Reaction of Human Myoglobin and Nitric Oxide

HEME IRON OR PROTEIN SULFHYDRYL (S) NITROSATION DEPENDENCE ON THE ABSENCE OR PRESENCE OF OXYGEN*

Paul K. WittingDagger , D. J. Douglas§, and A. Grant MaukDagger

From the Dagger  Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3 and the § Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada

Received for publication, June 30, 2000, and in revised form, October 12, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The amino acid sequence of human myoglobin (Mb) is similar to other mammalian Mb except for a unique cysteine residue at position 110 (Cys110). Anaerobic treatment of ferrous forms of wild-type human Mb, the C110A variant of human Mb or horse heart Mb, with either authentic NO or chemically derived NO in vitro yields heme-NO complexes as detected by electron paramagnetic resonance spectroscopy (EPR). By contrast, no EPR-detectable heme-NO complex was observed from the aerobic reactions of NO and either the ferric or oxy-Mb forms of wild-type human or horse heart myoglobins. Mass analyses of wild-type human Mb treated aerobically with NO indicated a mass increase of ~30 atomic mass units (i.e., NO/Mb = 1 mol/mol). Mass analyses of the corresponding apoprotein after heme removal showed that NO was associated with the apoprotein fraction. New electronic maxima were detected at A333 nm (epsilon  = 3665 ± 90 mol-1 cm-1; mean ± S.D.) and A545 nm (epsilon  = 44 ± 3 mol-1 cm-1) in solutions of S-nitrosated wild-type human Mb (similar to S-nitrosoglutathione). Importantly, the sulfhydryl S-H stretch vibration for Cys110 measured by Fourier transform infrared (nu  ~ 2552 cm-1) was absent for both holo- and apo- forms of the wild-type human protein after aerobic treatment of the protein with NO. Together, these data indicate that the reaction of wild-type human Mb and NO yields either heme-NO or a novel S-nitrosated protein dependent on the oxidation state of the heme iron and the presence or absence of dioxygen.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Endothelium-derived relaxant factor (EDRF)1 or endogenously derived nitric oxide (NO) (1) is formed enzymatically from L-arginine (2) and is an important mediator in a variety of physiologically relevant processes (3, 4). One important regulatory role is that of endothelium-dependent relaxation of vascular smooth muscle (5, 6), which affects vascular tone and ultimately circulating blood pressure (7). However, despite numerous studies describing the biological activity of EDRF/NO, there is no consensus regarding the biological half-life of NO (8, 9) or possible stabilization of NO by reaction with carrier molecule(s) in vivo that may result in a prolonged half-life with or without preservation of NO bioactivity (10, 11).

Among the biological targets of NO, low molecular weight reduced thiols, protein sulfhydryls (12-14), and heme and nonheme iron can be nitrosated (15-17). S-Nitrosothiols exhibit characteristic electronic maxima in the regions 330-350 and 530-550 nm (12), whereas heme-nitrosyl complexes can be readily detected directly by low temperature EPR spectroscopy (18, 19). The decomposition of S-nitrosothiols to regenerate NO is proposed to be a mechanism whereby NO may be either translocated or prevented from diffusing away from the site of synthesis (8).

Myoglobin is present in relatively high concentration in skeletal and cardiac muscle (2.1-6 mg/g of wet tissue (Refs. 20-22)), and myoglobin of identical sequence has recently been identified in human smooth muscle cells (23). Intracellular concentrations of ferric or metmyoglobin are strictly controlled in vivo by a "metmyoglobin reductase" that maintains the heme iron in the reduced form and able to bind dioxygen (24). Similar to oxyhemoglobin (25), oxymyoglobin (oxy-Mb) is able to oxidize NO to yield nitrate (NO3-) (26), and this process has been proposed as a mechanism to regulate NO in skeletal and cardiac muscle (9) and vascular smooth muscle (27).

The amino acid sequence of human Mb is unique among mammalian myoglobins in that it possesses a cysteine residue, Cys110 (28). Recently, we investigated the reaction of recombinant human Mb and hydrogen peroxide and showed that Cys110 is readily oxidized to a thiyl radical that promotes formation of a disulfide-linked dimeric Mb product (29). This reactivity of the sulfhydryl led us to hypothesize that human Mb may also react with NO to yield an S-nitrosothiol group at Cys110 of human Mb. If substantiated, such reactivity would endow Mb with the unique combination of abilities to preserve NO bioactivity through formation of a S-nitrosothiol group and to regulate NO in vivo by either formation of a nitrosyl Mb complex or oxidizing NO to NO3-. In the present work, we have studied the reaction of human Mb and its C110A variant to evaluate the relative reactivities of the thiol group at Cys110 and the heme iron in the in vitro reaction of wild-type human Mb with NO through the combined use of EPR, Fourier transform infrared (FTIR), electronic absorbance spectroscopy, and electrospray mass spectrometry.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials

2,2'-Dithiodipyridine (DTP), horse heart Mb, reduced glutathione (GSH), iodoacetamide, trypsin (type III, 10,200 units/mg of protein), urea, EDTA, 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), Tris buffer, and diethylenetriamine pentaacetic acid (DTPA) were obtained from Sigma. Diethylammonium(z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DeaNO, epsilon 260 ~ 6500 M-1 cm-1) was from Cayman Chemicals (Ann Arbor, MI). Electronic absorbance spectra of solutions of DeaNO at pH 10 and 7.4 indicated a single maximum at A260 nm consistent with that reported by the manufacturer, so the reagent was used without further purification. Concentrated stock solutions of DeaNO (up to 100 mM in 1 mM NaOH) were prepared immediately prior to use. NaNO2 (purity 97%) was from Eastman Kodak Co. Tryptone and yeast extract were from Becton Dickinson (Sparks, MD). NaCl (purity 99.5%) was obtained from Fisher Scientific (Fair Lawn, NJ). NO gas (purity 98.5%) was from Linde Union Carbide (Praxair, Vancouver, Canada). Where required, buffers were prepared from either glass-distilled water or glass-distilled water purified further by passage through a Barnstead Nanopure system. All buffers were stored over Chelex-100® (Bio-Rad) at 4 °C for at least 24 h to remove contaminating transition metals as verified by ascorbate autoxidation analysis (30). Organic solvents and all other reagents employed were of the highest quality available.

Preparation of Recombinant Proteins

Transformed bacteria (strain AR68) containing plasmids for the wild-type recombinant human myoglobin and the C110A variant (31) were obtained from Prof. S. G. Boxer. The procedures used here for the expression and purification of the various proteins has been described in detail elsewhere (29, 31). The respective recombinant myoglobins were isolated as the corresponding metmyoglobin. When required, proteins were concentrated by centrifugal ultrafiltration (Centriprep-10 concentrators, Millipore).

Samples of the various apo-Mbs were prepared by adjusting the pH of corresponding solutions of holo-Mb in Tris buffer (5 mM, pH 7.4) to pH = 2.5 by addition of 1 M HCl at 4 °C. Free heme was then removed from the mixture by repeated extraction (three times) of the acidified fraction into ice-cold 2-butanone as described previously (32). The acidified aqueous fraction was then dialyzed extensively against the buffer of choice (2, 20, and 50 mM phosphate buffer, pH 7.4, containing 100 µM DTPA), and the concentration of apo-Mb was determined from the A280 nm (epsilon 280 = 16 mM-1 cm-1). For all apoprotein preparations, no other absorption maxima were detected over A220-700 nm except for a residual Soret band (<2% of the initial Soret intensity) that could not be eliminated by further extraction into 2-butanone. All working solutions of apo-Mb were prepared immediately before use to avoid inadvertent oxidation of redox-sensitive amino acids (e.g. Cys110 in wild-type human Mb).

Reduction of Ferric Myoglobin(s) to Ferrous Myoglobin(s)

Samples of metmyoglobin were reduced to the corresponding ferrous protein with a 2-3-fold molar excess of Fe(EDTA)2- (33) under an atmosphere of nitrogen. Oxygen was removed from protein solutions either by gently purging solutions of the ferric or ferrous holo-Mb (or where applicable apo-Mb) with purified nitrogen gas that had previously passed through a vanadium(II)/mercury-zinc amalgam (34) or by alternately freezing and thawing the sample under vacuum with a dual manifold vacuum line. Anaerobic reactants and reaction mixtures were transferred with gas-tight syringes and placed in flasks fitted with rubber septa to avoid oxygen contamination. In some instances, dioxygen (99.0% purity, Praxair, Vancouver, Canada) was bubbled directly into samples of ferrous Mb, and then excess Fe(EDTA)2- and Fe(EDTA)- were removed by gel filtration (PD-10 G-25 column, Amersham Pharmacia Biotech, Uppsala, Sweden). Under these conditions, proteins were isolated as the corresponding ferrous oxy-Mb.

Preparation of S-Nitrosated and Heme-Nitrosyl Complexes

In this investigation, three sources of nitrosating reagents were employed throughout to yield nitroso complexes at either sulfhydryl (S) or heme (iron) centers. Each of the sources has been verified previously as established means of generating NO (35-38).

Method 1-- Solutions of GSH or apo-Mb (either modified with iodoacetamide or not for the case of wild-type human apo-Mb) were prepared in 0.5 M HCl, exposed to NaNO2 (NO:target sulfhydryl ~2 mol/mol) and the reaction mixture stirred for 30 min at 20 °C in either the presence or absence of air. Acidic solutions of NO2- yield NO gas in a stoichiometric reaction (18). The pH of the reaction mixtures was then raised to pH 7.4 by titration with phosphate buffer (final phosphate concentration 2, 25, or 50 mM and containing DTPA (100 µM)) and the protein was eluted over two gel filtration columns (PD-10) to remove excess nitrite/nitrate. As holo-Mb is sensitive to acidic conditions (<pH ~ 5.5), nitrosation of either ferric or ferrous holo-Mb was not performed by method 1 but by one of the following techniques.

Method 2-- Solutions of ferric or ferrous holo-Mb were degassed with purified nitrogen (where appropriate) and then treated with authentic NO gas in a dual manifold vacuum line. Prior to the introduction of nitric oxide gas into the vacuum line, the NO was first passed through an anaerobic column of solid KOH pellets at 5 p.s.i. and 20 °C and then introduced to the sample chamber. Protein solutions were then stirred continuously for 20 min under an atmosphere of NO gas (39) (concentration of dissolved NO ~2.1 mM at 20 °C, based on solubility (X) of NO = 3.78 × 10-5 mol of NO/mol of H2O at the prevailing atmospheric pressure). Finally, reaction mixtures were removed from the gas line, and excess low molecular weight contaminants were removed by gel filtration (PD-10) using columns pre-equilibrated with appropriately degassed buffers.

Method 3-- Solutions of apo-Mb or ferric or ferrous holo-Mb (each prepared in 2, 25, or 50 mM phosphate buffer, pH 7.4) were treated with alkaline solutions (1 mM NaOH) of concentrated DeaNO (at ratios of DeaNO:protein ~ 0-20 mol/mol). Concentrated stock solutions of DeaNO were used to minimize hydroxide addition to the buffered solutions and resulting changes in pH. Where appropriate, stock reagents and solutions containing the target protein were thoroughly degassed prior to mixing to yield anaerobic reaction mixtures. Reaction mixtures were then purified by gel filtration (PD-10) using columns that had been equilibrated with appropriately degassed buffers.

Regardless of the method used to generate the nitrosated product, DTPA (final concentration, 100 µM) was included in all reaction mixtures containing sufhydryls to minimize the possibility of either transition metal-mediated oxidation of the protein sulfhydryl or the metal-mediated decomposition of the corresponding S-nitrosothiol (12, 37). Method 3 proved to be the most convenient protocol for addition of precise amounts of NO to solutions of Mb and so was used for all concentration-dependent studies (see "Results").

EPR Spectroscopy

X-band EPR spectroscopy was carried out at 77 K with a Bruker model ESP 300e spectrometer equipped with a Hewlett Packard microwave frequency counter. For low temperature EPR, samples (250 µl) of reaction mixture were placed into a 3-mm quartz cell (Wilmad, Buena, NJ), snap-frozen in liquid nitrogen, and transferred to a finger Dewar insert (77 K) for EPR analysis. The time required to removing the sample from the reaction vial and freezing for EPR analyses was consistently <10 s. The limit of detection of a stable nitroxide (TEMPO) under identical conditions was determined to be ~50 nM. Unless stated otherwise, EPR spectra were obtained as an average of five scans with sweep time of 84 s. Microwave power, modulation amplitude, and scan range were as indicated in the legend to the figure(s).

Electrospray Ionization Mass Spectrometry (ESI-MS)

ESI-MS was performed with a triple quadrupole mass spectrometer described in detail elsewhere (40). Samples of Mb (± NO gas) were exchanged into water, diluted to a final concentration of 10 µM in heme (with methanol/water 1:10, v/v) and then infused continuously (flow rate of 1 µl/min) into the ion source at 20 °C. Experiments were performed without an internal standard. Multiply charged gas phase proteins were generated by pneumatically assisted ESI. In some experiments, the voltage difference between the orifice and skimmer was set at +100-110 V, conditions under which mass peaks for apo-Mb dominate (29, 40). Under these conditions, however, some minor contamination from intact holoprotein was detected. Estimates of mass for the holo-Mbs were performed with a voltage difference of 50-60 V to preserve the heme-protein complex and afford charge state distributions largely for the holo-Mb form of the protein. The ESI-MS system was mass-calibrated with solutions of CsI. Horse heart Mb (predicted mass of apoprotein = 16,950 atomic mass units) was employed as a standard to test the mass accuracy of the system prior to use. Mass values were obtained by standard fitting analyses of the various m/z distributions with "BioMultiView" software (Applied Biosystems, Foster City, CA). Mass determinations were performed for >= 2 independent protein preparations on different days. Mass values reported here refer to the mean ± S.D. from >= 5 analyses.

Electronic Absorption Spectra

Electronic absorption spectra were obtained with a Cary 300 UV-visible spectrophotometer at 25 °C. Mb solutions were diluted into 50 mM sodium phosphate buffer (pH 7.4) prior to analysis. Holo-Mb concentration was determined from the Soret maximum (epsilon 408 nm ~ 188,000 M-1 cm-1), while apo-Mb concentration was determined from the A280 nm (epsilon 280 nm ~ 16,000 M-1 cm-1). For the analyses of NO complexes, various Mb solutions were first exposed to NO using one of methods 1-3. The reaction mixture was then diluted into the appropriate degassed buffer, and the spectrum was recorded. By performing the nitrosation reactions in 2 mM phosphate buffer (pH 7.4) and subsequently diluting into 50 mM phosphate buffer (pH 7.4), the yields of the respective S-nitrosothiol were optimized. Under these conditions, steady state levels of S-nitrosothiols were monitored in the various reaction mixtures. In some experiments, wild-type human Mb was incubated with iodoacetamide (thiol-blocking reagent:protein = 10 mol/mol) to block the free thiol prior to reaction with NO. Carboxymethylation of the protein thiol was confirmed by determining residual thiol content before and after iodoacetamide treatment using DTP (molar ratio of DTP:protein = 10 mol/mol) and monitoring the reaction at A325 nm (29, 41). Concentrations of free reduced thiol were estimated by comparison with GSH standards. Consistent with our previous report (29), modification with iodoacetamide decreased the free thiol in wild-type human Mb by >85% (data not shown).

FTIR Measurements

FTIR spectra were recorded with a Perkin-Elmer System 2000 FTIR spectrometer equipped with a liquid nitrogen-cooled MCT detector. The IR cell consists of two CaF2 windows and a Teflon spacer (25 µm). The temperature of the sample was maintained at 20 °C with a water-jacketed cell holder and IR cells that have large contact surface to allow efficient thermal equilibration (Specac, Inc.). In addition, the sample compartment was purged continuously with dry nitrogen gas to minimize vapor absorption. Spectra were collected at an OPD velocity of 2 cm/s on both sides of the center burst and averaged over 500 scans. An apodization function was used to truncate the spectra, and the spectral resolution was 2 cm-1. Background absorption from the empty cavity was recorded under conditions identical to those used for the protein samples. As a result of the weak S-H stretching vibration for protein sulfhydryls in solution, relatively high concentrations of holo- or apoprotein (5-6 mM) were necessary to obtain useful data above the FTIR background for water.

Statistical Analyses

Statistical analyses were performed with Student's t test available in Excel (Microsoft), and significant difference was accepted at p < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Electronic Spectroscopy of Myoglobins following Exposure to NO-- Spectroscopic characteristics of a variety of metmyoglobins and their corresponding ferrous nitrosyl-heme complexes are collected in Table I. The ferric form of horse heart Mb, wild-type human Mb, and the C110A variant of human Mb each exhibited similar Soret and visible maxima, as expected from the similarity of the amino acid sequences for all mammalian Mb at the heme binding pocket (28, 42). In addition, each ferric protein exhibited a ASoret/A280 nm ratio > 5, consistent with the high purity of these preparations (not shown). In the absence of dioxygen, both the ferrous form of wild-type human Mb and the C110A variant of human Mb readily reacted with either authentic NO gas (method 2) or NO derived from the decomposition of DeaNO (method 3) to yield ferrous nitrosyl-heme complexes of the corresponding protein (Table I). Visible bands measured for solutions of the various human nitrosyl Mb complexes were similar to each other and distinct from those for the corresponding metmyoglobin. Furthermore, the visible bands for NO complexes of both wild-type human Mb and the C110A variant were nearly identical to those that are characteristic for the ferrous NO complex of horse heart Mb (Table I). Importantly, no significant increase in absorbance was detected between 330 and 350 nm for the ferrous nitrosyl-heme complexes of the various Mb proteins (Table I).


                              
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Table I
Electronic absorbance data obtained for various proteins before and/or after NO treatment

EPR of Myoglobins following Exposure to NO-- To verify the formation of ferrous heme-NO complexes of the various Mb proteins, EPR spectra were obtained at 77 K for samples from the reaction of authentic NO gas (NO:protein ~ 4 mol/mol) and the ferrous form of both horse heart Mb and wild-type human Mb performed under anaerobic conditions (Fig. 1). Consistent with the electronic spectra obtained for corresponding reaction mixtures, treatment of either ferrous horse heart Mb or ferrous wild-type human Mb with NO gas in the absence of dioxygen (method 2) resulted in a broad EPR absorption in the g ~ 2 region (Fig. 1, A and D). Each EPR signal exhibited characteristic components centered at g1 ~ 2.08, g2 ~ 2.01, and g3 ~ 1.98 that have been assigned previously as arising from the nitrosyl-heme complex of Mb (18, 19). Therefore, the EPR signals detected here were assigned to the ferrous nitrosyl-heme complex for the horse and wild-type human Mb proteins, respectively. Similarly, reaction of the ferrous form of either horse heart Mb or wild-type human Mb with NO (from reaction with DeaNO, method 3) also gave the corresponding ferrous nitrosyl-Mb (Fig. 1, B and E), indicating that NO derived from the decomposition of the DeaNO behaved in identical fashion to NO gas. Thus, the EPR data obtained from the reaction of various Mb with NO were in complete agreement with the optical data on the corresponding samples and confirmed the formation of a nitrosyl-heme complex in wild-type human Mb under anaerobic reaction conditions. Consecutive EPR spectra (77 K) measured for up to 30 min using the same solution of nitrosyl-Mb remained unchanged, consistent with the high affinity that NO shows for heme iron (dissociation constant Kd ~ 10-5 M) (13) (data not shown).



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Fig. 1.   EPR analyses of heme iron-nitroso complexes of horse heart- and wild-type human Mb. Ferrous nitrosyl complexes of horse heart Mb (spectra A-C) and human wild-type Mb (spectra D-F) were prepared from their respective proteins (~0.5 mM protein in 2 mM phosphate buffer, pH 7.4) using either method 2 (A and D) or method 3 (B and E) for anaerobic NO generation. After formation of the nitrosyl-Mb complex, a sample of reaction mixture was removed for EPR analyses at 77 K (see "Experimental Procedures"). In the presence of air, neither ferrous horse heart Mb (C) or ferrous wild-type human Mb (F) afforded EPR signals when exposed to aerobic NO generated by method 3. EPR parameters were as follows: microwave power, 5 milliwatts; modulation frequency, 100 kHz; and modulation amplitude, 0.4 millitesla. Spectra are an average of five scans with sweep time of 84 s. The data represent three independent analyses using different Mb preparations. mT, milliteslas.

In contrast to the relative ease of formation of ferrous nitrosyl-Mb complexes in the absence of dioxygen, aerobic treatment of oxy-Mb solutions (horse heart or wild-type human Mb) with authentic NO gas (method 1, ~4-fold excess) resulted in the conversion of oxy-Mb to metmyoglobin as judged by characteristic changes to the electronic absorbance spectra for these solutions (data not shown). Similarly, addition of a relatively low mole excess of NO to oxy-Mb (NO:protein ~ 2 mol/mol, method 3) also resulted in the oxidation of oxy-Mb to metmyoglobin (data not shown). Nitrosyl-heme was not detected by EPR spectroscopy upon aerobic exposure of either horse heart or wild-type human metmyoglobin to NO generated by method 2 (data not shown) or method 3 (Fig. 1, C and F). This latter observation contrasts with reports of Mb-NO formation in reactions of ferric myoglobin with NO under low oxygen tension (43, 44).

Characterization of S-Nitrosated Wild-type Human Mb-- The results above suggested that aerobic treatment of wild-type human oxy-Mb with NO yields only metmyoglobin without detectable generation of nitrosyl-Mb as judged by optical absorbance analyses. Additionally, aerobic reaction of metmyoglobin and NO (generated by method 2 or 3) did not yield nitrosyl-Mb as monitored directly by EPR. Therefore, we next investigated whether the apoprotein of wild-type human metmyoglobin was modified by exposure to NO in the presence of dioxygen. Treatment of ferric wild-type human Mb and NO (method 3) at mole ratios of NO:protein = 5 mol/mol resulted in an increase in A330-350 nm as well as an increase in the Soret band intensity (Fig. 2 and Table I). As the NO-treated protein showed changes in absorbance over a relatively wide range of the absorbance envelope, we next investigated whether NO treatment altered the visible region (450-700 nm) of the Mb solution spectrum. Importantly, no significant change was detected over A450-700 nm as judged from the treatment of relatively concentrated solutions of wild-type human holo-Mb with NO under identical conditions (Fig. 2A, inset). In contrast to the absorbance increase in the region 330-350 nm detected in reaction mixtures of wild-type-human Mb and NO, solutions of the ferric C110A variant of human Mb treated with NO afforded no increased absorbance in this region (Fig. 2B), nor were there observable changes between 450 and 700 nm (Fig. 2B, inset). Thus, these data support the conclusion that the increase in absorbance between 330 and 350 nm that results from the aerobic treatment of wild-type human metmyoglobin with NO may be derived from either direct modification of the protein or the overlap of multiple absorbing species.



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Fig. 2.   Changes to the optical absorbance spectrum of wild-type human Mb upon addition of NO. A solution of either wild-type human metMb (panel A) or C110A variant of human metMb (panel B) (each 5 µM protein in 2 mM phosphate buffer, pH 7.4) was analyzed before (solid line) and after (dashed line) exposure to NO (NO:protein = 5 mol/mol) generated by method 3. Note the increase in intensity for both the Soret band (A409 nm) and the region A330-350 nm. Insets to panels A and B show the respective visible regions (A450-700 nm) of concentrated solutions of either wild-type or C110A variant human Mb (each protein ~ 50 µM, NO:protein = 5 mol/mol) analyzed before (solid line) and after (dashed line) exposure to NO generated by method 3. The data represent three independent analyses using different preparations of Mb proteins.

To evaluate the possibility of multiple species, the electronic absorbance spectrum of the reaction mixture was measured before and after purification by gel filtration (2x PD-10 column). Importantly, the A408 nm/A350 nm ratio was virtually unchanged by this treatment, consistent with the notion that low-molecular weight contaminants did not contribute to the detectable changes in absorbance and that the protein was somehow modified by reaction with NO in the presence of air. The yield of adduct increased significantly above that for the corresponding control (i.e., addition of carefully matched volumes of 1 mM NaOH) with increasing concentration of NO (NO:protein = 0-5 mol/mol), as judged by an NO-dependent increase in A350 nm (Fig. 3A). At NO:protein ratios >5 mol/mol, no further increase in A350 nm was detected at least up to NO:protein ratios <=  20 mol/mol (Fig. 3A). In contrast, addition of chemically generated NO (Method 3) to the C110A variant of human Mb over the same range of NO:protein ratios resulted in no significant change in A350 nm after removal of low-molecular weight contaminants by gel filtration (Fig. 3A).



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Fig. 3.   Steady state measurements of changes to the optical absorbance measured at 350 nm as a function of NO concentration. Panel A, the change in optical absorbance for solutions of metMb (each 5 µM protein in 50 mM phosphate buffer, pH 7.4) were measured at A350 nm over a range of increasing NO:protein (method 3) for wild-type human Mb (filled squares), C110A variant of human Mb (filled circles), and vehicle (1 mM NaOH) alone added to wild-type human Mb (filled triangles) as a control. Panel B shows the changes in A350 nm for a solution of wild-type human Mb treated with NO (NO:protein ~ 5 mol/mol) in 2 mM (open squares), 20 mM (open circles), and 50 mM phosphate buffer (open triangles), respectively. Prior to analyses, proteins were diluted into 50 mM phosphate buffer, pH 7.4. Data represent mean ± S.D. of three independent analyses using different Mb preparations. The asterisk indicates significant difference from corresponding absorbance values for both the control and C110A data (p < 0.05).

The increase in A330-350 nm upon addition of NO to wild-type human metmyoglobin is similar to that observed for S-nitrosoglutathione (GS-NO) obtained from reaction of reduced GSH and NO generated from either method 1 or method 3 (Table I), or by comparison with data previously reported (36, 45). The relatively weak absorbance at ~544 nm (epsilon  < 50 M-1 cm-1) also associated with GS-NO formation (36, 45) was not observed in the corresponding reactions of NO and wild-type human Mb owing to the complication of overlap with the visible bands of the protein. Despite the inability to detect the weak absorption at 540-550 nm, we tentatively concluded that the aerobic reaction of human metmyoglobin with NO results in S-nitrosation of the human protein at Cys110. Similar to the case for GS-NO (45), the yield of S-nitrosated human Mb was dependent on the concentration of phosphate employed in the reaction mixture, as reflected by the decrease in A350 nm with increasing phosphate over the range 2-50 mM (Fig. 3B). Under physiological conditions (50 mM phosphate, pH 7.4), the extent of S-nitrosation was ~40-45% of that obtained at the lowest phosphate concentration employed here (i.e., 2 mM phosphate). Importantly, DeaNO solutions (<= 1 mM) did not exhibit any absorbance over the range 300-700 nm when added to the buffer (2, 20, or 50 mM phosphate, pH 7.4) alone to act as control (data not shown).

To eliminate interference from the delta, Soret, and visible bands of the holoprotein, we next investigated the electronic spectrum of apoprotein prepared from wild-type human Mb and the C110A variant before and after treatment with NO (method 1). Upon treatment of the wild-type human apo-Mb with NO (NO:protein = 2 mol/mol), the electronic absorbance spectrum of the reaction mixture exhibited new, broad maxima at 333 and 545 nm (see filled lines in Figs. 4, A and B, and respective insets). The broadened maxima at 333 nm in reaction mixtures of apo-Mb and NO may explain the wide range increase in absorbance detected at 333-350 and 409 nm for corresponding reactions of NO and holo-Mb (see above). These maxima were not observed prior to NO-treatment (data not shown) and are consistent with S-nitrosation of the wild-type human apo-Mb. By contrast, treatment of either apo-Mb from the C110A variant or wild-type human apo-Mb, previously modified with iodoacetamide, resulted in a significant decrease at 333 nm and near elimination of the broad band at 545 nm (dashed lines in Fig. 4 (A and B) and respective insets). Together, these data strongly support the argument that S-nitrosation occurs at Cys110 of wild-type human Mb. In the absence of the overlapping Soret band, the A333 nm/A545 nm ratio obtained for the S-nitrosated apo-Mb was 81 ± 5 (mean ± S.D., n = 3). The value of this ratio is similar to that (epsilon 335 nm (3869 M-1 cm-1)/epsilon 545 nm (47 M-1 cm-1) ~ 82) reported (36) for S-nitrosated bovine serum albumin, another protein with a single reactive sulfhydryl (29, 46, 47) that is readily S-nitrosated by aerobic addition of NO (36). Molar absorptivity values of epsilon 333 nm = 3667 ± 90 and epsilon 545 nm = 44 ± 3 M-1 cm-1 (mean ± S.D.) were obtained by plotting the apomyoglobin protein dependent change in A333 nm (e.g. Fig. 4C) and A545 nm (data not shown), respectively. Notably, employing epsilon 333 nm to the relative increase at A330-350 nm observed for solutions of untreated versus NO-treated holo-Mb (data taken from Fig. 2) yields ~1-1.2 NO/human Mb mol/mol, consistent with stoichiometric conversion to the NO adduct.



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Fig. 4.   Optical spectra from solutions of both wild-type and C110A variant of human apo-myoglobin after NO treatment. The change in A250-700 nm for solutions of apo-Mb (2 µM protein, 50 mM phosphate buffer, pH 7.4) after exposure to ~2 mol eq of NO (method 1). Panel A, spectra correspond to NO-treated wild-type human apo-Mb (solid line) and NO-treated apoprotein from the C110A variant (dashed line). Panel B shows NO-treated wild-type apo-Mb before (solid line) and after treatment with iodoacetamide (dashed line). Insets to panels A and B show visible electronic spectra (500-600 nm) from solutions of corresponding Mb at 50 µM protein concentration. Panel C shows the effect of increasing apo-Mb concentration (~0-80 µM) on formation of S-nitroso apo-Mb (NO:apo-Mb~2 mol/mol). Inset to panel C shows linear relationship between [apo-Mb] and A333 nm:epsilon 333 nm is determined as the slope of the line. The data represent three independent analyses using different Mb preparations.

FTIR Analyses of the Reaction Mixture from Wild-type Human Holo-Mb and NO-- As the evidence from electronic absorbance spectra from reaction mixtures of wild-type human Mb and NO supported the formation of S-nitrosated wild-type human Mb, we sought to confirm that NO reacted specifically with the sulfhydryl group at Cys110. The S-H stretching vibration of the sulfhydryl(s) in human hemoglobin is readily detected by IR spectroscopy in concentrated solutions of the protein, nu  ~ 2550-2580 cm-1 (48, 49). Thus, to confirm that the reaction of aerated solutions of ferric wild-type human Mb and NO (method 3) resulted in the modification of the protein at Cys110, solutions of wild-type and C110A variant of Mb were analyzed by FTIR spectroscopy before and after NO treatment (Fig. 5). In the absence of NO, aqueous solutions of wild-type human holo-Mb exhibited a weak and broad vibration at nu  ~ 2552 cm-1 with half-height peak width Delta nu 1/2 ~ 10-12 cm-1 (Fig. 5A). Concentrated solutions of human apo-Mb also exhibited a weak vibration at nu  ~ 2551 cm-1 with Delta nu 1/2 ~ 8-10 cm-1 (Fig. 5B), while the C110A variant of human Mb did not (Fig. 5E). Solutions of reduced-GSH (100 mM) measured under identical conditions as a control also exhibited a peak at nu  ~ 2550 cm-1 (data not shown). The band intensity for the wild-type human holo-Mb was severely reduced at pH 10, above the pKa for a reduced thiol (49) (data not shown). Together, these data support the assignment of an S-H stretching band for Cys110 in human Mb at 2551-2552 cm-1. After treatment of either wild-type human holo- or apo-Mb with NO (NO:protein = 5 mol/mol), the band intensity for the S-H stretch was significantly diminished (cf. Fig. 5, panels A and C and panels B and D, for holo- and apo-Mb, respectively). Overall, these data strongly support the conclusion that the sulfhydryl at Cys110 of human Mb can be detected directly by FTIR spectroscopy and that this residue is specifically modified by aerobic exposure to NO independent of the presence or absence of the heme prosthetic group.



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Fig. 5.   Infrared stretching vibration of the human Mb sulfhydryl group before and after treatment with NO. Aerobic solutions of pooled wild-type human holo-metMb or apo-Mb (each 5-6 mM protein in 2 mM phosphate buffer, pH 7.4) were analyzed by FTIR before (panels A and B) and after (panels C and D) exposure to NO (NO:protein = 5 mol/mol) generated by method 3, respectively. Panel E shows the corresponding IR region for a concentrated solution (6 mM) of the C110A variant of human Mb in the absence of NO treatment.

Mass Analyses of Reactions of Wild-type Human Holo- and Apo-Mb and NO-- To establish unambiguously that wild-type human Mb was S-nitrosated by reaction with NO in a 1:1 stoichiometric reaction, mass analyses were performed on holo- and apo-Mb in both the presence and absence of NO. The mass of wild-type human Mb was determined first by ESI-MS under ionization conditions that produce mass-to-charge (m/z) distributions for holo-Mb (Fig. 6). Consistent with our previous study (29), the mass determined for human holo-Mb was 17,670 ± 2 atomic mass units, which compares well with a mass of 17,669 atomic mass units predicted from the amino acid sequence (Fig. 6A, inset). The small shoulder on each peak in the m/z distribution of each reaction mixture results from residual phosphate contamination in the samples. Importantly, the peaks in the m/z distribution for the NO-treated sample increased by 2-3 units (cf. Fig. 6, A and B) to yield a mass for the nitrosated human Mb of 17,698 ± 5 atomic mass units, i.e. an increase of 28 ± 4 atomic mass units (mean ± S.D., n = 5) (cf. insets from Fig. 6, A and B). This increase in mass of ~30 units corresponds to the addition of one molecule of NO to each molecule of protein. No other significant adduct(s) was detected in the reaction mixture under conditions employed to optimize S-nitrosation of the wild-type human Mb (i.e. nitrosation reactions performed in low phosphate, 2 mM phosphate, pH 7.4), indicating near complete conversion to the S-nitrosated holoprotein.



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Fig. 6.   Mass analyses of the wild-type human holo-Mb before and after treatment with NO. Solutions of wild-type human holo-Mb (10 µM protein in 2 mM phosphate buffer, pH 7.4) were analyzed by ESI-MS and the mass-to-charge (m/z) ratios shown for the protein before (panel A) and after (panel B) exposure to NO (NO:protein = 5 mol/mol) generated by method 3, respectively. The respective insets show the deconvoluted mass value for the corresponding m/z distribution. The data represent five analyses using at least two different Mb preparations. amu, atomic mass units.

To determine whether the NO adduct was derived from sulfhydryl S-NO rather than a heme-NO complex, mass spectrometry was performed on preparations of wild-type human apo-Mb and S-nitrosated wild-type human apo-Mb (Fig. 7) under conditions that favor the detection of apo-Mb (39). The m/z distribution of authentic human apo-Mb, accumulated with an expanded scale (1400 <=  m/z <=  2200), exhibited relatively sharp peaks (Fig. 7A). The m/z distribution (m/z (charge in parentheses) = 1421 (12+), 1550 (11+), 1705 (10+), 1894 (9+), and 2131 (8+)) yielded a mass of 17,053 ± 1 atomic mass units (Fig. 7A, inset). This mass compares well with the predicted mass of 17,053 atomic mass units for wild-type human apo-Mb. The m/z distribution of dilute samples from the reaction of authentic apo-Mb and NO (method 1 or 3) were shifted 2-5 units compared with the untreated apo-Mb (m/z (charge in parentheses) = 1424 (12+), 1553 (11+), 1708 (10+), 1899 (9+), and 2136 (8+)) and yielded a mass of 17,082 ± 3 atomic mass units. Thus, the mass of the NO-modified apoprotein also corresponded to an increase in protein mass of ~29 ± 4 (mean ± S.D., n = 5) relative to wild-type human apo-Mb (cf. Fig. 7, A and B, and respective insets), indicating that the apoprotein was readily nitrosated in the absence of the heme prosthetic group. Finally, to show unambiguously that the NO adduct results from sulfhydryl (S-) rather than heme-nitrosation in the holoprotein, samples of reaction mixture from holo-Mb and NO (method 3) at mole ratios of NO:protein = 5 mol/mol, which showed a mass increase of ~30 atomic mass units, were adjusted to pH 2.5 with HCl so that heme could be extracted from the protein (32). Analysis of the resulting apoprotein fraction by ESI-MS yielded an m/z distribution and corresponding mass value identical to that for authentic wild-type human apo-Mb treated aerobically with NO (cf. Fig. 7, B and C, and respective insets). Together, these data strongly support the conclusion that the reaction of ferric wild-type human Mb and NO in the presence of air yields S-nitrosated Mb.



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Fig. 7.   Mass analyses of authentic wild-type human apo-Mb before and after treatment with NO. Solutions of authentic wild-type human apo-Mb (~10 µM protein in 2 mM phosphate buffer) were analyzed by electrospray mass spectrometry and the m/z ratios shown for the apoprotein (panel A) before and (panel B) after exposure to NO (NO:protein = 5 mol/mol) generated by method 1, respectively. Panel C shows the mass analyses performed on apoprotein derived from samples of holo-Mb after treatment with NO and removal of heme. Respective insets show the deconvoluted mass values for corresponding m/z distributions. The data represent five analyses using at least two different Mb preparations. amu, atomic mass units.

Other minor peaks were detected in the mass analyses of the reaction mixtures (Fig. 7, B and C, diamonds). The minor peaks are attributed to the incomplete conversion of holo-Mb to apo-Mb. As these peaks were resolved with low signal to noise ratios, they were not used to characterize the NO adduct. Mass peaks obtained from aerobic solutions of wild-type apo-Mb treated with NO were broader than those for the corresponding untreated apoprotein (Fig. 7, compare A with B and C). This peak broadening did not likely result from sample inhomogeneity but rather from accumulation of mass data with a lower resolution setting for NO-treated versus untreated apo-Mb, respectively. This change in resolution parameter was necessitated by the consistently lower peak intensities detected in the dilute samples of reaction mixture used for mass analyses of S-nitrosated apo-Mb. Solutions of S-nitrosated apo-Mb were not concentrated prior to mass analyses to minimize the potential for decomposition of any S-nitrosothiol protein subsequent to gel filtration.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The reactions of NO with heme iron, non-heme iron, and sulfhydryl groups are thought to be important mechanisms for regulating NO in vivo (see Introduction). Human Mb contains both a heme prosthetic group and a protein sulfhydryl at Cys110. Interestingly, human Mb differs from all other known Mbs by the presence of the sulfhydryl at Cys110. The sulfhydryl group in human Mb significantly modifies its reaction with hydrogen peroxide (29) and can be reasonably expected to modify the reaction of the protein with other reactive biological agents such as NO. In addition to the reaction of NO and ferrous Mb to yield heme-NO complexes (18, 19), and oxy-Mb induced oxidation of NO to NO3- (26), wild-type human Mb is now shown for the first time to react aerobically with NO in vitro to yield a derivative that is S-nitrosated at Cys110. The evidence we have obtained by combining FTIR, mass spectrometry, and optical absorbance spectroscopy together with EPR analyses of aerobic reaction mixtures of wild-type human Mb and NO strongly support this conclusion. Thus, it is conceivable that wild-type human Mb interacts with NO by a variety of mechanisms (and under a variety of conditions) to yield both heme iron and S-nitrosated protein and possibly modulate the biological activity of NO and/or the lifetime of NO under physiological conditions.

Glutathione is a widely distributed low molecular weight thiol that is present in high concentration (0.5-10 mM) inside cells (50). Kinetic analyses (45, 51, 52) of the aerobic nitrosation reaction of reduced GSH and NO have proven that the nitrosating reagent is not NO itself but rather a higher oxidation state of the gas. That GS-NO formation requires the presence of NO and oxygen supports this conclusion (45, 51). Furthermore, evidence that the rate-limiting process in the formation of GS-NO is actually the reaction of NO and dioxygen to yield N2O3 supports the conclusion that the likely nitrosylating reagent is N2O3 rather than NO (45). That anaerobic treatment of ferrous Mb with NO gave only the nitrosyl-Mb, and that S-nitrosation of the ferric- or apo-Mb required the presence of both NO and dioxygen, is completely consistent with the proposal that higher oxides of NO are responsible for the S-nitrosation of the human protein. The dependence of the overall yield of S-nitrosated Mb on the concentration of phosphate in the reaction buffer is also consistent with that previously reported for S-nitrosothiols (45).

S-Nitrosation of proteins, however, typically occurs with a rate constant ~10-fold lower than that for low molecular weight reduced thiols (51). Therefore, to compete with the S-nitrosation of reduced GSH and other low molecular weight reduced thiols, intracellular concentrations of protein sulfhydryl must be at least an order of magnitude greater than the corresponding GSH concentration. Since human Mb possesses only a single sulfhydryl per molecule of protein, the role of S-nitrosated human Mb may be limited to human cells where the concentration of Mb is sufficiently high that the reaction of NO and the protein sulfhydryl is favorable. Interestingly, Mb is reportedly present in relatively high concentration in human heart and skeletal tissue (2.1-6 mg/g of wet tissue (Refs. 20-22)). Although Mb is known to be present in human smooth muscle (23), the concentration of the protein in these cells is not known.

Both NO and N2O3 are reported (26) to be capable of oxidizing horse heart oxy-Mb to produce metmyoglobin and nitrate in both the presence and absence of dioxygen. Our observation that low molar excess of NO (NO:protein <=  5 mol/mol) readily oxidized wild-type human oxy-Mb to metmyoglobin in the presence of dioxygen is consistent with this report. Under physiological conditions, a majority of Mb is both reduced and oxygenated. Thus, it may be argued that the oxidation of human oxy-Mb to metmyoglobin by NO, NO2, or N2O3 may occur in preference to S-nitrosation of the protein. Despite the rapid rate of reaction of NO with oxy-Mb (k ~ 4 × 107 M-1 s-1 (Ref. 26)), N2O3-mediated oxidation of oxy-Mb is known to be relatively slow (k ~ 4 × 102 M-1 s-1 (Ref. 26)). Furthermore, N2O3 (generated from the relatively rapid reaction of NO and dioxygen (k ~ 2 × 106 (Ref. 45)) reacts with reduced thiols with rate constant k ~ 2 × 105-2 × 106 M-1 s-1 (45, 51). Together, these data suggest that formation of an S-nitrosothiol on human Mb may compete with the oxidation of oxy-Mb under conditions that favor the oxidation of NO to N2O3. Importantly, a similar argument can be made for the reaction of human oxy-hemoglobin with NO; however, we note that at present in vivo S-nitrosation is regarded by some (11, 52-54) but not others (55) to be a physiologically relevant reaction of hemoglobin. That wild-type human metmyoglobin may also be S-nitrosated in the presence of low molar excess of NO affords the intriguing possibility that this protein might regulate the availability of NO by several modes. Thus, human Mb may oxidize excess NO to nitrate while also preserving NO bio-activity through formation of the S-nitrosated protein.

As discussed in the Introduction, NO is responsible for a variety of physiological processes. Although S-nitrosothiols are stable under physiological conditions, they can decompose to yield the corresponding disulfide and NO (12) and thereby participate in the regulation of NO release and activity. Furthermore, the decomposition of S-nitrosothiols can be enhanced by increased temperature (12), photolysis (37, 56, 57), or reaction with transition metals (37). With our demonstration that human Mb can also yield an S-nitrosated protein in the presence of NO and dioxygen, this protein joins the S-nitrosoproteins, S-nitrosohemoglobin and S-nitrosoalbumin as a well characterized sulfhydryl-containing protein that may exhibit potent NO-mediated biological activity in vivo (36). Overall, combined data from FTIR spectroscopy, EPR spectroscopy, mass spectrometry, and electronic absorption spectroscopy strongly support the conclusion that both heme or sulfhydryl complexes of NO can form in human Mb. Whether S-nitrosated wild-type human Mb is capable of releasing biologically active NO is yet to be determined. However, if NO release from S-nitrosated human Mb can occur, then this form of the protein could represent a previously unrecognized pool of biologically active NO that may participate in the regulation of intracellular function of human tissues that contain Mb in abundance, e.g. human smooth muscle.


    ACKNOWLEDGEMENTS

We thank Professor Steven G. Boxer and Dr. Eun Sun Park for providing the plasmids used to express the wild-type and C110A variant of human Mb used in this study and for advice regarding purification of these proteins.


    FOOTNOTES

* This work was supported by Grant O 98S 0008 from the National Heart Foundation of Australia (to P. K. W.), an NSERC-SCIEX Industrial Chair (to D. J. D.), and Medical Research Council of Canada Grant MT-7182 (to A. G. M.).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.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, 2146 Health Sciences Mall, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. E-mail: mauk@interchg.ubc.ca.

Published, JBC Papers in Press, October 25, 2000, DOI 10.1074/jbc.M005758200


    ABBREVIATIONS

The abbreviations used are: EDRF, endothelium-derived relaxant factor; C110A human Mb, the cysteine 110 to alanine mutant of human myoglobin; DeaNO, diethylammonium(z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, DTP, 2,2'-dithiodipyridine; DTPA, diethylenetriamine pentaacetic acid; EPR, electron paramagnetic resonance spectroscopy; ESI, electrospray ionization; MS, mass spectrometry; FTIR, Fourier transform infrared spectroscopy; Mb, myoglobin; metMb, ferric myoglobin; nitrosyl Mb, the ferrous heme-NO complex of Mb; oxy-Mb, oxymyoglobin; TEMPO, 2,2,6,6-tetramethylpiperidine-N-oxyl.


    REFERENCES
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DISCUSSION
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