Nitric-oxide dioxygenase function of human cytoglobin with cellular reductants and in rat hepatocytes

Abstract

Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b(5) were found to support a high NOD activity. Cygb-NOD activity shows respective K(m) values for ascorbate, cytochrome b(5), NO, and O(2) of 0.25 mm, 0.3 microm, 40 nm, and approximately 20 microm and achieves a k(cat) of 0.5 s(-1). Ascorbate and cytochrome b(5) reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m(-1) s(-1) and 3 x 10(6) m(-1) s(-1), respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar k(cat) of 1.2 s(-1), a K(m)(NO) of 40 nm, and a k(cat)/K(m)(NO) (k'(NOD)) value of 3 x 10(7) m(-1) s(-1), demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O(2)] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b(5) as reductants.

FIGURE 1.

Catalytic NO metabolism by globins in vitro. Human Cygb (A), mouse Ngb (B), and sperm whale Mb (C) were measured for NO metabolic activity at 37 (●) or 20 °C (■) in a 2-ml reaction volume containing 10 mm l-ascorbate, 100 mm sodium phosphate, pH 7.0, 0.3 mm EDTA, and 11 μm MnSOD. The reactions were initiated with 400 nm NO, and the activities were determined for 100 nm NO. The concentrations of globins are relative to heme content.

FIGURE 2.

Cygb-catalyzed NO metabolism with ascorbate. A, Cygb activity was measured by varying concentrations of ascorbate with 200 μm O₂ and 200 nm NO. B, activity was measured with 200 μm O₂ for different [NO]. C, activity was measured at different [O₂] with 100 nm NO. D, activity was measured with 20 μm O₂, 150 nm NO, and varying [CO]. E, activity was measured for 100 nm NO with no addition (●), the addition of 250 μm sodium cyanide (○), or following a 7-min preincubation with 250 μm sodium cyanide (▵). All of the measurements were made with 22.5 nm Cygb (heme) at 37 °C in 100 mm sodium phosphate buffer, pH 7.0, containing 0.3 mm EDTA, 11 μm MnSOD, and 10 mm ascorbate, unless otherwise specified. The reactions were initiated with 400 nm NO.

FIGURE 3.

Anions competitively inhibit ascorbate-dependent NO metabolism. A, NO metabolism was measured with 7.5–30 nm Cygb (heme) at 37 °C in 10 mm sodium phosphate buffer with 0, 50, 100, and 200 mm KCl (lines 1–4, respectively) or 100 mm NaCl (line 5). B, K_m(ascorbate) values are plotted versus buffer conductivity. The values are for 10, 20, 50, or 100 mm sodium phosphate buffer (□), for 10 mm sodium phosphate buffer containing either 50, 100, or 200 mm KCl (●) or 100 mm NaCl (○), and for 25 mm sodium citrate buffer alone (▵) or containing 100 mm NaCl (◇) or 50 mm sodium phosphate (▴). All of the buffers were pH 7.0 and contained 0.3 mm EDTA and 11 μm MnSOD.

FIGURE 4.

Cygb-catalyzed NO metabolism with cellular reductants and reductases. A, activity of 15 nm Cygb in the presence of varying concentrations of rat cytochrome b₅, 100 μm NADPH, and 20 milliunits of ferredoxin-NADP⁺ oxidoreductase. B, activity of 15 nm Cygb with varying concentrations of soluble human CYPOR (2 milliunits/nmol) and 20 μm NADPH. C, activity of 30 nm Cygb with varying concentrations of NCB5OR and 100 μm NADH. D, activities of various concentrations of Cygb (●), Ngb (■), and Mb (▵) with 28 nm NCB5OR and 100 μm NADH. Globin concentrations represent heme content. The reactions in A–D were in 2-ml of 100 mm sodium phosphate buffer, pH 7.0, containing 0.3 mm EDTA, 11 μm MnSOD, and 200 μm O₂. The reactions were initiated with 400 nm NO, and all of the activities were measured at 150 nm NO. E, human microsomes (20 μl) were measured for NO metabolic activity at 1 μm NO with or without 100 μm NADPH and with or without 300 nm Cygb in 100 mm sodium HEPES buffer, pH 7.8, containing 0.25 m sucrose, 11 μm MnSOD, and 200 μm O₂ at 37 °C. The error bars represent the S.D. of three measurements.

FIGURE 5.

Cygb expression in cultured rat hepatocytes (K9), A549, and Caco-2 cells. Human Cygb was measured in K9, A549, and Caco-2 cell extracts (A) and in K9neo and K9Cygb cell extracts (B) by Western blot analysis as under “Materials and Methods.” The gel lanes were loaded with 200 (A) or 100 μg (B) of cell-free extract protein or the indicated amounts of human Cygb. The Cygb monomer (mono) and dimer (di) signals are labeled.

FIGURE 6.

NO consumption by K9neo and K9Cygb cells. Cellular NO consumption rates were measured for 200 nm NO in the presence of 200 μm O₂ (A). K9neo (white bars) and K9Cygb (black bars) NO consumption activities were measured at 10 nm NO with the indicated O₂ concentrations (B). NO metabolism was measured at 37 °C in Dulbecco's phosphate-buffered saline containing 5 mm glucose and 100 μg/ml cycloheximide. The error bars represent the S.D. of three measurements.

FIGURE 7.

Cygb-catalyzed NO consumption in rat hepatocytes. A, activity was measured at different NO concentrations with 200 μm O₂. B, activity was measured for 20 nm NO with varying O₂ concentrations in the absence (●) or presence of 20 μm CO (○). C, activities of K9Cygb and K9neo cells were measured in the absence (● and ■, respectively) and presence of 250 μm NaCN (○ and □, respectively). The activities were measured for 100 nm NO following repetitive additions of 200 nm NO in the presence of 200 μm O₂. D, activity of K9Cygb cells was measured in the presence of either Me₂SO solvent (0.1% v/v) (●) or 50 μm DPI in Me₂SO (0.1% v/v) (○). The activities were measured for 100 nm NO following repetitive additions of 200 nm NO with 200 μm O₂. All of the measurements were at 37 °C. The error bars represent the S.D. of three independent measurements and in some cases are within data points.

FIGURE 8.

Putative ascorbate-binding site in Cygb. Ascorbate hydrogen bonds Arg-172 and Lys-30 in ascorbate peroxidase (Protein Data Bank code 1OAF) (55, 60). Hydrogen bonding stabilizes the ene-diol(ate) intermediate and facilitates the transfer of an electron via the heme proprionate (top). The A subunit of human Cygb dimer (Protein Data Bank code 1UMO) (58) shows conserved ArgE10–84 and LysFG2–116 residues for potential binding and stabilization of ascorbate (bottom left). The B subunit (bottom right) shows an alternate orientation of ArgE10–84, LysFG2–116, and the heme proprionate. Distances are given in Å by the triangles where the vertices represent the proprionate carboxylate O-atom and the arginine or lysine side chain N-atoms.