids

Cited by 19 publications

References 9 publications

Characterization of three different flavodoxins from Azotobacter vinelandii

Characterization of three different flavodoxins from Azotobacter vinelandii

On the Efficiency of Oxidative Phosphorylation in Membrane Vesicles of Azotobacter vinelandii and of Rhizobium leguminosarum Bacteroids

The Effect of the Redox Potential on the Activity of the Nitrogenase and on the Fe-Protein of Azotobacter vinelandi

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