Propionate metabolism and its relationship to leghemoglobin biosynthesis in soybean nodules Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/b2773z862

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  • Experiments are reported which demonstrate that a cobalt deficiency in R. meliloti results in a decreased cytochrome content of bacterial cells. It is concluded that the effect of cobalt deficiency on cytochrome content of Rhizobium cells and on the leghemoglobin content of nodules possibly may be explained by an effect of cobalt deficiency on the utilization of propionate. Radiotracer experiments have provided evidence that propionate may be utilized for heme synthesis as well as for the maintenance of the citric acid cycle in nodules. When soybean nodules are incubated with propionate-2-C¹⁴, the intermediates of the citric acid cycle not only become labeled with C¹⁴ but also the heme moiety of leghemoglobin becomes labeled. The incorporation of propionate-2-C¹⁴ into heme is linear with time and it appears that propionate is utilized without a lag period. The rate of incorporation of propionate-2-C¹⁴ into heme is more rapid than the rate of incorporation of succinate-2-C¹⁴ and citrate-1, 5-C¹⁴, however, these rates of incorporation may be influenced by different endogenous pool sizes of organic acids. It can be concluded from additional radioactive tracer experiments that the supply of succinyl-CoA from propionate is competitive with the supply of succinyl-CoA from the citric acid cycle. It was observed that when the concentration of propionate was high in the incubation mixture, the rate of succinate-2-C¹⁴ incorporation into heme was inhibited. Furthermore, when a large amount of substrate (succinate or acetate) which can be utilized by the citric acid cycle enzymes is added to the incubation mixture using whole nodules, the rate of incorporation of propionate-2-C¹⁴ into heme is reduced. The addition of acetate to the incubation mixture reduced the rate of incorporation of propionate-2-C¹⁴ into heme by 33 percent, yet it stimulated the citric acid cycle activity and increased the rate of incorporation of succinate-2-C¹⁴ into heme by nearly 50 percent. Since C¹⁴-labeled metabolites were incorporated into the heme moiety of leghemoglobin, a method was developed for the isolation of pure heme from nodules. In brief, the heme was extracted with acid acetone and reextracted with chloroform. After separation and evaporation of the chloroform, the pyridine hemochromogen was isolated by column chromatography from a silicone impregnated cellulose column. The fact that propionate is readily utilized by bacteroids suggested that this compound may be a normal metabolite in nodules. No detectable pool size of propionate was found however, in either soybean nodules or in isolated bacteroids. These results indicated that propionate may be utilized as rapidly as it is formed. An investigation was initiated therefore to determine whether or not lactate could be a precursor of propionate in this symbiotic relationship. Tracer experiments have indicated that lactate-1-C¹⁴ and lactate-2-C¹⁴ are incorporated into the heme moiety of leghemoglobin at approximately equal rates. The rate of incorporation of lactate-C¹⁴ into heme is significantly decreased by the addition of non-radioactive propionate to the reaction mixture, and isolated propionate from this mixture shows that propionate becomes radioactive. Further experiments using a cell-free extract from nodule bacteroids demonstrated the direct conversion of lactate to propionate. The cofactor requirements for this enzymatic conversion are ATP, Mg⁺⁺, NADH and coenzyme A. The rate of C¹⁴ accumulation in propionate from lactate-1-C¹⁴ is inhibited by the addition of nonradioactive a.crylate, suggesting but not proving that acrylate may be an intermediate in the reaction.
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