EPR spectroscopy of nitrite complexes of methemoglobin

Abstract

The chemical interplay of nitrogen oxides (NO's) with hemoglobin (Hb) has attracted considerable recent attention because of its potential significance in the mechanism of NO-related vasoactivity regulated by Hb. An important theme of this interplay-redox coupling in adducts of heme iron and NO's-has sparked renewed interest in fundamental studies of FeNO(x) coordination complexes. In this Article, we report combined UV-vis and comprehensive electron paramagnetic resonance (EPR) spectroscopic studies that address intriguing questions raised in recent studies of the structure and affinity of the nitrite ligand in complexes with Fe(III) in methemoglobin (metHb). EPR spectra of metHb/NO(2)(-) are found to exhibit a characteristic doubling in their sharper spectral features. Comparative EPR measurements at X- and S-band frequencies, and in D(2)O versus H(2)O, argue against the assignment of this splitting as hyperfine structure. Correlated changes in the EPR spectra with pH enable complete assignment of the spectrum as deriving from the overlap of two low-spin species with g values of 3.018, 2.122, 1.45 and 2.870, 2.304, 1.45 (values for samples at 20 K and pH 7.4 in phosphate-buffered saline). These g values are typical of g values found for other heme proteins with N-coordinated ligands in the binding pocket and are thus suggestive of N-nitro versus O-nitrito coordination. The positions and shapes of the spectral lines vary only slightly with temperature until motional averaging ensues at approximately 150 K. The pattern of motional averaging in the variable-temperature EPR spectra and EPR studies of Fe(III)NO(2)(-)/Fe(II)NO hybrids suggest that one of two species is present in both of the alpha and beta subunits, while the other is exclusive to the beta subunit. Our results also reconfirm that the affinity of nitrite for metHb is of millimolar magnitude, thereby making a direct role for nitrite in physiological hypoxic vasodilation difficult to justify.

Figure 1

X-Band EPR spectra of solutions of metHb:NO₂⁻ in HEPES (—) or PBS (− − −) buffers at 20 K. The samples included metHb at 0.5M and twenty-fold molar excess (per heme) of NaNO₂; the buffer concentration was 0.1M and the pH 7.4. EPR spectra were obtained with the following spectrometer parameter values: 0.5 s time constant, 5 G modulation amplitude, 16.67 G/s sweep rate, 5 mW microwave power, and 9.24 GHz microwave frequency.

Figure 2

EPR spectra of solutions of metHb:NO₂⁻ in H₂O and D₂O in boiling nitrogen (76K). The protein concentration was 0.5M in the H₂O sample and 0.4M in the D₂O sample. Both samples contained a twenty-fold molar excess (per heme) of NaNO₂ m, and buffered at an effective pH of 7.4 in HEPES. Spectral amplitudes were corrected for difference in concentration. Both EPR spectra were recorded at 9.12 GHz with a 16.67 G/s sweep rate, a 0.5 s time constant, 5 G modulation amplitude, and 10 mW of microwave power.

Figure 3

S-Band EPR spectrum of solutions of metHb:NO₂⁻. The sample was prepared in pH 7.4 HEPES buffer with a protein concentration of 0.75M and a twenty-fold molar excess (per heme) of NaNO₂. The spectrum was obtained with the sample at ~40K , and at a frequency of 3.98 GHz , with a a sweep rate of 17.88 G/s , a time constant of 0.328 s, 5 G modulation amplitude, and 9.7 mW microwave power.

Figure 4

Variable Temperature EPR spectra of solutions of metHb:NO₂⁻ in HEPES buffer at pH 7.4. The sample included metHb at 0.65M and a twenty-fold molar excess (per heme) of NaNO₂⁻. Spectra were obtained at 22, 32, 43, 54, 65, 76, 86, and 97 K. The EPR spectra were recorded at 9.24 GHz with a 16.67 G/s sweep rate, a 0.5 s time constant, 5 G modulation amplitude, and 5 mW of microwave power.

Figure 5

Variable Temperature EPR spectra of solutions of metHb:NO₂⁻ in HEPES buffer at pH 7.4. The sample included metHb at 0.65M and a twenty-fold molar excess (per heme) of NaNO₂⁻. Spectra were obtained at the indicated temperatures in the range 100K to 175K. EPR spectra were recorded at 9.1 GHz with a 16.67 G/s sweep rate, a 0.5 s time constant, 5 G modulation amplitude, and 10 mW of microwave power.

Figure 6

EPR spectra of Fe(II)NO/Fe(III)NO₂⁻hybrids and related hemoglobins. For detail, only the variable, low-field portion of the spectrum is shown. (A) metHb:NO₂⁻ in HEPES, pH 7.4; (B) β-Fe(II)NO/α-Fe(III)NO₂⁻ in HEPES, pH 7.4; (C) SNO-metHb:NO₂⁻ in HEPES pH 7.4; (D) α-Fe(II)NO/β-Fe(III)NO₂⁻ hybrid in HEPES pH 7.4; (E) metHb:NO₂⁻ in PBS pH 7.4. All spectra were recorded at 20 K with a frequency of 9.24 GHz, 0.5 s time constant and sweep rate of 16.67 G/s. For spectra A and E the modulation amplitude was 5 G and the microwave power was 5 mW. Spectra B-D were recorded with 1 G modulation amplitude and 10 mW microwave power.

Figure 7

EPR spectra of Fe(II)NO of Fe(II)NO/Fe(III)NO₂⁻ hybrid hemoglobins Only the central region of the spectrum, featuring the the ferrous nitrosyl EPR signal is shown. (A) β-Fe(II)NO/α-Fe(III)NO₂⁻, as in fig. 6B. (B) α-Fe(II)NO/β-Fe(III)NO₂⁻ hybrid as in fig.6D. Spectra were obtained with the sample in boiling nitrogen (76 K), at a microwave frequency of 9.12 GHz, 10mW microwave power, 5 G modulation amplitude, a sweep rate of 3.33 G/s, and a time constant of 0.128 s.

Figure 8

pH dependence of EPR spectrum of metHb:NO₂⁻. Solutions of metHb in PBS poised at pH values as indicated in figure, 0.5mM in protein and 40mM in NaNO₂ for all samples. The EPR spectra were recorded at 20 K with a microwave frequency of 9.24 GHz, a 16.67 G/s sweep rate, 0.5 s time constant, 5 G modulation amplitude, and 5 mW of microwave power.

Figure 9

Exemplary Hill plot summarizing the titration of metHb with NaNO₂. The species concentrations used in the plot were obtained from detailed simulation of experimental EPR spectra. For the particular trial depicted in this plot, the protein concentration was 0.5 mM (pH 7.4 HEPES), with NaNO₂ at, alternatively, one-, five-, ten- and twenty-fold excess over heme. Supporting data are provided in supplementary figures.