The oxyhemoglobin reaction of nitric oxide

Abstract

The oxidation of nitric oxide (NO) to nitrate by oxyhemoglobin is a fundamental reaction that shapes our understanding of NO biology. This reaction is considered to be the major pathway for NO elimination from the body; it is the basis for a prevalent NO assay; it is a critical feature in the modeling of NO diffusion in the circulatory system; and it informs a variety of therapeutic applications, including NO-inhalation therapy and blood substitute design. Here we show that, under physiological conditions, this reaction is of little significance. Instead, NO preferentially binds to the minor population of the hemoglobin's vacant hemes in a cooperative manner, nitrosylates hemoglobin thiols, or reacts with liberated superoxide in solution. In the red blood cell, superoxide dismutase eliminates superoxide, increasing the yield of S-nitrosohemoglobin and nitrosylated hemes. Hemoglobin thus serves to regulate the chemistry of NO and maintain it in a bioactive state. These results represent a reversal of the conventional view of hemoglobin in NO biology and motivate a reconsideration of fundamental issues in NO biochemistry and therapy.

Figure 1

Production of iron nitrosylHb by addition of NO to variously oxygenated Hb. (A) EPR spectra of iron-nitrosyl Hb derivatives formed by incubation of 19 μM NO with 393 μM Hb at various degrees of oxygen saturation in 10 mM phosphate buffer, pH 7.4. The oxygen saturations for the largest through smallest EPR signals are 5.5, 32, 50, and 69%, respectively. Spectra show predominantly six coordinate α and β nitrosyl hemes, as typically observed for Hb in R state. (B) EPR spectra of iron-nitrosyl Hb derivatives formed by incubation of 55 μM NO with 380 μM Hb at various degrees of oxygen saturation in 100 mM phosphate, pH 7.4. The oxygen saturations for the largest through smallest EPR signals are 1, 15, 41, 60, and 80%, respectively. Spectra show a significant component of five coordinate α nitrosyl hemes (triplet structure) associated with Hb in T state. (C) Trials conducted with Hb in 10 mM phosphate, pH 7.4. The symbols are experimental results and the solid lines represent a best fit to the functional form for cooperative NO binding. Open diamonds, 393 μM Hb incubated with 19 μM NO; open circles, 350 μM Hb incubated with 15 μM NO plus 0.05% borate (added to bring the buffer concentration to 100 mM as in D); open squares, 365 μM Hb incubated with 15 μM NO and 1,190 units/ml SOD. (D) Trials conducted with Hb in 100 mM phosphate, pH 7.4. The symbols are experimental results, and the lines represent a best fit to the functional form for simple competition between oxidation and NO addition reactions (Eq. 4). Filled circles, 380 μM Hb incubated with 55 μM NO; filled squares, 375 μM Hb incubated with 7 μM NO. Application of the simple competition function to data of C or the cooperativity function to data of D gives an order of magnitude increase in χ².

Figure 2

Production of metHb by reaction of NO is disfavored with increasing oxygen saturation. The samples used in Fig. 1 were assayed for metHb production by UV-visible difference spectroscopy. The data are normalized to added [NO]. As in Fig. 1, open diamonds, 10 mM phosphate; open circles, 10 mM phosphate plus borate; open squares, 10 mM phosphate plus SOD; filled circles and filled squares, 100 mM phosphate. The dotted (10 mM phosphate) and dashed (100 mM phosphate) lines are calculated by using Eq. 5 and Fe(II)NO yields in Fig. 1 C and D, respectively. Data show metHb to be disfavored in low phosphate, particularly at high oxygen saturation. Deviations of the data points below the curves suggest the presence of additional reactions for NO. Systematic deviations are most pronounced in low phosphate at high oxygen saturation—i.e., under physiological conditions.

Figure 3

NO addition under normoxic conditions (≈99% O₂ saturation) produces nitrosylated Hb. (A) Nitrosyl content of oxyHb (10 mM phosphate/100 μM DTPA, pH 7.4) after exposure to 1.2 μM NO, as measured by photolysis-chemiluminescence (26). Nitrosyl yield increases as a function of Hb concentration (P < 0.05). Solid symbols, absolute yield of NO bound to Hb (FeNO plus SNO); open symbols, percentage of NO added. Data shown are the average of 7 to 19 experiments ±SE. (B) Standard difference spectra of metHb (solid line), deoxyHb (dotted line), and iron nitrosylHb (dashed line) vs. oxyHb (see Materials and Methods for conditions). (C) Difference spectra generated from the exposure of NO to normoxic (≈99% oxygen sat.) Hb. NO was added (in 10 aliquots totaling 2.2 μM) to 33 μM Hb in 100 mM phosphate (solid line) or 10 mM phosphate (dotted line) or 10 mM phosphate plus 0.05% borate (dashed line). Notably, the spectrum in 100 mM phosphate shows the formation of metHb (e.g., peak at 630 nm; see B for comparison); the spectrum in 10 mM phosphate shows formation of iron nitrosyl Hb and some metHb [e.g., peak at 595 nm (nitrosyl) and small peak at 630 nm (met); see B for comparison]; and the spectrum in 10 mM phosphate plus borate shows predominantly iron nitrosylHb (e.g., peak at 595 nm; see B for comparison). (D) Calculated fits for difference spectra shown in C, demonstrating simple (noncooperative) competition between NO binding and oxidation reactions in high phosphate (solid line, 95% metHb) and cooperative binding in low phosphate (dotted line, 54% iron nitrosylHb; only 50% of the added NO accounted for) and low phosphate plus borate (dashed line; 85% iron nitrosylHb). Specifically, spectra in C were fitted, by a least-squares process, to either the simple competition model or the cooperativity model without a mass balance constraint.

Figure 4

S-nitrosoHb and iron nitrosylHb formed under various physiological air-oxygenated conditions. (A) SOD increases the yield of NO bound to Hb. The experiments in Fig. 3A were repeated in the absence (solid line; 1.2 μM NO) or presence (dashed line; 1.5 μM NO) of 1,190 units/ml of SOD, which enhances the yield of nitrosyl species to approximately 100% of the NO added. Similar nitrosyl yields were obtained by using stroma-free Hb (25 μM), which is enriched in endogenous SOD (open circle). Data shown are the average of five to nine experiments ±SE. (B) EPR spectrum of a DNIC formed by exposure of oxyHb (≈99% sat; 3.93 mM) to NO (36 μM). (C) S-nitrosoHb and iron nitrosylHb formed by exposure of oxyHb (≈99% sat., 48 μM) to NO (1.2 μM). SNO (hatched bar) and FeNO (solid bar) were measured by photolysis-chemiluminesence (26). Data shown are the average of 12 experiments ±SE. (D) Measurement of intraerythrocytic S-nitrosoHb and iron nitrosylHb formed by exposure of oxygenated RBCs (mean [Hb], 25 μM) to 1 μM NO. Isolation of Hb and measurements were as previously described (26). Data are the mean of 12 experiments ±SE.