Hydrogen Inhibition of Nitrogen Reduction by Nitrogenase in Isolated Soybean Nodule Bacteroids

Abstract

Dihydrogen, a by-product of biological nitrogen fixation, is a competitive inhibitor of N2 reduction by nitrogenase. To evaluate the significance of H2 inhibition in vivo, we have measured the apparent inhibition constant for H2 inhibition of N2 reduction in Bradyrhizobiumjaponicum bacteroids isolated from soybean nodules. The rate of N2 reduction was measured as ammonia production by bacteroids incubated in a buffer containing 200 micromolar leghemoglobin and 10 millimolar succinate under 0.02 atmosphere O2 and various concentrations of N2 and H2. The apparent inhibition constant for H2 under these conditions was determined to be approximately 0.03 atmosphere. This relatively low value strengthens the proposal that H2 inhibition of N2 reduction may be a significant factor in lowering the efficiency of nitrogen fixation in legume nodules.Dihydrogen is a by-product of biological nitrogen fixation and inhibits the reduction of N2 by nitrogenase (15). The production of H2 from protons and electrons appears to be intrinsic to the mechanism of N2 fixation (15,26), and a minimum of 25% of the electron flux through purified nitrogenase is allocated to H2 production (25). A survey of legume nodules found that approximately 40 to 60% of the electrons allocated to nitrogenase were utilized in the production of H2 (23). However, nodules evolving little or no H2 have been identified, and bacteroids isolated from these nodules have been shown to contain a H2-oxidizing hydrogenase (10). Field and greenhouse studies comparing soybean plants inoculated with HUP+2 versus HUP-strains of Bradyrhizobium japonicum (formerly Rhizobium japonicum) have suggested a beneficial effect of hydrogenase on plant productivity (1,13,14).Release of H2 inhibition of N2 reduction by nitrogenase has been proposed as one mechanism by which hydrogenase may increase the efficiency of N2 fixation in legume nodules (9). This proposal is derived from the observation that H2 is a competitive inhibitor of N2 reduction by nitrogenase (15,29 coefficient, H2 would diffuse through the nodule rapidly enough to prevent accumulation of inhibitory levels within the nodule (9). However, later evidence demonstrated that the cortex of some legume nodules contains a layer of cells with few or no intercellular spaces, and that this layer provides a diffusion barrier to gas exchange (11, 27). Based on H2 evolution rates from nodules and the assumption that H2 must diffuse through an aqueous layer, Dixon et al. (11) estimated that the H2 concentration in the center of legume nodules could reach as high as 200 uM (0.3 atm). More recent calculations using computer models of nodule structure and physiology have predicted a H2 concentration of up to 114 jM (about 0.2 atm) in the center of soybean nodules (18).From these estimates and published values of the Kj(H2) for purified nitrogenase from free-living bacteria (0.1-0.5 atm, see Table I), it was concluded that H2 concentrations in infected cells could indeed build up to levels which inhibit N2 fixation (1 1, 18).D...

A Model of the Regulation of Nitrogenase Electron Allocation in Legume Nodules (I. The Diffusion Barrier and H2 Inhibition of N2 Fixation)

“…The predicted EAC values from this equation are shown in Figure 1, assuming values for K,,,(Nz) and Ki(Hz) of 1.66 and 1.25 mo1 m-3 in the gas phase, respectively (Rasche and Arp, 1989). At 32.4 mo1 m-3 of N2 (the atmospheric pNz) with no H2 present in the atmosphere (Hi = O), the predicted EAC is 0.713.…”

“…Similarly, the Kil[Hz) values were chosen to represent the range of values measured on Rkizobium or Bradyrhizobium nitrogenase. A Ki(Hz) of 0.14 atm has been measured in whole clover plants (Wilson, 1940), and Rasche and Arp (1989) reported Ki(Hz) values between 0.022 and 0.032 atm H2 in Bradyrkizobium japonicum bacteroids. To cover this range, two Ki(H2) values were modeled:…”

“…HZ acts as a competitive inhibitor of NZ fixation (Wilson, 1940;Bums, 1985;Rasche and Arp, 1989), and, thereiore, a11 rates of N2 fixation and H2 evolution can be calculated from the Lineweaver-Burk transformation of the MichaelisMenten equation for competitive inhibition (Williams ei: al., 1967):…”

“…The Km(Nz) values used in the simulations presented here were set at 1.66 and 2.91 mo1 m-3 (0.04 and 0.07 atm, respectively) to account for the range of K,(Nz) values reported in previous studies (Wilson, 1940;Bergersen and Turner, 1968;Rasche and Arp, 1989). Similarly, the Kil[Hz) values were chosen to represent the range of values measured on Rkizobium or Bradyrhizobium nitrogenase.…”

“…More recently, Rasche and Arp (1989) estimated that the intemal pHz of soybean nodules was 0.02 to 0.027 atm (15-20 p~ in solution at 2OoC), and Witty (1991) used an Hz microelectrode to measure a pH2 in the interior of soybean nodules of approximately 0.01 to 0.02 atm Hz (about 7.5-15 p~ in solution). Estimates of the Ki(Hz) for Hz inhibition of NZ fixation range between 0.02 and 0.14 atm (about 15-105 PM in solution) of HZ (Wilson, 1940;Rasche and Arp, 1989). Given these values, the measured or theoretical Hz concentration in the nodule central zone would be in the range of the apparent Ki(H2) for the inhibition of N2 fixation, indicating that HZ has the potential to affect the in vivo regulation of…”