Expressed Soybean Leghemoglobin: Effect on Escherichia coli at Oxidative and Nitrosative Stress

Leghemoglobin (Lb) is an oxygen-binding plant hemoglobin of legume nodules, which participates in the symbiotic nitrogen fixation process. Another way to obtain Lb is its expression in bacteria, yeasts, or other organisms. This is promising for both obtaining Lb in the necessary quantity and scrutinizing it in model systems, e.g., its interaction with reactive oxygen (ROS) and nitrogen (RNS) species. The main goal of the work was to study how Lb expression affected the ability of Escherichia coli cells to tolerate oxidative and nitrosative stress. The bacterium E. coli with the embedded gene of soybean leghemoglobin a contains this protein in an active oxygenated state. The interaction of the expressed Lb with oxidative and nitrosative stress inducers (nitrosoglutathione, tert-butyl hydroperoxide, and benzylviologen) was studied by enzymatic methods and spectrophotometry. Lb formed NO complexes with heme-nitrosylLb or nonheme iron-dinitrosyl iron complexes (DNICs). The formation of Lb-bound DNICs was also detected by low-temperature electron paramagnetic resonance spectroscopy. Lb displayed peroxidase activity and catalyzed the reduction of organic peroxides. Despite this, E. coli-synthesized Lb were more sensitive to stress inducers. This might be due to the energy demand required by the Lb synthesis, as an alien protein consumes bacterial resources and thereby decreases adaptive potential of E. coli.

Effect of the synthesis of rice non-symbiotic hemoglobins 1 and 2 in the recombinant Escherichia coli TB1 growth

Non-symbiotic hemoglobins (nsHbs) are widely distributed in land plants, including rice. These proteins are classified into type 1 (nsHbs-1) and type 2. The O2-affinity of nsHbs-1 is very high mostly because of an extremely low O2-dissociation rate constant resulting in that nsHbs-1 apparently do not release O2 after oxygenation. Thus, it is possible that the in vivo function of nsHbs-1 is other than O2-transport. Based on the properties of multiple Hbs it was proposed that nsHbs-1 could play diverse roles in rice organs, however the in vivo activity of rice nsHbs-1 has been poorly analyzed. An in vivo analysis for rice nsHbs-1 is essential to elucidate the biological function(s) of these proteins. Rice Hb1 and Hb2 are nsHbs-1 that have been generated in recombinant Escherichia coli TB1. The rice Hb1 and Hb2 amino acid sequence, tertiary structure and rate and equilibrium constants for the reaction of O2 are highly similar. Thus, it is possible that rice Hb1 and Hb2 function similarly in vivo. As an initial approach to test this hypothesis we analyzed the effect of the synthesis of rice Hb1 and Hb2 in the recombinant E. coli TB1 growth. Effect of the synthesis of the O2-carrying soybean leghemoglobin a, cowpea leghemoglobin II and Vitreoscilla Hb in the recombinant E. coli TB1 growth was also analyzed as an O2-carrier control. Our results showed that synthesis of rice Hb1, rice Hb2, soybean Lba, cowpea LbII and Vitreoscilla Hb inhibits the recombinant E. coli TB1 growth and that growth inhibition was stronger when recombinant E. coli TB1 synthesized rice Hb2 than when synthesized rice Hb1. These results suggested that rice Hb1 and Hb2 could function differently in vivo.

Effect of the synthesis of rice non-symbiotic hemoglobins 1 and 2 in the recombinant Escherichia coli TB1 growth

Non-symbiotic hemoglobins (nsHbs) are widely distributed in land plants, including rice. These proteins are classified into type 1 (nsHbs-1) and type 2. The O2-affinity of nsHbs-1 is very high mostly because of an extremely low O2-dissociation rate constant resulting in that nsHbs-1 apparently do not release O2 after oxygenation. Thus, it is possible that the in vivo function of nsHbs-1 is other than O2-transport. Based on the properties of multiple Hbs it was proposed that nsHbs-1 could play diverse roles in rice organs, however the in vivo activity of rice nsHbs-1 has been poorly analyzed. An in vivo analysis for rice nsHbs-1 is essential to elucidate the biological function(s) of these proteins. Rice Hb1 and Hb2 are nsHbs-1 that have been generated in recombinant Escherichia coli TB1. The rice Hb1 and Hb2 amino acid sequence, tertiary structure and rate and equilibrium constants for the reaction of O2 are highly similar. Thus, it is possible that rice Hb1 and Hb2 function similarly in vivo. As an initial approach to test this hypothesis we analyzed the effect of the synthesis of rice Hb1 and Hb2 in the recombinant E. coli TB1 growth. Effect of the synthesis of the O2-carrying soybean leghemoglobin a, cowpea leghemoglobin II and Vitreoscilla Hb in the recombinant E. coli TB1 growth was also analyzed as an O2-carrier control. Our results showed that synthesis of rice Hb1, rice Hb2, soybean Lba, cowpea LbII and Vitreoscilla Hb inhibits the recombinant E. coli TB1 growth and that growth inhibition was stronger when recombinant E. coli TB1 synthesized rice Hb2 than when synthesized rice Hb1. These results suggested that rice Hb1 and Hb2 could function differently in vivo.