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

Protective Effect of Dinitrosyl Iron Complexes Bound with Hemoglobin on Oxidative Modification by Peroxynitrite

Dinitrosyl iron complexes (DNICs) are a physiological form of nitric oxide (•NO) in an organism. They are able not only to deposit and transport •NO, but are also to act as antioxidant and antiradical agents. However, the mechanics of hemoglobin-bound DNICs (Hb-DNICs) protecting Hb against peroxynitrite-caused, mediated oxidative modification have not yet been scrutinized. Through EPR spectroscopy we show that Hb-DNICs are destroyed under the peroxynitrite action in a dose-dependent manner. At the same time, DNICs inhibit the oxidation of tryptophan and tyrosine residues and formation of carbonyl derivatives. They also prevent the formation of covalent crosslinks between Hb subunits and degradation of a heme group. These effects can arise from the oxoferryl heme form being reduced, and they can be connected with the ability of DNICs to directly intercept peroxynitrite and free radicals, which emerge due to its homolysis. These data show that DNICs may ensure protection from myocardial ischemia

Antiglycation and Antioxidant Effect of Nitroxyl towards Hemoglobin

Donors of nitroxyl and nitroxyl anion (HNO/NO−) are considered to be promising pharmacological treatments with a wide range of applications. Remarkable chemical properties allow nitroxyl to function as a classic antioxidant. We assume that HNO/NO− can level down the non-enzymatic glycation of biomolecules. Since erythrocyte hemoglobin (Hb) is highly susceptible to non-enzymatic glycation, we studied the effect of a nitroxyl donor, Angeli’s salt, on Hb modification with methylglyoxal (MG) and organic peroxide―tert-butyl hydroperoxide (t-BOOH). Nitroxyl dose-dependently decreased the amount of protein carbonyls and advanced glycation end products (AGEs) that were formed in the case of Hb incubation with MG. Likewise, nitroxyl effectively protected Hb against oxidative modification with t-BOOH. It slowed down the destruction of heme, formation of carbonyl derivatives and inter-subunit cross-linking. The protective effect of nitroxyl on Hb in this system is primarily associated with nitrosylation of oxidized Hb and reduction of its ferryl form, which lowers the yield of free radical products. We suppose that the dual (antioxidant and antiglycation) effect of nitroxyl makes its application possible as part of an additional treatment strategy for oxidative and carbonyl stress-associated diseases

Role of Nitric Oxide-Derived Metabolites in Reactions of Methylglyoxal with Lysine and Lysine-Rich Protein Leghemoglobin

Carbonyl stress occurs when reactive carbonyl compounds (RCC), such as reducing sugars, dicarbonyls etc., accumulate in the organism. The interaction of RCC carbonyl groups with amino groups of molecules is called the Maillard reaction. One of the most active RCCs is α-dicarbonyl methylglyoxal (MG) that modifies biomolecules forming non-enzymatic glycation products. Organic free radicals are formed in the reaction between MG and lysine or Nα-acetyllysine. S-nitrosothiols and nitric oxide (•NO) donor PAPA NONOate increased the yield of organic free radical intermediates, while other •NO-derived metabolites, namely, nitroxyl anion and dinitrosyl iron complexes (DNICs) decreased it. At the late stages of the Maillard reaction, S-nitrosoglutathione (GSNO) also inhibited the formation of glycation end products (AGEs). The formation of a new type of DNICs, bound with Maillard reaction products, was found. The results obtained were used to explain the glycation features of legume hemoglobin—leghemoglobin (Lb), which is a lysine-rich protein. In Lb, lysine residues can form fluorescent cross-linked AGEs, and •NO-derived metabolites slow down their formation. The knowledge of these processes can be used to increase the stability of Lb. It can help in better understanding the impact of stress factors on legume plants and contribute to the production of recombinant Lb for biotechnology

Role of Nitric Oxide-Derived Metabolites in Reactions of Methylglyoxal with Lysine and Lysine-Rich Protein Leghemoglobin

Carbonyl stress occurs when reactive carbonyl compounds (RCC), such as reducing sugars, dicarbonyls etc., accumulate in the organism. The interaction of RCC carbonyl groups with amino groups of molecules is called the Maillard reaction. One of the most active RCCs is α-dicarbonyl methylglyoxal (MG) that modifies biomolecules forming non-enzymatic glycation products. Organic free radicals are formed in the reaction between MG and lysine or Nα-acetyllysine. S-nitrosothiols and nitric oxide (•NO) donor PAPA NONOate increased the yield of organic free radical intermediates, while other •NO-derived metabolites, namely, nitroxyl anion and dinitrosyl iron complexes (DNICs) decreased it. At the late stages of the Maillard reaction, S-nitrosoglutathione (GSNO) also inhibited the formation of glycation end products (AGEs). The formation of a new type of DNICs, bound with Maillard reaction products, was found. The results obtained were used to explain the glycation features of legume hemoglobin—leghemoglobin (Lb), which is a lysine-rich protein. In Lb, lysine residues can form fluorescent cross-linked AGEs, and •NO-derived metabolites slow down their formation. The knowledge of these processes can be used to increase the stability of Lb. It can help in better understanding the impact of stress factors on legume plants and contribute to the production of recombinant Lb for biotechnology