Studies on the relationship between cell synthesis and energy utilization in Hydrogenomonas eutropha have shown that the amount of oxidative energy required for synthetic reactions depends on the conditions of growth. The energy of hydrogen oxidation was most efficiently used when growth conditions were optimal (continuous culture, cells in exponential growth phase) and when the rate of growth was limited by H2 or 02 supply. Under these conditions, 2 to 2.5 atoms of oxygen were consumed by the oxyhydrogen reaction for the concomitant conversion of 1 mole of CO2 to cell matter. This conversion efficiency, expressed as the O/C energyyield value, was observed with continuous cultures. A less efficient conversion was found with batch cultures. With limiting concentrations of CO2 the rate of hydrogen oxidation was relatively high, and the O/C value was dependent on the growth rate. With nonlimiting concentrations of CO2, the rate of hydrogen oxidation was strictly proportional to the rate of CO2 fixation, and the O/C value was independent of growth rate. This proportionality between the rate of H2 oxidation and the rate of CO2 fixation suggested that energy supply regulates the (maximum) rate of growth. From the energy-yield measurements, we concluded that the oxidation of 1 mole of H2 yields the equivalent of 2 moles of adenosine triphosphate for H. eutropha, and that at least 5 moles of this high-energy phosphate is required for the conversion of 1 mole of CO2 into cellular constituents.
The electron-transport system of cell-free extracts obtained from Hydrogenomonas H-20 has been studied with particular reference to phosphorylation associated with the oxyhydrogen reaction. Cell-free preparations of this organism exhibit oxidative phosphorylation with hydrogen and succinate as electron donors. This activity could be uncoupled with a number of agents. Ratios of phosphorylative activity to oxidative activity observed varied from 0.2 to 0.7. Factors affecting the efficiency of phosphorylation were examined. Inhibitor and spectrophotometric studies indicated that phosphorylation with hydrogen as electron donor occurs exclusively at a site in an abbreviated electron transport chain between H 2 and cytochrome b . The possible occurrence of a cytochrome b oxidase and the requirement for a quinone are discussed, as well as the correlation between the abbreviated pathway and the energy generation by the cell. Evidence is presented which indicates that nicotinamide adenine dinucleotide does not participate in the hydrogen oxidation path which is coupled to adenosine triphosphate formation.
Molar growth yields were determined from chemostat cultures of Hydrogenomonas eutropha on succinate and on fumarate. The yields from culture on succinate were about 12 g higher than on fumarate. Assuming this difference to be equivalent to 1 molecule of adenosine triphosphate, it is concluded that the oxidation by oxygen of the Hydrogenomonas cytochrome b yields 1 molecule of adenosine triphosphate.The ratio of H2 oxidation (gram-atoms of oxygen utilized in oxidation of H2) to CO2 fixation (gram-atoms of carbon converted into cellular matter), defined as the O/C value, was found to vary appreciably in autotrophically grown Hydrogenomonas eutropha. The observed variation appeared dependent on the availability of inorganic nutrients and the supply rate of H2, 02, and CO2 (3). Since the amount of adenosine triphosphate (ATP), or equivalent, made available per consumed atom of oxygen was not precisely known, the extent of coupling between energy supply and energy utilization could not be evaluated.Our earlier observations (2) from experiments with cell-free preparations substantiated the occurrence of oxidative phosphorylation. Measurements of ratios of phosphorylative activity (gram-atoms of phosphorus converted to ATP) to oxidative activity (gram-atoms of oxygen utilized in oxidation of substrate) suggested a respiratory chain with a single coupling site between H2 and the Hydrogenomonas cytochrome b (P/O = 1). On the other hand, our (3) efficient growth yields (O/C -2.5) are difficult to explain unless one assumes the operation of an additional coupling site between cytochrome b and 02 (P/O 2 2). To test the presence of such an additional coupling site in the respiratory chain, we used a procedure suggested by Gunsalus and Shuster (4). The procedure is based on measuring the difference in cell yield between succinate and fumarate and assuming that some 10 to 12 g of cells are synthesized per mole of ATP (1).H. eutropha was cultivated in a chemostat (Pyrex, 1,000 ml liquid volume) under aerobic conditions with either succinate or fumarate as the sole energy and carbon source. The mineral medium used for these experiments was described previously (2). It contained 1.5 X 102 M NH4Cl as a nitrogen source. The culture chamber and mineral medium were autoclaved. The organic substrates were filter-sterilized (Nalgene, 0.20-,Am membrane filter) and added to the mineral medium prior to use. Substrate utilization was determined from the difference in organic carbon content of the diluent medium and the supernatant effluent liquid. Cells were rapidly removed by filtration through a membrane filter (Millipore Corp., Bedford, Mass., HA 0.45 pAm type). The content of organic carbon was determined by a method described by Van Hall et al. (10), and cell concentration was measured as described previously (2). The molar growth yields (grams of cells per gram-mole of utilized substrate) obtained with these two substrates are recorded in Table 1.The yield values reported in Table 1 were obtained during steady-state growth. The diluti...
SUMMABYKinetic studies were made of the photochemical reduction of nitrate and nitrite to am monia and correlations to the photosynthetic CO2 assimilation investigated. Two molecules of O2 are evolved per molecule of nitrate reduced to ammonia and 1% molecules of O2 per molecule of nitrite.In the rate-limiting light intensity region, the rate of oxygen production was found to be independent of the nature of the oxidant (CO2, NO 3" or NO2 -) ; the quantum yield of nitrate reduction is equal to that of CO2 reduction. Hence the energetic efficiency of nitrate and nitrite reduction is only about '3 of the efficiency of the CO2 assimilation.Under light saturation the rate of oxygen evolution in CO2 containing media is not increased by a simultaneously occurring NO;; -and N02~ reduction.
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