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

Abstract

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 ...

Uptake hydrogenase activity and ATP formation in Rhizobium leguminosarum bacteroids

“…Our estimated Km for dissolved 02 of 0.5 ,uM was a little lower than that of 1 ,uM reported for R. japonicum bacteroids (14,17). The experimental difficulties in obtaining accurate estimates of H2 uptake at low H2 and 02 concentrations have been discussed (7,9,17). With more sensitive methods, apparent Km values for H2 of 0.05 ,uM (9) and for 02 of 10 nM (7) in R. japonicum bacteroids have been obtained.…”