The reaction of oxyhemoglobin with nitric oxide: EPR evidence for an iron(III)-nitrate intermediate

Inorganica Chimica Acta 436 (2015) 179–183

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The reaction of oxyhemoglobin with nitric oxide: EPR evidence for an iron(III)-nitrate intermediate Radu Silaghi-Dumitrescu a,b,⇑, Florina Scurtu b, Maria G. Mason a, Dimitri A. Svistunenko a, Michael T. Wilson a, Chris E. Cooper a a b

School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK Department of Chemistry, ‘Babes-Bolyai’ University, 1 Mihail Kogalniceanu Str., RO-400084 Cluj-Napoca, Romania

a r t i c l e

i n f o

Article history: Received 3 December 2014 Received in revised form 21 July 2015 Accepted 24 July 2015 Available online 3 August 2015 Keywords: Hemoglobin Nitric oxide Stopped-flow Peroxynitrite EPR

a b s t r a c t The reaction of oxyhemoglobin with nitric oxide is known to occur in vivo, with direct medical and pathophysiological relevance. The mechanism has been proposed to involve a transient iron(III)-peroxynitrite intermediate, the formation of which would be rate-limiting and the decay of which would yield iron(III)-aqua/iron(III)-hydroxo hemoglobin. Reported here are rapid-freeze-quench EPR (RFQ-EPR) spectroscopy data on the reaction of oxyhemoglobin with nitric oxide; no direct evidence is seen for an iron(III)-peroxynitrite intermediate. These findings are consistent with theoretical considerations according to which such an intermediate does form but is too short-lived to be detectable. Instead, iron(III) low-spin and high-spin transient species are detected in the RFQ-EPR experiments, which can be explained as arising from interactions of nitrate with methemoglobin. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction The almost diffusion-controlled reaction of oxyhemoglobin with NO occurs in vivo and defines a physiological function for flavohemoglobins [1] and perhaps other globins [2]. The proposed mechanism [2–13] involves initial attack of NO on the iron(III)-superoxo heme, yielding an iron(III)-peroxynitrite species, [Fe–OONO]2+ which would then yield nitrate by isomerisation, cf. Reaction (1). Indirect evidence for a ferric-peroxynitrite adduct was also found when reacting ferric myoglobin with peroxynitrite [14].

FeðIIÞ-O2 þ NO ! fFeðIIIÞ-OONO g ! fFeðIIIÞ-NO3 g ! FeðIIIÞ þ NO3

ð1Þ

An alternative mechanism, with O2 attacking Fe(II)-NO to yield an N-bound iron(III)-peroxynitrite intermediate [15], was disfavoured by DFT calculations [2,16]. Furthermore, in myoglobin the Fe(II)-NO + O2 reaction is rate-limited by dissociation of NO [17]. However, the ‘Fe(II)-NO + O2’ mechanism was recently supported by atomistic simulations showing a large barrier for the ‘Fe(II)-O2 + NO’ mechanism but a low barrier for the ‘Fe(II)-NO + O2’ mechanism [18]. ⇑ Corresponding author at: Department of Chemistry, ‘Babes-Bolyai’ University, 1 Mihail Kogalniceanu Str., RO-400084 Cluj-Napoca, Romania. E-mail address: [email protected] (R. Silaghi-Dumitrescu). http://dx.doi.org/10.1016/j.ica.2015.07.037 0020-1693/Ó 2015 Elsevier B.V. All rights reserved.

Stopped-flow UV–Vis spectra were reported for a short-lived species in the Fe(II)-O2 + NO reaction in hemoglobin, suggestive of a hexacoordinate S = 5/2 iron(III) heme [3,4] and interpreted as an iron(III)-peroxynitrite adduct; this was more readily observable at basic pH, where it rapidly converts into the resting iron(III) form, liberating nitrate [5,19–22]. A high-spin signal was accordingly detected in EPR samples frozen 6 ms after mixing oxy-hemoglobin with NO at basic pH; the low-spin region of these EPR spectra was obscured by impurities [10]. However, the (presumably) iron(III)nitrate species obtained when mixing ferryl myoglobin with NO2 decays at the same rate as the oxymyoglobin + NO intermediate [23,24]. Furthermore, basic thermodynamic arguments place the lifetime of an iron(III) peroxynitrite adduct below 1 ls [24], in agreement with density functional (DFT) calculations [2,16]. Recently the vibrational spectrum of the putative ferric-peroxynitrite intermediate was reported, showing nitrate, not peroxynitrite, bound to iron [25]. A peroxynitrite intermediate stable on the ms time scale therefore appears difficult to reconcile with recent theoretical and experimental data. These findings suggest a re-evaluation is needed of the rapid kinetic UV–Vis and EPR studies of the reaction of oxyhemoglobin with NO. Reported here are rapid-freeze-quench EPR data, indicating that the reaction of iron(II)-oxy hemoglobin with NO produces new EPR signals, from S = 1/2 and S = 5/2 species. These same signals are seen upon manual mixing of iron(III) hemoglobin with nitrate suggesting a common origin.

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2. Experimental

3. Results and discussion

2.1. Protein purification

Fig. 1 shows UV–Vis spectra of oxyhemoglobin mixed with NO in stopped-flow experiments at pH 9.5 and at pH 7.4. Consistent with previous observations [3–5,10–13], a transient species (previously assigned as iron(III)-peroxynitrite) forms at both pH values, with maxima at 539 and 575 nm (indicative of low-spin character) but also at 500 and 634 nm (indicative of high-spin character); the nature of this intermediate appears pH-independent. However, it decays to yield different optical species: high-spin iron(III) hemoglobin at pH 7.4 (note in particular the 630-nm band) and low-spin iron(III) hemoglobin at pH 9.5 [26]. The UV–Vis spectrum of the alkaline product is very different from that of the intermediate (542 and 575 nm, with no maxima at 500 and 630 nm), which is why the intermediate was first detected at alkaline pH [3]. However, although at neutral pH the decay of the intermediate is somewhat faster [5], the difference in rates is not as large as might be expected if a peroxynitrite intermediate were being protonated. Fig. 2 shows EPR spectra collected for samples frozen 13 ms and 207 ms, respectively, after mixing oxyhemoglobin with excess NO at pH 9.5. Several features not present prior to NO addition are seen immediately after mixing (13 ms) and disappear after 207 ms. These features (cf. Fig. 2) have g-values of 6.12 and 5.47, characteristic of S = 5/2 hemes, and g = 2.78, 2.29, typical for S = 1/2 hemes. Other signals observed in both the 13 ms and 207 ms EPR spectra (Fig. 2) are readily assignable to unreacted NO (g = 1.97) [32] or the stable products of the reaction – high-spin resting iron(III) hemoglobin (iron(III)-aqua, g = 5.95, 5,76) and low-spin resting iron(III) hemoglobin (iron(III)-hydroxo, g = 2.59, 2.19, 1.84).

Hemoglobin was purified following a variation of the general protocol of Antonini and Brunori [26]. Blood (regardless of source – human or bovine), freshly drawn on citrate, was centrifuged 15 min at 5000 rpm (g) to separate the red blood cells, which were then washed three times with 5 mM phosphate pH 7.4+150 mM NaCl. Hemoglobin concentrations in text are given per heme rather than per tetramer. The met forms of hemoglobin and myoglobin were obtained by treatment of the oxy form of protein with ferricyanide followed by aerobic passage over a PD-10 desalting column as previously described [27–29].

2.2. DEA NONOate solution Stock solutions (25 mM) of DEA NONOate (diethyl ammonium (Z)-1-(N,N-diethyl amino) diazen-1-ium-1,2-40 diolate, Cayman Chemicals, Inc.) were prepared in 0.01 M NaOH. DEA NONOate is stable at high pH but decomposes to release NO gas (1.5 mol NO/1 mol DEA NONOate) when added to assay mixtures.

2.3. Stopped-flow measurements Were performed using an Applied Photophysics (Leatherhead, Surrey, U.K.) model SX-18MV instrument. Diode-array-collected data were analysed within the Pro-K II software (Applied Photophysics). Nitric oxide solutions for stopped-flow experiments were prepared by diluting appropriate amounts of the NO donor, ProliNONOate (Cayman Chemical) in anaerobic 20 mM phosphate, pH 7.4, from a stock solution of 12 mM ProliNONOate whose concentration was verified spectrophotometrically at 252 nm (e = 8400 cm1 M1).

2.4. EPR sample preparation and measurement EPR samples of the hemoglobin reacting with NO and nitrate, respectively were prepared made by the rapid freeze quenched (RFQ) method. The data reported here were collected using an Update Instruments (Madison, WI) mixing machine. Additionally, the same experiments were also repeated with a prototype apparatus for freezing the reaction mixtures on the surface of a rapidly rotating aluminium disk maintained at liquid nitrogen temperature [30]. EPR spectra were recorded at 10 K in a Bruker EMX EPR spectrometer (X-band) with a spherical high quality Bruker resonator SP9703 and an Oxford Instruments liquid helium system. Instrument conditions were: microwave frequency 9.47 GHz, microwave power 3.18 mW, modulation frequency 100 kHz, modulation amplitude 5 G, sweep rated 22.6 G/s; time constant 81.92 ms, single sweep for each spectrum. For an (approximate) spin quantitation basis spectra of met Hb species at acidic and basic pH values, measured under the same power/temperature/concentration conditions, were employed.

Fig. 1. UV–Vis spectra during stopped-flow mixing of oxyhemoglobin with NO at pH 7.4 and 9. The solid gray trace can be simulated (not shown) as a weighted sum of the solid black trace and dashed gray trace, i.e. the same intermediate species is produced at both pH values. Spectra shown in dashed traces are identical to iron(III) hemoglobin (gray – low-spin at basic pH, black – high-spin at neutral pH).

2.5. Density functional calculations (UBP86/6-31G**) were performed following protocols previously applied to S = 1/2 and S = 3/2 heme-peroxynitrite adducts [16,31]. For the non-heme peroxynitrite models, UB3LYP/6-31G** yields similar results (not shown).

Fig. 2. EPR spectra of the rapid-freeze-quenched oxyhemoglobin reacting with NO (100 lM heme, 400 lM NO, pH 9.5; black trace – 13 ms, grey trace – 207 ms). The g-values of the signals are indicated.

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It is reasonable to assign the transient features in the 13 ms spectrum to the transient species previously detected in stoppedflow UV–Vis experiments [3] (‘‘the oxy-NO intermediate”). The presence of both high-spin and low-spin features (65 lM and 11 lM, respectively) is consistent with electronic absorption spectra (Refs. [3,5] and Fig. 1), which showed peaks at 630 nm and 500 nm indicative of a high-spin state as well as peaks at 535 and 585 nm suggestive of a low-spin component. The decay of the oxy-NO intermediate as monitored optically can be fitted well as a single reaction, A ? B, suggesting that the transient S = 5/2 and S = 1/2 signals probably arise from different spin states/conformers of the same intermediate (cf. Ref. [3], differences in rates but not mechanism are seen between hemoglobin alpha and beta chains). The S = 5/2 signal is split into two components at g1 = 6.12 and g2 = 5.47, which is an indication of a rhombic distortion of an otherwise axially symmetrical (g1 = g2  g\  6) high-spin iron (III) heme complex. Such splitting is known for the fluoride adduct of myoglobin but is more common with non-globin hemoproteins [33]. The g = 2.78 and 2.29 features are reminiscent of bis-histidinyl globin adducts [32]. Nevertheless, accumulation of a bishistidinyl adduct would imply a minimal reaction scheme A (high-spin) ? B(low-spin, bis-histidyl) ? C(low-spin, hydroxo), which is at odds with the simple A ? B scheme supported by stopped-flow UV–Vis data wherein A is a mixture of low-spin and high-spin [3,5]. To verify whether the transient EPR signals in the 13 ms sample arise from a iron(III)-nitrate adduct, we studied the nitrate adducts directly. Due to the high concentrations necessary to observe binding to the heme (2–4 M), we first compared sodium nitrate to sodium chloride binding using UV–Vis optical changes. There was no significant absorbance change following chloride addition, whereas nitrate showed changes similar in nature to the peroxynitrite adduct. Fig. 3 shows the EPR spectra of iron(III) hemoglobin mixed with nitrate at pH 9.5 and 7.4. This confirms that nitrate

Fig. 3. EPR spectra of 100 lM iron(III) hemoglobin at pH 7.4 and 9.5, manually mixed with nitrate (4 and 2 M, respectively).

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indeed binds at the heme, generating a split of the g  6 signal (S = 5/2, in the pH 7.4 sample) as well as low-spin signals (g = 2.78 and g = 2.27), all of which are the hallmarks of the oxy-NO intermediate trapped in the freeze-quench experiment (cf. Fig. 2). We therefore assign the ‘oxy-NO intermediate’ to be an iron(III)-nitrate adduct. The ratio of high-spin to low-spin species is somewhat higher in the manually mixed samples of Fig. 3 compared to the freeze-quenched samples of Fig. 2 (65:11 lM in the pH 9.5 RFQ, 70:5 lM in the pH 9.5 manually-mixed sample); we attribute this to an incomplete equilibrium between the two spin states in the kinetic experiment. A nitrate ligand directly bound to iron in hemoglobin would incur sterical clashes with the distal histidine residue and/or with nearby hydrophobic side-chains (cf. Fig. 4), which should weaken/destabilize the iron-nitrate bond, favouring the S = 5/2 state over S = 1/2 as the former entails a more loosely-bound nitrate ligand than the latter, and also favouring dissociation of nitrate from the iron. The accumulated evidence now suggests that the sole intermediate detected so far in the reaction of oxy-globins with nitric oxide is an iron(III)-nitrate adduct, present as a mixture of low- and highspin conformers/isomers, rather than a ferric-peroxynitrite adduct. We have previously reported S = 1/2 heme ferric-peroxynitrite adducts [Fe-O-O-N-O]2+ to be energetically favoured over S = 3/2 [16]; we now report that with the same methodology (BP86/ 6-31G**) the S = 5/2 state is also disfavoured, by 25 kcal/mol, over S = 1/2. Thus, should a ferric-peroxynitrite intermediate ever be detected, theory at this stage predicts a low-spin state. The OONO moiety in the S = 5/2 isomer appears structurally identical to that in the previously-reported S = 1/2 counterpart (details in Fig. 5) [16]. Incidentally, there are two other isomers possible for a ferric-peroxynitrite heme complex. The N-bound isomer [Fe-N (O)-O-O]2+ was previously reported [16] to essentially contain an Fe(II)-NO adduct non-covalently associated with an oxygen molecule, which among other things served to further argue against direct reaction between heme Fe(II)-NO and dioxygen [15], as opposed to the diffusion-controlled reaction between Fe(II)-O2 and NO [3]; we now report that the S = 5/2 N-bound isomer features similar structural features (Fig. 5), reinforcing these conclusions. The Fe-O-O-N-O isomer is only 1 kcal/mol more stable than Fe-N(O)-O-O. The Fe-O(NO)-O S = 5/2 isomer is calculated to involve no well-defined OO–NO bond; this isomer is 15 kcal/mol less stable than Fe-O-O-N-O. The failure to detect an iron(III)-peroxynitrite intermediate in globins, as well as the predicted lifetime of less than 1 ls [24] for such an intermediate, would argue against metal-peroxynitrite adducts as isolatable species. An example of an isolatable metal-peroxynitrite adduct is nevertheless [Co(CN)5(OONO)]3, the stability of which was linked to charge donation by the cyano ligands [17]. Indeed, using the same density functional (DFT)

Fig. 4. Spacefilling views of two possible conformations of a heme-nitrate adduct of hemoglobin, illustrating sterical clashes with His 58 and Leu29 (left panel) or with Phe42 and Val62 (right panel).

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Fig. 5. Isomers for the putative S = 5/2 state of a globin ferric-peroxynitrite adduct. Key bond lengths (Å) are indicated.

Fig. 6. Geometry-optimized Co and Cr peroxynitrite models. Key bond lengths (Å) are indicated (Co/Cr–O, O–O, N–O, and O–O). The N–O bond length in models where the OO–NO bond has dissociated (>1.9 Å) is clearly indicative of NO+ [35].

methodology previously applied to heme peroxynitrite models [16] Fig. 6 shows that in [Co(CN)5(OONO)]3 an intact peroxynitrite ligand is obtained, whereas a putative [Co(OH2)5(OONO)]2+ adduct is calculated not to be a stable species, in the sense that the predicted CoOO–NO bond is far beyond the sum of the oxygen + nitrogen covalent radii, featuring instead an NO+ loosely associated with a Co(III)-peroxo adduct – in excellent agreement with experiment. The related [Cr(OH2)5(OONO)]2+, also invoked as a possible transient species [34], similarly produces NO+ and a metal-peroxo species simply upon geometry optimization. Based on these and previous [16] observations, the likelihood of OO–NO bond cleavage (or, outer-sphere oxidation of an incoming NO molecule by a metal-superoxo adduct) thus appears to be smaller with electron-richer metal centres. Olson and co-workers reported that the ability of oxyhemoglobin to affect arterial blood pressure correlates with the rate of nitric oxide reacting with oxyhemoglobin [36]. The data shown here suggests that the overall rate-limiting step of this process is the decay of the iron(III)-nitrate intermediate. Olson and co-workers also reported on a hemoglobin mutant, rHb4 [36], whose reaction with NO was significantly slower than in native Hb; in fact, this rate in rHb4 was also smaller than the rate of decay of the iron(III)-nitrate intermediate in the wildtype, implying that initial formation of the iron(III)-peroxynitrite intermediate may have become the slowest step of the process (presumably due to slower diffusion of NO into the heme pocket). Such shifts in rate-limiting processes may need to be considered in rationally designing a successful hemoglobin-based blood substitute. Lastly, we note that the findings reported herein on hemoglobin need not necessarily apply in identical manner to other hemoproteins, where a peroxynitrite adduct may in principle still be observed, or where other reaction mechanisms may be at work.

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