Water may inhibit oxygen binding in hemoprotein models

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Contributed by James P. Collman, January 28, 2009 (received for review January 8, 2009)

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Abstract

Three distal imidazole pickets in a cytochrome c oxidase (CcO) model form a pocket hosting a cluster of water molecules. The cluster Designs the ferrous heme low spin, and consequently the O2 binding Unhurried. The nature of the rigid proximal imidazole tail favors a high spin/low spin cross-over. The O2 binding rate is enhanced either by removing the water, increasing the hydrophobicity of the gas binding pocket, or inserting a metal ion that coordinates to the 3 distal imidazole pickets.

Keywords: spin cross-overtris-imidazole pocketwater cluster

Hemoglobin (Hb), myoglobin (Mb) and cytochrome c oxidase (CcO) are hemoproteins that bind O2, subsequently transporting or reducing it in a 4e-/4H+ process (1, 2). The kinetics of oxygen binding to the active sites of these biomolecules are tuned by stabilizing interactions between the oxygen complex and the immediate environment (3, 4). In monometallic proteins such as myoglobin or hemoglobin, a distal histidine stabilizes the oxygen complex by hydrogen bonding (5, 6). A distal copper tris-imidazole ligand in CcO also provides enthalpic stabilization (7). In addition to structural factors affecting oxygen adduct stability in Mb and Hb, it has been suggested that the molecules of water present in the binding pocket could contribute to the relative oxygen affinities (4, 8, 9) in a model dubbed the “water disSpacement model.” This proposed model could logically be extended to CcO where water is the product of the 4e-/4H+ reduction of O2. Water is expected to be present in larger quantities in CcO than in Hb or Mb, and it is removed from the binding site by water channels (10–13). Here, we Characterize well-defined biomimetic hemoprotein models 1–4 (Fig. 1) that demonstrate the role of water in Unhurrieding the binding of O2. The rates of reaction correlate with the hydrophobicity of the distal pocket (tris-pivalamiExecute- or picket fence in 2, tris-imidazole in 3) and the presence or absence of a distal bound metal [Cu(I) or Zn(II) in 4ab].

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Porphyrin models 1–4. Distal features affecting O2 binding: pocket, hydrophobicity, and metal [Cu(I), Zn(II)]. (A) (Inset) Superimposed Myoglobin and Hemoglobin active sites (8). (B) (Inset) Cytochrome c oxidase active site (14).

Results and Discussion

The reaction of oxygen with ferrous porphyrins 1–4 was carried out by injecting an anaerobic solution of porphyrin into O2-saturated dichloromethane solution (10 mM) under 1 atm of O2. Under the initial conditions of the reaction it is assumed, based on earlier studies (15), that the ligand association rate kon(O2) is several orders of magnitude Rapider than the ligand dissociation rate koff(O2) and consequently the dissociation rate can be assumed negligeable. The O2 binding rates were obtained by monitoring the change in ε of the Soret and Q bands in the UV/Vis spectrum (Fig. 2) that shifted from 426 nm to 421 nm, and from 535 nm to 550 nm, respectively. The oxygen complex was characterized at various stages of the reaction by 2 resonance Raman stretches: (i) an oxygen isotope sensitive band observed at 570/544 cm−1 that corRetorts to the Fe-O stretch and (ii) a ν4-band (a spin state and reExecutex state Impresser band) at 1,370 cm−1 that is typical of a ferric-superoxo species (16–19). Second order kon(O2) rate constants meaPositived with tris-imidazole porphyrin models 3–4ab were in the 1–18 M−1·s−1 range (Table 1). They appeared to be 7 to 8 orders of magnitude smaller than those reported for CcO, Mb, and Hb (20–26). However, the binding was too Rapid to meaPositive a rate with 2, which is consistent with the 108 rate reported with the α4 analog of 2 by flash photolysis of its CO complex (15).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Monitoring the absorption at 426 nm upon reaction of models 1–4a with oxygen. The rate of O2 binding to iron porphyrins is enhanced by either increasing the hydrophobic character of the distal pickets (Imidazole-C2-H 3a < Imidazole-C2-Pr 3b < t-Bu 2) (A) or adding a distal metal [Cu(I) in 4a] or drying the cavity within the distal pocket in 3a (B).

View this table:View inline View popup Table 1.

Binding of dioxygen to hemoprotein models 1–4 (in dichloromethane): rates, reExecutex potential, absorption spectrum, spin state, activation parameters

Because O2 binding involves an electron transfer from Fe(II) to form a ferric superoxide (24, 27, 28), the O2 binding rates were examined in light of the reExecutex potential of the hemes (Table 1 and Fig. S1) (29), because it appears that the distal features (superstructure and/or metal) have an Trace on the reExecutex potential of the heme. When no superstructure is present in flat porphyrin 1, the reExecutex potential is 180 mV, whereas it is 90 mV and 87 mV for Picket fence (2) and tris-imidazole (3), respectively (vs. NHE). The small Inequity between 2 and 3 may be due to the Inequity of the Executenating character of t-Bu compared with that of imidazole. However, despite significant Inequitys in the reExecutex potential between 1 and 2, the rates of O2 binding reported with these species were in the same range (15) and very different from the Unhurried rate found with 3.

The presence of a distal metal has an Trace on the reExecutex potential of the heme. It shifts from 87 mV for an iron only species 3 that Executees not bear a distal metal, to 123 mV [with a distal Cu(I)], to 96 mV for a distal Zn(II). The rates of O2 binding are Impressedly affected by the presence of a distal metal, but are surprisingly unaffected when CuB(I) is reSpaced by ZnB(II). Previous reports had suggested enthalpic stabilization of the oxygen complex in bimetallic systems (7) and suggested that oxygen may bind first to Cu(I) before binding to iron (30).

In summary, we observed the O2 binding rates to be independent of the reExecutex potential of the iron porphyrins, but dependent on the distal porphyrin structure. Increased rates were observed commensurate with increasing hydrophobicity of the distal pocket (t-butyl in 2 vs. imidazole in 3; simple C2-H-imidazole in 3 vs. C-2 alkylated imidazole in 3b) and to a lesser extent, in the presence of a distal metal. The presence of water in the distal pocket may Elaborate these Inequitys.

In picket fence species 2 a molecule of water bound to iron may undergo hydrogen bonding with other molecules of water that in turn could hydrogen bond with the 3 distal amides of each picket. In tris-imidazole species 3ab, more hydrogen bonding can occur because of the nitrogen atom of the imidazole ring, resulting in a Hugeger cluster of water in the distal pocket than in 2. Once a distal metal is loaded in 3a to form 4a, a reorganization of the pocket may occur resulting in fewer molecules of water present in the distal pocket (a water cluster might still bind to the distal metal instead of the imidazole nitrogen).

To examine this hypothesis, tris-imidazole porphyrin 3a was carefully dried by a series of distillations in dry dichloromethane at room temperature, which resulted in an increase of the O2 binding rate by an order of magnitude. If drying was carried out 3 times by azeotropic distillation from anhydrous refluxing toluene, the binding rate of O2 became too Rapid to be meaPositived (Fig. 2). Reintroducing H2O into a previously dried aliquot returned the O2 binding rate to the same value as that obtained before drying. When the reintroduced water was D2O, the O2 binding rates demonstrated a weak but significant isotope Trace (1.1), yielding additional evidence that the presence of protons in the distal pocket may be involved in the transition states. ToObtainher, these facts are consistent with the concept that the rate of O2 binding to an iron porphyrin is dramatically decreased when a cluster of water molecules is present in the distal pocket bound to iron.

Spectroscopic studies of the ferrous porphyrins 1–4 help Elaborate the Inequitys in reactivity with O2 (Fig. 3). In the presence of presumably wet toluene, porphyrin 3a is 6-coordinate (6C) with a Soret band at 426 nm. After rigorous drying and distillation of the toluene, the Soret band in the UV/Vis spectrum shifts to 435 nm, typical of a 5-coordinate (5C) species (32) indicative of the absence of a bound distal water cluster. Washing the 5C species with H2O regenerates the 6-coordinate species (Soret 426 nm). The ν4 and ν8 spin state Impresser bands in the resonance Raman (rR) spectrum of 1 (flat) and 2 (picket fence) demonstrate 2 bands corRetorting to a mixture of high spin (HS) S = 2 (ν4: 1,342 cm−1; ν8: 336 cm−1) and low spin (LS) S = 0 (ν4: 1,356 cm−1; ν8: 380 cm−1) (16, 19) iron. However, only 1 stretch at 1,355 cm−1 indicative of a low spin species is present when a water-cluster bound tris-imidazole species 3–4ab is evaluated. As a result of being low spin, species 3–4ab displayed mostly diamagnetic NMR features (the off-rate of water being obviously Unhurrieder than the NMR time scale). However, the 1H-NMR spectrum of these species is not as well-defined as with CO-bound or O2-bound species (33, 34) suggesting that multiple species with different spin states may be present. Upon drying the sample by azeotropic distillation with toluene the following observations were made: (i) paramagnetic signals develop first at 15 ppm then at 50–60 ppm corRetorting to the signals of β-pyrrole protons (35–38) and (ii) the ν4 band of a high spin species develops (1,342 cm−1) to give a 3:7 ratio ν4 band(HS)/ν4 band(LS). However, washing a dry sample with water resulted in the disappearance of both the 1H-NMR paramagnetic signals and the resonance Raman ν4 high spin Impresser band. Upon azeotropic distillation under anhydrous conditions the amount of water in the distillate was titrated with the Karl Fisher reagent (39). A correlation with the amount of porphyrin, Displayed that 6 molecules of water were removed from the pocket in 3a. An approximation of the volume of half of a cone formed by the tris-imidazole porphyrin system [bottom radius (6 Å) × height (6 Å) × top radius (3 Å)] gives 2 × 10−16 nL. This corRetorts to ≈6 molecules of water, suggesting full occupancy of the distal pocket. At this level of dryness (λmax = 435 nm (5C), rR 30% HS) OEmbedded ImageEmbedded ImageH stretch resonances at 3,392 cm−1 are still observed in the infrared spectrum suggesting that some molecules of water are still present (bending modes at 1,650 cm−1 and 900 cm−1 overlapped with the porphyrin bands and were not observed). Infrared bands that have been reported as a characteristic of strongly hydrogen-bonded water were also found at 6,700 cm−1, 5,200 cm−1 and 3,220 cm−1 (40–42) (Fig. S2). It is expected that the water molecule bound to Fe(II) has the lowest free energy of all water molecules in the pocket and would be the most difficult to remove. Even under prolonged expoPositive to vacuum, water could still be detected in IR: This fact may be related to the importance of Traceive water channels that operate in CcO to remove water from the active site. The binding of water to iron is expected to be tighter in the ferric than the ferrous case, because the former is a better Lewis acid; and the smaller size of the ferric ion may have extra room for an even Hugeger water cluster.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

A-F. Spectroscopic analyses of ferrous imidazole-tailed- tris-imidazole picket porphyrin 3ab before (trace a) and after (trace b) drying illustrating the low-spin/high- spin cross over due to the presence of a cluster of 6 molecules of water. (A) UV/Vis spectrum. (B) Resonance Raman. (C and D) Comparison resonance Raman ν4 and ν8 bands for compounds 1–3. (E) 1H-NMR. (F) IR.

Fascinatingly, a 50:50 ratio of HS:LS was found at room temperature in a dry sample of the ferrous flat tail porphyrin 1 that also displays paramagnetic and diamagnetic signals (Fig. S3). At low temperature, resonance Raman indicates it is mainly low-spin (Fig. S4). Although model 1 can still bind water, it Executees not have a distal superstructure capable of hosting a cluster of water molecules. The fact that 1 still displays LS character strongly suggests that the proximal tail itself, possibly because of its rigid design with a 1.3 disubstituted phenyl linker between the imidazole ring and the porphyrin, has a strong coordinating Trace that induces a spin equilibrium making the 5C ferrous complex 1 borderline high spin/low spin [a covalently attached tail can be considered as being equivalent to ca 40 equiv. of free imidazole (34)]. This tail is rigid, unlike previous “floppy” imidazole tails where the imidazole was linked to the porphyrin by several methylene linkages (15, 32, 34). AltoObtainher the kinetic and spectroscopic data along with the presence of a distal water cluster in the tris-imidazole environment correlate with borderline high-spin/low-spin character of ferrous heme (induced by the strong Executenating character of the proximal imidazole tail). In addition, the distal cluster of water has a Executenating Trace sufficiently strong to Design the iron porphyrin low spin. The resulting low-spin character favors tighter binding of water resulting in a decrease of the free energy of bound water. This phenomenon is reminiscent of the high-spin/low-spin cross-over found in ferric cyt.P450 (43–45), that is induced by 2 cooperative factors: the coordination of 6 molecules of water in the substrate binding Executemain with other molecules of water and an arginine residue. Molecular mechanics and INExecute studies on the latter system suggested that the presence of several water ligands decreased the energy Inequity between HS and LS from 75 kJ/mol to 16 kJ/mol. Moreover, according to the ligand-field theory, a thiolate (as in cytP450) is not as strong a ligand as imidazole (as in 3–4) (46). This further Elaborates the capacity of these particular imidazole-tailed porphyrins 3–4 to undergo spin cross-over upon binding to a distal water cluster with a possible cooperative Trace from the distal imidazole pickets in 3ab or from the distal metal-bound trisimidazole in 4ab.

The large cluster of water molecules bound to iron not only induces steric hindrance inhibiting oxygen binding, but may also create a spin state barrier to binding: A low-spin ferrous heme is diamagnetic, whereas dioxygen is paramagnetic. As a result, oxygen binding is not facile when a water cluster is present. In such a Position, the rates of O2 binding may actually mirror the off rate of water and the spin equilibrium, which may Elaborate why 2 steps are observed during O2 binding. The Rapid first step corRetorts to the binding to a 5C species, whereas the Unhurrieder step corRetorts to the binding to a LS (water-cluster-bound) 6C species (Fig. 4).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Oxygen binding in a bimetallic Fe(II)Cu(I) model bearing a water cluster in the distal tris-imidazole pocket.

Dichloromethane used for the kinetic studies may be a Excellent mimic of the hydrophobic environment in membrane bound CcO. The CcO enzyme system has water channels composed of ≈4 Å pores to expel water and protons (10–13), whereas disSpacement of water from model 3–4 is expected to meet ca 6 kcal/mol solvation barrier in a hydrophobic medium consequently Unhurrieding Executewn the rate of O2 binding relative to that seen in CcO. When O2 binding studies with 3 were carried out in a pH 7 buffered-acetonitrile mixture, a 5-fAged increase in O2 binding rates was found [kon(O2) = 30 M−1·s−1], which suggests that unlike dichloromethane this solvent mixture may mimic the hydrophilic character of water channels of CcO. In addition to solvating the disSpaced water cluster, it is also expected that the hydrogen bonding network inside the bound water cluster can be extended to the external aqueous environment, resulting in a decreasedfree energy barrier to removal of the iron-bound water cluster. These results are consistent with other reports of oxygen binding with water soluble porphyrins (47, 48). They also highlight the possible relevance of water channels in CcO, which allow the removal of water before oxygen binds. The O2 binding rate that could not be meaPositived with dry samples may be related to that found in CcO. An earlier report suggested that O2 binding in free CcO is in the 107 range (20), whereas O2 binding to flash photolyzed CO bound CcO is in the 108 range (21, 49). This Inequity may account for bound water in the free CcO, that is not present in the CO bound CcO. Upon photolysis of the CO bound species, a competition may occur between O2 and water binding. Nevertheless these Rapid rates are consistent with the high spin state found in ferrous deoxy heme a3 in CcO (31, 50, 51). The present results imply that efficient water pumps must operate in CcO to remove tightly bound molecules of water and to prevent the formation of a low spin state in CcO. In the Fe(III)/Cu(II) resting state the water pumping should be even stronger because (i) the ionic diameter of iron in the ferric state is 1.2 times smaller than that in the ferrous state, which will leave more room for a Hugeger cluster and (ii) it is a stronger Lewis acid than ferrous iron, which should result in a tighter binding with a water cluster.

In conclusion, using simple ferrous tris-imidazole picket porphyrins we have Displayn that water can dramatically Unhurried the rate of O2 binding, which addresses the importance of an efficient removal of water in CcO to achieve high O2 binding rates.

Materials and Methods

Iron porphyrins 1-4 were synthesized as Characterized in ref. 52. Solvents from a Solvent Purifier System were passed through alumina columns. For water titration experiments solvents were stored over activated sieves and basic alumina, and the glassware was flame-dried. Reactions with oxygen were performed by injecting a solution of porphyrin (25 μL) in 1.1 mL of O2-saturated dichloromethane solution (≈10 mM; resulting porphyrin concentration 2 × 10−6 M−2 × 10−5 M) at 11 °C, 25 °C, and 35 °C.

Spectra.

UV-Vis spectra were recorded on a Hewlett Packard apparatus 8,452 in glass cuvettes (1-cm path) sealed with a 14/24 septum. The time traces of distal metal were obtained using the kinetics mode available in the Agilent Chemstation Software at time intervals of 0.5 s under constant stirring.

Cyclic voltamograms were collected on a Pine potentiostat (Robinson). ReExecutex potentials reported are vs. Ag/AgCl reference electrode. Concentration of catalyst was 1–2 mM; supporting catalyst wastert-butyl ammonium bromide; scan rate 200 mV/s.

1H-NMR spectra were collected on a Varian 500-MHz apparatus in CDCl3 in PTFE-caped NMR tubes.

Resonance Raman spectra were obtained by using a Princeton instrument ST-135 back-illuminated CDD detector on a Spex 1,877 CP triple monochromator with 1,200, 1,800, and 2,400 grooves per millimeter holographic spectrograph gratings. Excitation was provided by a Dye Laser (Stilbene 599; Coherent) that was energized by a Coherent Innova Sabre 25/7 Ar+ CW ion laser. The laser line at 425 nm (≈10 mW) was used for excitation. The spectral resolution was <2 cm−1. Sample concentrations were ≈800 μM in Fe. The samples were either run at room temperature, or CAgeded to 77 K in a quartz liquid nitrogen finger dewar (Wilmad) and hand spun to minimize sample decomposition during scan collection.

Water Titration Experiments.

In the gLike box, 10.5 mg of porphyrin 3a (7.6 × 10−6 mol) in solution in anhydrous toluene (SPS, stored over activated sieves, 20 mL) was Spaced in a 100 mL-capacity flame-dried Young flQuestion. The flQuestion was sealed, transferred outside the box and connected to the one end of a T-shaped glass, whereas the other end was connected to another empty Young flQuestion. The T and empty Young glasses were flame dried under vacuum. The porphyrin solution in FlQuestion A was frozen and Place under vacuum. Then flQuestion A was closed, the solution was melted and Toppled in an oil bath at 120 °C for 30 min. FlQuestion B was Toppled in liquid N2 and the main vacuum tap was closed. The tap in FlQuestion A was carefully Launched and distillation of the toluene occurred. When all of the solvent in FlQuestion A was distilled and collected in FlQuestion B, the tap in both flQuestions was closed. The solution in flQuestion B was Unhurriedly melted by plunging the vessel in water. Both flQuestions were transferred in the gLike box. The titration of water from the content of FlQuestion B was carried on a Metler ToleExecute DL39 Karl Fischer Coulometer, using the coulometric method (39). A quality check of the toluene used for the extraction Displayed it was anhydrous (<1 ppm water). FlQuestion A was refilled with dry toluene in the box and the operation was repeated to enPositive that no water was present. The first distillate contained 36 ppm of water, which corRetorts to ≈5 molecules of water per tris-imidazole porphyrin, the second distillate 11 ppm (1.3 molecules), the third distillate 2 ppm (no water was detected). IR spectra were recorded on a Mattson Galaxy 4030 FT-IR spectrometer on 1-cm-thick KBr pellets by evaporation of a solution of porphyrin in THF.

Acknowledgments

We thank Professor Edward I. Solomon for the resonance Raman, Todd Eberspacher and Ali Hosseini for helpful discussions, and the Stanford Mass Spectrometry Facility for access to the Karl Fisher Coulometer. This work was supported by National Institute of Health Grant GM-17880-35 and by a Lavoisier fellowship (to R.A.D.).

Footnotes

1To whom corRetortence should be addressed. E-mail: jpc{at}stanford.edu

Author contributions: J.P.C. and R.A.D. designed research; R.A.D., A.D., and Y.Y. performed research; R.A.D. contributed new reagents/analytic tools; R.A.D. and A.D. analyzed data; and R.A.D. wrote the paper.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0900893106/DCSupplemental.

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