The nature of heme/iron-induced protein tyrosine nitration

Abstract

Recently, substantial evidence has emerged that revealed a very close association between the formation of nitrotyrosine and the presence of activated granulocytes containing peroxidases, such as myeloperoxidase. Peroxidases share heme-containing homology and can use H(2)O(2) to oxidize substrates. Heme is a complex of iron with protoporphyrin IX, and the iron-containing structure of heme has been shown to be an oxidant in several model systems where the prooxidant effects of free iron, heme, and hemoproteins may be attributed to the formation of hypervalent states of the heme iron. In the current study, we have tested the hypothesis that free heme and iron play a crucial role in NO(2)-Tyr formation. The data from our study indicate that: (i) hemeiron catalyzes nitration of tyrosine residues by using hydrogen peroxide and nitrite, a reaction that revealed the mechanism underlying the protein nitration by peroxidase, H(2)O(2), and NO(2)(-); (ii) H(2)O(2) plays a key role in the protein oxidation that forms the basis for the protein nitration, whereas nitrite is an essential element that facilitates nitration by the heme(Fe), H(2)O(2), and the NO(2)(-) system; (iii) the formation of a Fe(IV) hypervalent compound may be essential for heme(Fe)-catalyzed nitration, whereas O(2)(*-) (ONOO(-) formation), (*)OH (Fenton reaction), and compound III are unlikely to contribute to the reaction; and (iv) hemoprotein-rich tissues such as cardiac muscle are vulnerable to protein nitration in pathological conditions characterized by the overproduction of H(2)O(2) and NO(2)(-), or nitric oxide.

Scheme 1

Figure 1

Heme-catalyzed protein nitration. Immunoblotting for NO₂-Tyr in heme-catalyzed BSA. (A) BSA (2 mg/ml) nitration was detected while incubating with heme (25 μM), nitrite (1 mM), and H₂O₂ (1 mM) in 0.1 M sodium phosphate buffer (pH 7.4) at 37°C for 30 min. (B) The combination of a H₂O₂ generator glucose/glucose oxidase (10 mM/100 units) with the same concentration of heme and nitrite also induced BSA nitration. Tyrosine nitration catalyzed by heme is comparable with that induced by 5 mM ONOO⁻.

Figure 2

Effect of nitrite and hydrogen peroxide on protein nitration and oxidation in the heme/NO/H₂O₂ system. Protein nitration (○) was detected spectrophotometrically by monitoring pH-dependent NO₂-Tyr absorbance (n = 3). Protein oxidation (carbonyls; ▵) was determined spectrophotometrically by measuring formation of the 2,4-dinitrophenylhydrazone (n = 3). (A) Reaction mixtures (in 0.1 M phosphate buffer) containing 2 mg/ml BSA, 25 μM heme, 1 mM H₂O₂, and different concentrations of NO were incubated at 37°C for 30 min. Correlation coefficients (r) were 0.98 for NO₂-Tyr formation and ≈0 for carbonyl formation. (B) The reaction mixtures (in 0.1 M phosphate buffer) containing 2 mg/ml BSA, 25 μM heme, 1 mM NO, and different concentrations of H₂O₂ were incubated at 37°C for 30 min. Both protein nitration and oxidation depend on H₂O₂ with the optimal concentration of 0.5 mM. Higher concentrations of H₂O₂ significantly attenuated the level of BSA tyrosine nitration while exhibiting less interference of the carbonyl formation.

Figure 3

Effects of SODs and catalase on heme and MPO-catalyzed nitration. (A) The scheme shows the effects of SOD and catalase on the reactive oxygen species and reactive nitrogen species generating system. (B) Immunoblotting for NO₂-Tyr that was catalyzed by 25 μM heme or 2 units/ml MPO while incubating with NO (1 mM) and H₂O₂ (1 mM) in 0.1 M sodium phosphate buffer (pH 7.4) at 37°C for 30 min. Treatment of SOD (10 and 100 units/ml) had no inhibitory effect on BSA nitration. In contrast, the application of catalase (500 units/ml) abolished both heme and MPO-induced protein tyrosine nitration.

Figure 4

Iron-catalyzed protein nitration. Immunoblotting for NO₂-Tyr in BSA (2 mg/ml) that was incubated with either ferrous (Fe²⁺; 0.1 mM) or ferric (Fe³⁺; 0.1 mM) ions plus NO(1 mM) and H₂O₂ (1 mM) in 0.1 M sodium phosphate buffer (pH 7.4) at 37°C for 16 h. To study the mechanism, cyanide (CN⁻; 0.8 mM) and EDTA (0.4 mM) were used as chelators. The labeling with the frame indicates the mixture of iron with chelator (CN⁻ or EDTA) before addition to the reaction buffer. The labeling without frame shows that iron and chelator (CN⁻ or EDTA) were added to the BSA-containing reaction buffer in a sequential manner.

Figure 5

Protein nitration in heme-rich organs. (A) Immunoblotting for NO₂-Tyr in tissue homogenates from brain, heart, liver, kidney, and skeleton muscle was compared. In the presence of heme (25 μM), H₂O₂ (1 mM), and NO (1 mM), protein nitration was observed in heart and skeleton muscle but not in the brain, liver, and kidney. (B) With increases of exogenous heme concentration to 50 μM, however, nitration in the brain was observed. (C) Immunoblotting for NO₂-Tyr in incubations of heart homogenate with or without exogenous heme. A significant nitration could be detected only by application of nitrite (1 mM) and H₂O₂ (1 mM) into the cardiac muscle homogenate, and addition of exogenous heme did not further increase the level of NO₂-Tyr formation.