Experimental Determination of the Respiration Associated with Soybean/Rhizobium Nitrogenase Function, Nodule Maintenance, and Total Nodule Nitrogen Fixation

Abstract

The total metabolic cost of soybean (Glycine max L. Mer Clark) nodule nitrogen fixation was empirically separated into respiration associated with electron flow through nitrogenase and respiration associated with maintenance of nodule function.Rates of CO2 evolution and H2 evolution from intact, nodulated root systems under Ar:02 atmospheres decreased in parallel when plants were maintained in an extended dark period. While H2 evolution approached zero after 36 hours of darkness at 22°C, CO2 evolution rate remained at 380 of the rate measured in light. Of the remaining CO2 evolution, 62% was estimated to originate from the nodules and represents a measure of nodule maintenance respiration. The nodule maintenance requirement was temperature dependent and was estimated at 79 and 137 micromoles CO2 (per gram dry weight nodule) per hour at 22°C and 30°C, respectively.The cost of N2 fixation in terms of CO2 evolved per electron pair utilized by nitrogenase was estimated from the slope of H2 evolution rate versus CO2 evolution rate. The cost was 2 moles CO2 evolved per mole H2 evolved and was independent of temperature.In this symbiosis, nodule maintenance consumed 22% of total respiratory energy while the functioning of nitrogenase consumed a further 52%. The remaining respiratory energy was calculated to be associated with ammonia assimilation, transport of reduced N, and H2 evolution.The ability of legume/Rhizobium symbioses to reduce dinitrogen has led to a large effort to understand processes regulating and affecting the efficiency of this process (12,15,18,24). Symbiotic N2 fixation has been shown to be sensitive to photosynthate supply (7) (14), a strong correlation between e flow through nitrogenase and respiration has been shown. This relationship led Mahon (13) to propose that the total respiration of an intact nodulated root system could be partitioned according to the following equation:where R is total respiration, W is nodule dry weight, t is time, N2-ase is nitrogenase activity, and RM, RG, and RFJx are maintenance, growth, and fixation coefficients, respectively. Based on this equation, nodule respiration becomes linearly related to nitrogenase activity if the growth and maintenance components remain constant. In mature nodule systems, assuming no growth, the slope of this relationship could be considered as the CO2 respired/e pair reduced and the intercept would be associated with nodule maintenance.Exploiting this relationship between nodule respiration and nitrogenase activity, the cost of total nodule function, as well as the individual cost of nitrogenase operation and nodule maintenance, were experimentally determined. In this report, H2 and CO2 evolution under varied plant light regimes was measured, while in another study (8)

Calorimetry of Nitrogenase-Mediated Reductions in Detached Soybean Nodules

“…The values from all our H2 evolution and acetylene reduction experiments average just over 1.9 cal/h .g fresh weight. This can be converted to about 110 Mmol C02/h-g dry weight, which agrees reasonably with a recent study of nodules in situ (16). This maintenance level of heat production is approximately 25 to 30% of the maximal heat evolution by fresh fixing nodules.…”

“…Our measurements of heat production associated with activation of the nitrogenase system clearly substantiate the high metabolic cost ofdriving this enzyme. This has been implied by previous estimates of fixation cost (7,10,16) and has often been regarded as being far greater than theory would predict (3,12). The HEYTLER AND HARDY heat associated with transfer of 1 mol of electron pairs is equivalent to the metabolism of 0.29 mol glucose.…”

Biosynthesis of (Bacterio)chlorophylls

“…Correlating this experimental value to Pchlide reduction, a ratio of ϳ14 mol of ATP hydrolyzed per mol of Chlide formed was deduced. Analogously, for the related nitrogenase system an in vitro consumption of up to 36 ATP per reduced N 2 versus an apparent minimal theoretical consumption of 16 ATP was observed (31)(32)(33). According to this, the minimal ATP consumption of DPOR catalysis might be in the range of 4 mol of ATP hydrolyzed per mol of Chlide formation.…”

The bioenergetics of electron transport to nitrogenase

“…This interaction is possibly not restricted to A. vinelandii. From the literature, such a relationship can be proposed for soybean bacteroids [109,110] and Anabaena species, when fixing nitrogen in the dark [111,112].…”