Root nodules, leghemoglobin

The symbiotic nitrogen-fixing system in root nodules contains the dioxygen-binding hemoprotein, leghemoglobin, but its function is still uncertain. 

Suganuma, N.M., Kitou, M. Yamamoto, Y. (1987). Carbon metabolism in relation to cellular organization of soybean root nodules and respiration of mitochrondria aided by leghemoglobin. Plant Cell Physiology 28, 113-22. 

Verma, D.P.S. Bal, A.K. (1976). Intracellular site of synthesis and localization of leghemoglobin in root nodule. Proceedings of the National Academy of Sciences (USA) 73, 3843-7. 

Nitrogen fixation is the conversion of N2 gas into ammonia, a process carried out by some soil bacteria, cyanobacteria and the symbiotic bacteria Rhizobium that invade the root nodules of leguminous plants. This process is carried out by the nitrogenase complex, which consists of a reductase and an iron-molybdenum-containing nitrogenase. At least 16 ATP molecules are hydrolyzed to form two molecules of ammonia. Leghemoglobin is used to protect the nitrogenase in the Rhizobium from inactivation by 02. 

O Brian MR, Kirshbom PM, Maier RJ (1987) Bacterial heme synthesis is required for expression of the leghemoglobin protein but not the apoprotein in soybean root nodules. Proc Natl Acad Sci USA 84 8390-8393... 

Ion focusing of ferric leghemoglobins from soybean root nodules... 

Reducing nitrogen (as occurs in nitrogen fixation), requires energy and electrons with low-potential (electron carriers with very low EO ). The enzymes involved are very sensitive to oxygen and must be studied only under anaerobic conditions. In root nodules of plants, the anaerobic environment is provided by the protein leghemoglobin, which binds any 02 that makes its way into the nodules. 

Fig. 6. Current concepts of bacteroid metabolism relating to N2 fixation and ammonia production in legume root nodules. The relationships to nitrogenase activity of carbohydrate utilization, electron transport, and H2 cycling, are idicated. Nodules without an uptake hydro-genase lose energy through Hj evolution. Leghemoglobin, LHb flavodoxin hydroquinone, FldHj flavodoxin semiquinone, FldH. (Based on Evans et al. (1979) and reproduced with permission.)...

Fig. 9. Proposed model for the cellular compartmentalization of the reactions of nitrogen fixation, ammonium assimilation, purine synthesis, and ureide biogenesis in infected and uninfected cells of soybean root nodules. Uncertainty still exists with respect to the nature of the intermediate (e.g., IMP, XMP, xanthine, glutamine ) transported from the infected cell to the uninfected cell as well as the site of purine synthesis. In addition, as discussed in the text the site(s) of PRPP synthesis (plastid and/or cytosolic) and the path and site of synthesis (de novo from the PPP or via salvage) of tibose S-phosphate (R-S-P) are s not defined, lliese uncertainties are indicated with question marks and/or dashed lines. Lb, leghemoglobin.

Soybean plants are grown, and after at least 3 weeks growth, the nodules are removed from the roots. The nodules are crushed, and the leghemoglobins extracted with pH 7 phosphate buffer. The debris is centrifuged away, and 15 pL are placed on top of a polymerized acrylamide gel containing an 8 1 ratio of pH 4.2 to 4.6 and 4.0 to 6.5 ampholines. 

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