Uptake hydrogenase activity and ATP formation in Rhizobium leguminosarum bacteroids

Abstract

The role of uptake hydrogenase was studied in Rhizobium leguminosarum bacteroids from the nodules of Pisum sativum L. cv. Homesteader. Uptake hydrogenase activity, measured by the 3H2 uptake method, was dependent on C( consumption and was similar to H2 uptake measured by gas chromatography. z Km for 02 of 0.0007 atm (0.0709 kPa) and a Km for H2 of 0.0074 atm (0.7498, kPa) were determined. H2 increased the rate of endogenous respiration by isolates with uptake hydrogenase (Hup+) but had no effect on an isolate lacking uptake hydrogenase (Hup-). A survey of 14 Hup+ isolates indicated a wide range of H2 uptake activities. Four of the isolates tested had activities similar to or higher than those found in two Hup+ Rhizobium japonicum strains. H2 uptake was strongly coupled to ATP formation in only 5 of the 14 isolates. H2 increased the optimal 02 level for C2H2 reduction by 0.01 atm and permitted enhanced C2H2 reduction at 02 levels above the optimum in both a coupled and an uncoupled isolate. At suboptimal 02 concentrations a small enhancement of C2H2 reduction by H2 was seen in two out of three isolates in which H2 oxidation was coupled to ATP formation. Thus, the main function of uptake hydrogenase in R. leguminosarum appears to be in the protection of nitrogenase from 02 damage.

Hydrogen metabolism and energy costs of nitrogen fixation

“…Hydrogen enhances respiration in R. japonicum bacteroids [62]. In bacteroids of Hup + R. leguminosarum strains [63] and in bacteroids of R. japonicum [64], hydrogen increased the level of oxygen, optimal for acetylene reduction. These authors conclude that a major function of hydrogen oxidation is to provide respiratory protection of the nitrogenase proteins against oxygen.…”

Characterization of hup-specific DNA in Rhizobium leguminosarum strains of different origin

“…Hup determinants of these strains may, therefore, differ significantly from the "R. japonicum-type" hup sequences found in the other Hup ÷ strains of R. leguminosarum. These two strains, as well as 128C13, which hybridized only weakly to the hupspecific probes, have all been shown to couple H 2 oxidation to ATP formation in bacteroid preparations [5]. The only coupled strain tested which showed strong hybridization was 175R1.…”

“…Only a few strains of R. leguminosarum appear to be Hup+, and in these there is insufficient activity to recycle all the H 2 produced during N 2 fixation [3,4]. A physiological characterization of a numer of Hup + strains of R. leguminosarum indicated that Hup activity varied among strains and that H 2 oxidation was strongly coupled to ATP formation in only a few strains [5].…”

Repression of growth in Rhizobium japonicum 3Ilb 110 by molecular hydrogen

“…Weber, USDA, Beltsville, MD, U.S.A. Strain 6-8 was one of several Hup -mutants isolated after ethyl methane sulfonate (EMS) mutagenesis of 3Ilb 110 using a procedure similar to that described by Maier [6] for the isolation of Rhizobium japonicum mutants unable to grow chemolithotrophically on H 2. This strain did not show any hydrogenase activity when tested using the tritium uptake assay [7,8] and H 2 uptake when monitored by gas chromatography [7].…”

“…H 2 uptake was monitored using the tritium 0378-1097/83/0000-0000/$03.,00 © 1983 Federation of European Microbiological Societies uptake assay as described previously [7,8], and also by monitoring H 2 consumption using gas chz,~matography (Varian Aerograph model 920, equipped with a thermal conductivity detector and molecular sieve column (0.5 nm)) with N 2 as the carrier gas.…”