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

Abstract

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.

Keywords: Escherichia coli; dinitrosyl iron complexes; electron paramagnetic resonance; leghemoglobin; nitrosative stress; oxidative stress; protein expression; spectrophotometry.

Figure 1

The absorption spectra of E. coli cells washed from medium, representing the difference between spectra of actively synthesizing Lba cells (C_Lb++) and nonsynthesizing Lb ones (C_Lb−): the spectrum of C_Lb++ cells minus the spectrum of C_Lb− cells.

Figure 2

Growth curves of E. coli (C_Lb++) at different concentrations of S-nitrosoglutathione (GSNO) (a), tert-butyl hydroperoxide (t-BOOH) (b), and benzylviologene (Bv) (c). Inducers of oxidative and nitrosative stress were added after 3 h of E. coli growth. Structural formulas of GSNO, t-BOOH, and Bv are shown on (d).

Figure 3

The growth of the bacterial culture, in percentage of the control culture growth (without adding inducers of oxidative and nitrosative stress). Inducers of oxidative and nitrosative stress were added after 3 h since the flasks sowing in concentrations: GSNO—0.2 mM, t-BOOH—0.04 mM, Bv—0.125 mM. Picture panels: GSNO—(a), t-BOOH—(b), Bv—(c), GSNO + t-BOOH—(d), and GSNO + Bv—(e).

Figure 4

The concentration of heme—(a), and the total peroxidase activity—(b) of the cell lysate proteins in the middle of the logarithmic growth phase (4 h after the addition of the inducers of oxidative and nitrosative stress—7 h of growth).

Figure 5

The dependence of the leghemoglobin (Lb) peroxidase reaction rate on the concentration of the substrate (t-BOOH). The insert shows the dependence in the Linuiver–Burke coordinates. Peroxidase activity was measured in the presence of 8 µM Lb and 0.8 mM o-dianisidine.

Figure 6

The dependence of initial rate of the peroxidase reaction catalyzed by Lba and by horseradish root peroxidase (HRP) on the concentration of t-BOOH. Peroxidase activity was measured in the presence of 8 µM Lb or 0.8 µM HRP and 0.8 mM o-dianisidine.

Figure 7

Absorption spectra of recombinant Lba—the black curve—and its complex with DNIC—the red curve. The insert shows the spectrum of dinitrosyl iron complexes (DNICs) with phosphate ligands.

Figure 8

Electron paramagnetic resonance (EPR) spectra of DNICs bound with human methemoglobin (metHb) and soybean metLb. (a): 1—the spectrum of DNICs with phosphate ligands used to produce protein DNIC; 2—spectrum of Hb-bound DNICs (Hb-DNICs). (b): 1—spectrum of Lb-bound DNICs (Lb-DNIC); 2—the same as (1) + 1 mM t-BOOH. Spectra recorded at room temperature.

Figure 9

Low-temperature EPR spectra of E. coli cells: C_Lb− (a) and C_Lb++ (b). 1—untreated bacteria; 2—bacteria after incubation with t-BOOH; 3—after incubation with GSNO; 4—after incubation with GSNO + t-BOOH. The registration was carried out at −170 °C.