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* This work was supported by the Natural Sciences and Engineering Research Council of Canada, the Canadian Institutes for Health Research, and the Fonds pour la Formation des Chercheurs et L'Aide à la Recherche (to A. M. E. and J. A. C.) and by a Graduate Fellowship from Concordia University (to A. A. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
NO reactions with hemoglobin (Hb) likely play a role in blood pressure regulation. For example, NO exchange between Hb and S-nitrosoglutathione (GSNO) has been reported in vitro. Here we examine the reaction between GSNO and deoxyHb (HbFeII) in the presence of both Cu(I) (2,9-dimethyl-1, 10-phenanthroline (neocuproine)) and Cu(II) (diethylenetriamine-N,N,N′,N“,N”-pentaacetic acid) chelators using a copper-depleted Hb solution. Spectroscopic analysis of deoxyHb (HbFeII)/GSNO incubates shows prompt formation (<5 min) of ∼100% heme-nitrosylated Hb (HbFeIINO) in the absence of chelators, 46% in the presence of diethylenetriamine-N,N,N′,N“,N”-pentaacetic acid, and 25% in the presence of neocuproine. Negligible (<2%) HbFeIINO was detected when neocuproine was added to copper-depleted HbFeII/GSNO incubates. Thus, HbFeIINO formation via a mechanism involvingfree NO generated by Cu(I) catalysis of GSNO breakdown is proposed. GSH is a source of reducing equivalents because extensive GSSG was detected in HbFeII/GSNO incubates in the absence of metal chelators. No S-nitrosation of HbFeII was detected under any conditions. In contrast, the NO released from GSNO is directed to Cysβ93 of oxyHb in the absence of chelators, but only metHb formation is observed in the presence of chelators. Our findings reveal that the reactions of GSNO and Hb are controlled by copper and that metal chelators do not fully inhibit NO release from GSNO in Hb-containing solutions.
Fourier transform infrared
HbS-nitrosated at Cysβ93
inductively coupled plasma
sodium phosphate buffer
red blood cell
Possible exchange of NO between thiols and hemoglobin (Hb)1 in red blood cells (RBCs) has been the focus of intense interest recently (
). Hence, it was proposed that most of the NO released from Cysβ93 is actually captured by HbFeII. Capture of NO released from GSNO is also possible and would compete with the exit of NO from the RBC. In fact, Spencer et al. (
). Therefore, we considered it likely that neocuproine would also inhibit NO release from GSNO in solutions containing HbFeII. To distinguish between direct reductive cleavage of GSNO by HbFeII(Reaction 2) and Cu(I)-catalyzed release (Reaction 4), it is necessary to remove all trace copper or prevent its turnover via redox cycling using GSH (Reaction 5) or another donor in the Hb-containing solutions.
Here we report the results of a detailed examination of HbFeII/GSNO incubates after 5 min in the presence of preferential chelators of Cu(I) (neocuproine) and Cu(II) (DTPA). Solutions of HbFeII that were not dialyzed and solutions that underwent exhaustive dialysis versus EDTA were used. Our direct spectroscopic and ESI-MS analyses reveal that NO release from GSNO is <2% in the presence of neocuproine in HbFeII/GSNO solutions containing dialyzed Hb. HbFeIINO formation, and hence GSNO breakdown, is ∼100% within 5 min in the absence of chelators. HbFeIINO formation is decreased by ∼50–75% in the presence of DTPA and neocuproine and in solutions containing dialyzed Hb without neocuproine. Because trace copper was found in all reagents by ICP-MS, these observations are consistent with Cu(I)-catalyzed release of NO (Reaction 4).
The source of the reducing equivalents for the prompt Cu(I)-catalyzed reductive cleavage of GSNO is also of interest. Extensive GSSG was formed in HbFeII/GSNO incubates in the absence of metal chelators, indicating that GSH is the main source of reducing equivalents under these conditions (Reaction 5). We detected less GSSG than expected by ESI-MS in HbFeII/GSNO incubates containing DTPA, although we observed ∼50% GSNO breakdown. This suggested HbFeII as a possible additional source of reducing equivalents to [Cu(II)(DTPA)]2− because related EDTA complexes were shown to be redox-active with Hb (
). Careful examination of the absorption (optical and FTIR) spectra revealed the presence of HbFeIII. Thus, our results indicate that the direct reduction of GSNO by HbFeII is unlikely to play a role in NO transport in RBCs. Nonetheless, HbFeII may indirectly provide a source of electrons for the reductive cleavage of GSNO or other S-nitrosothiols in vivo.
The realization that commonly used Cu(I) (neocuproine) and Cu(II) chelators (EDTA and DTPA) may not always prevent copper turnover is an important consideration in deciphering mechanisms ofS-nitrosothiol signaling and NO biochemistry in general. Although neocuproine, a tight binding Cu(I) chelator (Kd = 1.2 × 10−19m2) (
), is a better inhibitor than DTPA of GSNO breakdown (Reaction 4) in Hb-containing solutions, dialysis of the Hb samples and neocuproine addition were necessary to obtain negligible GSNO breakdown.
Finally, the prompt changes in HbFeIIO2 on incubation with GSNO are compared with those observed for the HbFeII incubates. The key results of this comparison are that in the absence of chelators, S-nitrosation of Cysβ93 of the oxy protein is extensive, and this competes with NO capture by the FeIIO2 heme to form HbFeIII and NO. In contrast, no S-nitrosation of the deoxy protein is detected, suggesting that all of the NO released from GSNO is captured by the FeII heme.
Human hemoglobin A was obtained from Sigma and used without further purification. Nanopure water (specific resistance, 18mΩ-cm) obtained from a Millipore Simplicity water purification system and treated with Chelex-100 (Sigma) to remove trace metal ions was used to prepare all H2O solutions. The reactions were carried out in 200 mm sodium phosphate buffer, pH 7.2 (NaPi), prepared from sodium phosphate salts (Fisher) in nanopure H2O. Stock solutions of 15 mmdiethylenetriamine-N,N,N′,N“,N”-pentaacetic acid (DTPA; ICN) and 650 μm2,9-dimethyl-1,10-phenanthroline (neocuproine; Sigma) were prepared in NaPi. Stock solutions of 250 mm GSNO (Cayman) in NaPi were prepared just before use in a glove bag (Aldrich) under nitrogen, and the GSNO concentrations were determined spectrophotometrically (ε333.5 nm = 774 m−1cm−1) (
Preparation and Optical Spectroscopy of Hb Samples
Typically 1 g of lyophilized metHb (HbFeIII) from the bottle was dissolved in 2 ml of NaPi. After 2 min of centrifugation at 12,000 rpm, the precipitate was discarded, and the dark red solution of HbFeIII was stored at 4 °C prior to use. An aliquot (10 μl) of the Hb solution was pipetted onto a 13-mm CaF2 window of a dismountable FTIR type cell (Harrick). The cell was immediately assembled using a 6-μm Teflon spacer (Harrick) and placed in a custom-made bracket in a Beckman DU 650 UV-visible spectrophotometer. HbFeIIIconcentrations were found to be 32 mm in heme assuming ε500 nm = 10 mm−1cm−1/heme and ε630 nm = 4.4 mm−1 cm−1/heme (
). This was confirmed by diluting the samples 103-fold, adding potassium ferricyanide and excess KCN (BDH chemicals), and reading the absorbance of the CN− adduct at 540 nm (ε540 nm = 11.0 mm−1cm−1/heme) in a 1-cm cuvette on a Beckman spectrophotometer.
HbFeII was prepared in the glove bag under N2by treating HbFeIII with equimolar sodium dithionite (Fisher) (
). HbFeIIO2 was prepared from HbFeIIby introducing a small volume of air into the sample using a syringe; for example, 720 μl of air was added to 200 μl of a 32 mm heme sample. A single addition of O2 in slight excess (8.5 μmol of O2/8 μmol of heme) yielded fully oxygenated Hb as indicated by the Soret spectrum recorded following a 5-min equilibration. HbFeIINO and HbFeIIINO were prepared from HbFeII and HbFeIII on exposure to NO gas. The optical spectra were recorded using a scan time of 1200 nm/min. Use of the FTIR type cell to record the optical spectra of the products formed in the HbFeII/GSNO incubates allowed measurements to be made at close to physiological concentrations of Hb (2–4 mm) (
Approximately 500 mg of HbFeIII was dissolved in 5 ml of 100 mmNa2EDTA (Sigma), pH 7.0, and allowed to stand for 30 min. This solution was dialyzed at 4 °C versus 500 ml of 10 mm Na2EDTA, pH 7.0, which was replaced with fresh solution six times in 24 h. The dialysis was continuedversus EDTA-free H2O, which was replaced with fresh solution 12 times in 48 h. After dialysis, HbFeIII was lyophilized and dissolved in NaPi to a concentration of 30 mm heme.
A PE Sciex Elan 6000 ICP-MS with a cross-flow nebulizer and a Scott type spray chamber was used to determine the amount of copper in the Hb and GSNO samples and in the buffers (see Table I). The RF power was 1000 W, and the argon flow was 0.85 liter/min, which gave the best sensitivity as determined by the recommended optimization procedure. The optimum lens voltage was determined by maximizing rhodium sensitivity, and the data were acquired in the pulse count mode (
). Stock Hb solutions in NaPi were added to 50 μl of 30% H2O2 (ACP Chemicals, Inc.) and 500 μl of concentrated HNO3(OmniTrace Ultra high purity, EM Science) to give a final heme (iron) concentration of 4.3 mm, and the samples were ashed using a Bunsen burner. The residue was dissolved in 10 ml of 5% (v/v) HNO3, and ICP-MS analyses for copper and iron were performed. An internal standard of 9 nm (0.500 ppb) manganese prepared from a 1000 ppm manganese standard solution (ACP Chemicals, Inc.) was added to all of the solutions. The standard curves were prepared by diluting 1000 ppm copper and iron standard solutions (ACP Chemicals, Inc.) in 5% (v/v) HNO3 and nanopure water to give 0–8 μm (0–0.500 ppm) copper and 0–9 μm (0–0.500 ppm) iron. All of the reported ICP-MS data are the results of at least triplicate experiments, and in all cases the standard deviations were <5%.
Table IICP-MS analysis of copper in stock solutions
Approximately 20 μl of Hb (28 mm heme) in NaPi was added by syringe onto a 13-mm CaF2 window in the glove bag under N2 where necessary. The FTIR cell was immediately assembled with a 250-μm Teflon spacer (Harrick), and the spectra were recorded at 25 °C on a Nicolet Magna-IR 550 spectrometer with a MCT detector cooled to 77 K and purged with dry air from a Whatman FTIR Purge (model 75-52). All of the reported spectra are averages of 500 scans recorded in 5.52 min at a resolution of 2 cm−1 using a Happ-Genzel apodization with a velocity and aperture of 4.4303 cm/s and 2, respectively. Omnic (Nicolet) software was used for subtraction, base-line correction, smoothing, and Fourier transform self-deconvolution employing a half-width-at-half-height of 0.6 cm−1 and an enhancement (K factor) of 1. Subtraction of water vapor absorption from the spectra was performed by the method of Dong et al. (
Stock Hb solutions (28 mm heme) in NaPi were diluted 10−3-fold with H2O to give ∼0.5 μg/μl protein. The aliquots were infused into the ESI source of the mass spectrometer (ThermoFinnigan SSQ 7000) by flow injection from the high performance liquid chromatography (Agilent 1090) using a 100-μl loop (but no column) at 50 μl/min with 75% CH3CN (0.05% trifluoroacetic acid) as a mobile phase. Stock (250 mm) GSH, GSNO, and GSSG solutions in 200 mm NaPi, pH 7.2, were diluted 500-fold with H2O and 10-fold to 50 μm with 75% CH3CN (0.05% trifluoroacetic acid), and their mass spectra were obtained as for the Hb solutions.
Multi-component Analysis of the Optical Spectra of Incubates
The absorbance at a given wavelength is the sum of the absorbances (A) of all species at that wavelength:A = Ax +Ay + Az + … = εxb [X] + εyb [Y] + εzb [Z] + … , where εx is the molar extinction of X at the selected wavelength, b is the pathlength (6 μm), and [X] is the molar concentration of X. Assuming a limited number of oxidation and coordination states for iron of Hb, the spectra recorded for the incubates were mathematically disassembled into those of their components. Absorbances at the Soret maxima of the Hb species were used to generate n Beer's law expressions, from which the concentrations ([X], [Y], [Z], etc.) of the nspecies present in the incubates were obtained. Solutions to then equations with n unknowns were obtained using a program at www.1728.com/indexalg.htm. For the HbFeII/GSNO incubates, absorbances were recorded at the Soret maxima of the expected heme species HbFeIII, HbFeIIINO, HbFeIINO, and HbFeII. The millimolar extinction coefficients (ε in mm−1 cm−1) and wavelengths (nm; in parentheses) used are the following: 169 (405), 110 (416), 97 (418), 50 (430) for HbFeIII; 95.5 (405), 129 (416), 130 (418), 95 (430) for HbFeIINO; 113 (405), 137 (416), 136 (418), and 64 (430) for HbFeIIINO and 62 (405), 92 (416), 99 (418), and 133 (430) for HbFeII. The corresponding values for the expected iron states in the HbFeIIO2/GSNO incubates are: 169 (405) and 116 (415) for HbFeIII and 102 (405) and 125 (415) for HbFeIIO2. The spectra of the components and mixtures were graphed using Origin 6.0 software (Microcal).
), we monitored HbFeII/GSNO incubates by optical and FTIR spectroscopies to probe changes occurring at the heme and Cysβ93 centers, respectively. In addition, the incubates were analyzed by ESI-MS to examine changes in the mass of the protein and to determine the low molecular weight species produced.
The optical spectra shown in Fig. 1 clearly reveal that trace Cu(I) is required for the release of NO from GSNO (Reaction 4). Essentially no HbFeIINO is formed in the HbFeII/GSNO incubate containing both copper-depleted Hb (i.e. dialyzed Hb in Table I) and neocuproine. The combined data in Tables I and II indicate that total inhibition of Cu(I) catalysis of GSNO breakdown requires removal of most of the copper as well as neocuproine addition to Hb-containing solutions. Thus, the results summarized in Table II are not consistent with direct reduction of GSNO by HbFeII (
) (Reaction 2) when equimolar heme and GSNO are present. Given the rapid (<5 min) Cu(I)-catalyzed release of NO from GSNO (Fig. 1b), it is likely that copper-catalyzed reductive cleavage of GSNO also occurs in vivo.
The next question that arises is, of course, what is the source of reducing equivalents to regenerate Cu(I) following electron transfer to GSNO to release NO (Reaction 4)? Possible electron donors to Cu(II) are GSH (Reaction 5) and HbFeII. With GSH as a donor, two molecules of NO would be released or two heme FeIINO adducts would be formed per molecule of GSSG produced. The mass spectrum reveals extensive formation of GSSG in the HbFeII/GSNO incubates in the absence of chelators (Fig.6b). Although a peak corresponding to its Na+adduct occurs at m/z 635, the expected GSSG peak at m/z 613 is not clearly evident in HbFeII/GSNO incubates containing DTPA. Nonetheless, GSNO does release NO, as evidenced by the loss of GSNO peak intensity relative to the base peak at m/z 638 (Fig. 6,c versus d) and the formation of HbFeIINO (Fig. 1c and Table II). Thus, reduction of [Cu(II)(DTPA)]2− by HbFeII was considered, which would account for the HbFeIII absorbance seen in Fig. 2a. From Table II, 7.13 mm NO is released (6.97 mm HbFeIINO and 0.16 mm HbFeIIINO) from the 15 mm GSNO present in the HbFeII/GSNO/DTPA incubate. If all of the reducing equivalents were to come from GSH oxidation (Reaction 5), then 3.56 mm GSSG should be produced. However, HbFeII oxidation provides 1.44 milliequivalents of reductant (1.28 mm HbFeIII and 0.16 mm HbFeIIINO), which would decrease the concentration of GSSG produced to 2.84 mm. This is approximately one-third of the GSSG produced in HbFeII/GSNO incubates where a strong GSSG peak is observed atm/z 613. The lower than expected intensity of the GSSG (m/z 613) peak in Fig. 6c is attributed to ion suppression. The production of HbFeIII in the HbFeII incubates in the presence but not the absence of DTPA (Table II) suggested that [Cu(II)(DTPA)]2− may accept an electron from HbFeII, which was confirmed. The addition of authentic [Cu(II)(DTPA)]2− to HbFeII led to oxidation of 21% of the heme (Fig. 7). Electron transfer between [Fe(II)(EDTA)]2− and HbFeIII has been reported previously (
No loss of ν(SH) intensity was detected by FTIR when HbFeII was treated with GSNO with or without metal chelators (Fig. 4a). This is consistent with the proposal that the allosteric transition of Hb controls Cysβ93S-nitrosation (
). The FTIR results also show that Cu(I) is required for HbFeIINO formation because neocuproine inhibited blue shifting of the Cysβ93 ν(SH) peak (Fig.4a, spectra 2 and 3) that accompanies heme nitrosylation (
). The reactions in the HbFeII/GSNO incubates reported here are shown in Schemefs1. Clearly the oxidation of HbFeII by [Cu(II)(DTPA)]2+ is unlikely to be of importance in vivo, but the requirement of copper for NO release from GSNO shown in Scheme FS1 could well have physiological relevance.
The key result that copper is also required forS-nitrosation of Cysβ93 of HbFeIIO2 (
) can be deduced from Figs.4b and 5b. S-Nitrosation of Cysβ93 is observed only in the absence of metal chelators (Fig. 4b, spectrum 1, and Fig. 5b). Thus, we propose Scheme FS2 for the reactions of NO with HbFeIIO2. NO is targeted to Cysβ93 in the absence of chelators, but in the presence of DTPA, all of the NO released from GSNO is targeted to the FeIIO2 heme (Figs. 3 and 4b) and converted to NO (Reaction 6). SchemeFS2 predicts that efficient Cu(II)–catalyzed S-nitrosation of Cysβ93 will preserve the biological activity of NO by preventing its conversion to nonvasoactive NO. The capture of NO by HbFeII (Scheme FS1) should not lead to loss of its vasoactive power as long as FeII-NO adduct formation is reversible on a physiologically relevant time scale. It has been proposed that the FeII heme centers of partially oxygenated Hb, as found in the RBC (
). Thus, copper control of NO reactivity with Hb, plus weaker binding of NO to HbFeIIin vivo thanin vitro, could explain why RBC Hb does not act a sink for most of the NO produced in the vascular system. A possible copper catalyst in vivo is copper-zinc superoxide dismutase, which is abundant in the RBC (
It is of interest to compare the extent of the prompt changes in HbFeII and HbFeIIO2 on incubation with GSNO. When 15 mm GSNO is incubated with 15 mm (heme) HbFeII in the absence of metal chelators, 99% is converted to HbFeIINO within 5 min, whereas only 46% of HbFeIIO2 is converted to HbFeIII under the same conditions. This is easy to understand when we consider that ∼7.5 mm NO released from GSNO was targeted to Cysβ93 in HbFeIIO2 but not in HbFeII (Figs. 4and 5). In the DTPA incubates 44% HbFeIIversus77% HbFeIIO2 remain, and in neocuproine incubates 75% HbFeIIversus 88% HbFeIIO2 remain after the same 5-min period. Clearly, the increased stability of the Hb reactants in the presence of metal chelators is due to inhibition of GSNO breakdown, but the lower consumption of HbFeIIO2 relative to HbFeII cannot be attributed to NO trapping by Cysβ93 in the former because this does not occur in the presence of chelators (Fig. 4b). More efficient NO trapping by HbFeII (
) would give rise to the higher HbFeII consumption in the incubates. Also, the reaction of NO with any free O2 in the HbFeIIO2 incubate would decrease the amount of HbFeIIO2 consumed.
In this work we also examined the spectra of the HbFeII/GSNO incubates after 1 h. The results obtained indicate that the prompt products undergo further reaction over longer times. For example, the Soret maximum after 1 h of HbFeII/GSNO incubate still shows a maximum at 418 nm but with decreased intensity (data not shown). Because the spectrum of authentic HbFeIINO in the absence of any glutathione-derived species is stable for >1 h, the HbFeIINO initially formed must react with some reagent in the incubate.2 The Soret maximum of the HbFeII/GSNO/DTPA incubate after 1 h (data not shown) is blue-shifted (409 nm) from that at 5 min (411 nm) (Fig.1c). We initially attributed this blue shift to increased HbFeIINO formation caused by NO-driven reduction of HbFeIII using reducing equivalents from GSH. However, the addition of GSH to authentic HbFeIIINO also gave rise to a Soret maximum at 411 nm and not the expected HbFeIINO peak at 418 nm. The biological relevance of these slow reactions is questionable because, for example, any HbFeIII formed in the RBC would be rapidly converted to HbFeII by methemoglobin reductase (
The results reported here provide insight into the mechanism of heme nitrosylation following mixing of HbFeII with GSNO. The data presented are inconsistent with direct reduction of GSNO by HbFeII but are consistent with catalysis of GSNO breakdown by trace copper and free NO generation in the reactions. Because HbFeII is stable in the presence of GSNO when free copper is rigorously excluded from the system, direct reduction of GSNO by HbFeII is unlikely to be of biological significance. Trace copper also controls targeting of NO released from GSNO to the Cysβ93 and FeIIO2centers of HbFeIIO2. Thus, we conclude that copper catalysis of S-nitrosation andS-denitrosation plays a key role in preserving the vasoactivity of NO in the RBC.
We thank Professor Eric Salin and Dr. John Tromp (Department of Chemistry, McGill University) for helping with the ICP-MS measurements and for the use of the ICP-MS instrumentation.
Proc. Natl. Acad. Sci. U. S. A.1999; 96: 9967-9969