Water May Inhibit Oxygen Binding in Hemoprotein Models

Water may inhibit oxygen binding in hemoprotein models

James P. Collman1, Richard A. Decre´ au, Abhishek Dey, and Ying Yang

Department of Chemistry, Stanford University, Stanford, CA, 94305

Contributed by James P. Collman, January 28, 2009 (sent for review January 8, 2009) Three distal imidazole pickets in a cytochrome c oxidase (CcO) was too fast to measure a rate with 2, which is consistent with the model form a pocket hosting a cluster of water molecules. The 108 rate reported with the ␣4 analog of 2 by flash photolysis of cluster makes the ferrous heme low spin, and consequently the O2 its CO complex (15). binding slow. The nature of the rigid proximal imidazole tail favors Because O2 binding involves an electron transfer from Fe(II) a high spin/low spin cross-over. The O2 binding rate is enhanced to form a ferric superoxide (24, 27, 28), the O2 binding rates were either by removing the water, increasing the hydrophobicity of the examined in light of the redox potential of the hemes (Table 1 gas binding pocket, or inserting a metal ion that coordinates to the and Fig. S1) (29), because it appears that the distal features 3 distal imidazole pickets. (superstructure and/or metal) have an effect on the redox potential of the heme. When no superstructure is present in flat spin cross-over ͉ tris-imidazole pocket ͉ water cluster porphyrin 1, the redox potential is 180 mV, whereas it is 90 mV and 87 mV for Picket fence (2) and tris-imidazole (3), respec- emoglobin (Hb), myoglobin (Mb) and cytochrome c oxidase tively (vs. NHE). The small difference between 2 and 3 may be due to the difference of the donating character of t-Bu compared H(CcO) are hemoproteins that bind O2, subsequently trans- porting or reducing it in a 4e-/4Hϩ process (1, 2). The kinetics with that of imidazole. However, despite significant differences of oxygen binding to the active sites of these biomolecules are in the redox potential between 1 and 2, the rates of O2 binding tuned by stabilizing interactions between the oxygen complex reported with these species were in the same range (15) and very and the immediate environment (3, 4). In monometallic proteins different from the slow rate found with 3. CHEMISTRY such as myoglobin or hemoglobin, a distal histidine stabilizes the The presence of a distal metal has an effect on the redox oxygen complex by hydrogen bonding (5, 6). A distal copper potential of the heme. It shifts from 87 mV for an iron only tris-imidazole ligand in CcO also provides enthalpic stabilization species 3 that does not bear a distal metal, to 123 mV [with a (7). In addition to structural factors affecting oxygen adduct distal Cu(I)], to 96 mV for a distal Zn(II). The rates of O2 stability in Mb and Hb, it has been suggested that the molecules binding are markedly affected by the presence of a distal metal, of water present in the binding pocket could contribute to the but are surprisingly unaffected when CuB(I) is replaced by relative oxygen affinities (4, 8, 9) in a model dubbed the ‘‘water ZnB(II). Previous reports had suggested enthalpic stabilization displacement model.’’ This proposed model could logically be of the oxygen complex in bimetallic systems (7) and suggested extended to CcO where water is the product of the 4e-/4Hϩ that oxygen may bind first to Cu(I) before binding to iron (30). In summary, we observed the O2 binding rates to be indepen- 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 dent of the redox potential of the iron porphyrins, but dependent binding site by water channels (10–13). Here, we describe on the distal porphyrin structure. Increased rates were observed well-defined biomimetic hemoprotein models 1–4 (Fig. 1) that commensurate with increasing hydrophobicity of the distal pocket (t-butyl in 2 vs. imidazole in 3; simple C2-H-imidazole in demonstrate the role of water in slowing the binding of O2. The rates of reaction correlate with the hydrophobicity of the distal 3 vs. C-2 alkylated imidazole in 3b) and to a lesser extent, in the pocket (tris-pivalamido- or picket fence in 2, tris-imidazole in 3) presence of a distal metal. The presence of water in the distal and the presence or absence of a distal bound metal [Cu(I) or pocket may explain these differences. Zn(II) in 4ab]. In picket fence species 2 a molecule of water bound to iron may undergo hydrogen bonding with other molecules of water that in Results and Discussion turn could hydrogen bond with the 3 distal amides of each picket. The reaction of oxygen with ferrous porphyrins 1–4 was carried In tris-imidazole species 3ab, more hydrogen bonding can occur out by injecting an anaerobic solution of porphyrin into O - because of the nitrogen atom of the imidazole ring, resulting in 2 a bigger cluster of water in the distal pocket than in 2. Once a saturated dichloromethane solution (10 mM) under 1 atm of O2. Under the initial conditions of the reaction it is assumed, based distal metal is loaded in 3a to form 4a, a reorganization of the pocket may occur resulting in fewer molecules of water present on earlier studies (15), that the ligand association rate kon(O2)is several orders of magnitude faster than the ligand dissociation in the distal pocket (a water cluster might still bind to the distal rate k (O ) and consequently the dissociation rate can be metal instead of the imidazole nitrogen). off 2 To examine this hypothesis, tris-imidazole porphyrin 3a was assumed negligeable. The O2 binding rates were obtained by monitoring the change in ␧ of the Soret and Q bands in the carefully dried by a series of distillations in dry dichloromethane UV/Vis spectrum (Fig. 2) that shifted from 426 nm to 421 nm, at room temperature, which resulted in an increase of the O2 and from 535 nm to 550 nm, respectively. The oxygen complex binding rate by an order of magnitude. If drying was carried out was characterized at various stages of the reaction by 2 resonance 3 times by azeotropic distillation from anhydrous refluxing Raman stretches: (i) an oxygen isotope sensitive band observed Ϫ1 at 570/544 cm that corresponds to the Fe-O stretch and (ii)a Author contributions: J.P.C. and R.A.D. designed research; R.A.D., A.D., and Y.Y. performed Ϫ1 ␯4-band (a spin state and redox state marker band) at 1,370 cm research; R.A.D. contributed new reagents/analytic tools; R.A.D. and A.D. analyzed data; that is typical of a ferric-superoxo species (16–19). Second order and R.A.D. wrote the paper. kon(O2) rate constants measured with tris-imidazole porphyrin The authors declare no conﬂict of interest. Ϫ1 Ϫ1 models 3–4ab were in the 1–18 M ⅐s range (Table 1). They 1To whom correspondence should be addressed. E-mail: [email protected]. appeared to be 7 to 8 orders of magnitude smaller than those This article contains supporting information online at www.pnas.org/cgi/content/full/ reported for CcO, Mb, and Hb (20–26). However, the binding 0900893106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900893106 PNAS Early Edition ͉ 1of5 Downloaded by guest on September 30, 2021 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).

toluene, the binding rate of O2 became too fast to be measured a result of being low spin, species 3–4ab displayed mostly (Fig. 2). Reintroducing H2O into a previously dried aliquot diamagnetic NMR features (the off-rate of water being obviously 1 returned the O2 binding rate to the same value as that obtained slower than the NMR time scale). However, the H-NMR before drying. When the reintroduced water was D2O, the O2 spectrum of these species is not as well-defined as with CO- binding rates demonstrated a weak but significant isotope effect bound or O2-bound species (33, 34) suggesting that multiple (1.1), yielding additional evidence that the presence of protons species with different spin states may be present. Upon drying in the distal pocket may be involved in the transition states. the sample by azeotropic distillation with toluene the following Together, these facts are consistent with the concept that the rate observations were made: (i) paramagnetic signals develop first at of O2 binding to an iron porphyrin is dramatically decreased 15 ppm then at 50–60 ppm corresponding to the signals of when a cluster of water molecules is present in the distal pocket ␤-pyrrole protons (35–38) and (ii) the ␯4 band of a high spin bound to iron. species develops (1,342 cmϪ1) to give a 3:7 ratio ␯4 band(HS)/␯4 Spectroscopic studies of the ferrous porphyrins 1–4 help explain band(LS). However, washing a dry sample with water resulted in 1 the differences in reactivity with O2 (Fig. 3). In the presence of the disappearance of both the H-NMR paramagnetic signals presumably wet toluene, porphyrin 3a is 6-coordinate (6C) with a and the resonance Raman ␯4 high spin marker band. Upon Soret band at 426 nm. After rigorous drying and distillation of azeotropic distillation under anhydrous conditions the amount the toluene, the Soret band in the UV/Vis spectrum shifts to 435 of water in the distillate was titrated with the Karl Fisher reagent nm, typical of a 5-coordinate (5C) species (32) indicative of the (39). A correlation with the amount of porphyrin, showed that absence of a bound distal water cluster. Washing the 5C species 6 molecules of water were removed from the pocket in 3a.An with H2O regenerates the 6-coordinate species (Soret 426 nm). approximation of the volume of half of a cone formed by the The ␯4 and ␯8 spin state marker bands in the resonance Raman tris-imidazole porphyrin system [bottom radius (6 Å) ϫ height (rR) spectrum of 1 (flat) and 2 (picket fence) demonstrate 2 (6 Å) ϫ top radius (3 Å)] gives 2 ϫ 10Ϫ16 nL. This corresponds bands corresponding to a mixture of high spin (HS) S ϭ 2(␯4: to Ϸ6 molecules of water, suggesting full occupancy of the distal Ϫ1 Ϫ1 1,342 cm ; ␯8: 336 cm ) and low spin (LS) S ϭ 0(␯4: 1,356 pocket. At this level of dryness (␭max ϭ 435 nm (5C), rR 30% HS) cmϪ1; ␯8: 380 cmϪ1) (16, 19) iron. However, only 1 stretch at OOH stretch resonances at 3,392 cmϪ1 are still observed in the 1,355 cmϪ1 indicative of a low spin species is present when a infrared spectrum suggesting that some molecules of water are water-cluster bound tris-imidazole species 3–4ab is evaluated. As still present (bending modes at 1,650 cmϪ1 and 900 cmϪ1

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 Ͻ Imidazole-C2-Pr 3b Ͻ t-Bu 2)(A) or adding a distal metal [Cu(I) in 4a] or drying the cavity within the distal pocket in 3a (B).

2of5 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900893106 Collman et al. Downloaded by guest on September 30, 2021 Table 1. Binding of dioxygen to hemoprotein models 1–4 (in Interestingly, a 50:50 ratio of HS:LS was found at room dichloromethane): rates, redox potential, absorption spectrum, temperature in a dry sample of the ferrous flat tail porphyrin 1 spin state, activation parameters that also displays paramagnetic and diamagnetic signals (Fig. 0 S3). At low temperature, resonance Raman indicates it is mainly konO2, E (mV) UV/Vis Spin Model MϪ1⅐sϪ1 MeCN vs NHE Deoxy,nm state* Ref(s). low-spin (Fig. S4). Although model 1 can still bind water, it does not have a distal superstructure capable of hosting a cluster of 15ϫ 107 180 428 HS/LS 15 water molecules. The fact that 1 still displays LS character ␣ 2( 3) Too fast 90 427–429 HS/LS This study strongly suggests that the proximal tail itself, possibly because of ␣ ϫ 8 ( 4) 4.3 10 — 426 HS 15 its rigid design with a 1.3 disubstituted phenyl linker between the 3a (H) Wet 1 87 426 LS This study imidazole ring and the porphyrin, has a strong coordinating Dry Too fast — 435 HS/LS effect that induces a spin equilibrium making the 5C ferrous 3b (Pr) 18 — 426 LS This study complex 1 borderline high spin/low spin [a covalently attached 4a Cu(I) Wet 6.0 123 426 LS This study tail can be considered as being equivalent to ca 40 equiv. of free 4b Zn(II) 6.5 96 426 LS This study imidazole (34)]. This tail is rigid, unlike previous ‘‘floppy’’ Hb, Mb 107 to 108 — 435 HS 24–26 imidazole tails where the imidazole was linked to the porphyrin CcO 107 to 108 — 444 HS 20, 21, 31 by several methylene linkages (15, 32, 34). Altogether the kinetic Comparison with hemoproteins Hb, Mb (in buffer), and CcO. *, as shown by and spectroscopic data along with the presence of a distal water Resonance Raman and 1H-NMR (Fig. 3 A–F). cluster in the tris-imidazole environment correlate with borderline high-spin/low-spin character of ferrous heme (induced by the strong donating character of the proximal imidazole tail). In addition, the distal cluster of water has a donating effect overlapped with the porphyrin bands and were not observed). sufficiently strong to make the iron porphyrin low spin. The Infrared bands that have been reported as a characteristic of resulting low-spin character favors tighter binding of water strongly hydrogen-bonded water were also found at 6,700 cmϪ1, Ϫ1 Ϫ1 resulting in a decrease of the free energy of bound water. This 5,200 cm and 3,220 cm (40–42) (Fig. S2). It is expected that phenomenon is reminiscent of the high-spin/low-spin cross-over the water molecule bound to Fe(II) has the lowest free energy found in ferric cyt.P450 (43–45), that is induced by 2 cooperative of all water molecules in the pocket and would be the most factors: the coordination of 6 molecules of water in the substrate CHEMISTRY difficult to remove. Even under prolonged exposure to vacuum, binding domain with other molecules of water and an arginine water could still be detected in IR: This fact may be related to residue. Molecular mechanics and INDO studies on the latter the importance of effective water channels that operate in CcO system suggested that the presence of several water ligands to remove water from the active site. The binding of water to iron decreased the energy difference between HS and LS from 75 is expected to be tighter in the ferric than the ferrous case, kJ/mol to 16 kJ/mol. Moreover, according to the ligand-field because the former is a better Lewis acid; and the smaller size theory, a thiolate (as in cytP450) is not as strong a ligand as of the ferric ion may have extra room for an even bigger water imidazole (as in 3–4) (46). This further explains the capacity of cluster. these particular imidazole-tailed porphyrins 3–4 to undergo spin

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 ␯4 and ␯8 bands for compounds 1–3.(E) 1H-NMR. (F) IR.

Collman et al. PNAS Early Edition ͉ 3of5 Downloaded by guest on September 30, 2021 more room for a bigger 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 shown that water can dramatically slow 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 Fig. 4. Oxygen binding in a bimetallic Fe(II)Cu(I) model bearing a water Iron porphyrins 1-4 were synthesized as described in ref. 52. Solvents from a cluster in the distal tris-imidazole pocket. 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 ␮ cross-over upon binding to a distal water cluster with a possible performed by injecting a solution of porphyrin (25 L) in 1.1 mL of O2- Ϸ cooperative effect from the distal imidazole pickets in 3ab or saturated dichloromethane solution ( 10 mM; resulting porphyrin concentration 2 ϫ 10Ϫ6 MϪ2 ϫ 10Ϫ5 M) at 11 °C, 25 °C, and 35 °C. from the distal metal-bound trisimidazole in 4ab. The large cluster of water molecules bound to iron not only Spectra. UV-Vis spectra were recorded on a Hewlett Packard apparatus 8,452 induces steric hindrance inhibiting oxygen binding, but may also in glass cuvettes (1-cm path) sealed with a 14/24 septum. The time traces of create a spin state barrier to binding: A low-spin ferrous heme is distal metal were obtained using the kinetics mode available in the Agilent diamagnetic, whereas dioxygen is paramagnetic. As a result, oxygen Chemstation Software at time intervals of 0.5 s under constant stirring. binding is not facile when a water cluster is present. In such a Cyclic voltamograms were collected on a Pine potentiostat (Robinson). situation, the rates of O2 binding may actually mirror the off rate of Redox potentials reported are vs. Ag/AgCl reference electrode. Concentration water and the spin equilibrium, which may explain why 2 steps are of catalyst was 1–2 mM; supporting catalyst wastert-butyl ammonium bro- mide; scan rate 200 mV/s. observed during O2 binding. The fast first step corresponds to the 1 H-NMR spectra were collected on a Varian 500-MHz apparatus in CDCl3 in binding to a 5C species, whereas the slower step corresponds to the PTFE-caped NMR tubes. binding to a LS (water-cluster-bound) 6C species (Fig. 4). Resonance Raman spectra were obtained by using a Princeton instrument Dichloromethane used for the kinetic studies may be a good ST-135 back-illuminated CDD detector on a Spex 1,877 CP triple monochro- mimic of the hydrophobic environment in membrane bound mator with 1,200, 1,800, and 2,400 grooves per millimeter holographic spec- CcO. The CcO enzyme system has water channels composed of trograph gratings. Excitation was provided by a Dye Laser (Stilbene 599; Ϸ4 Å pores to expel water and protons (10–13), whereas Coherent) that was energized by a Coherent Innova Sabre 25/7 Arϩ CW ion displacement of water from model 3–4 is expected to meet ca 6 laser. The laser line at 425 nm (Ϸ10 mW) was used for excitation. The spectral Ͻ Ϫ1 Ϸ ␮ kcal/mol solvation barrier in a hydrophobic medium conse- resolution was 2cm . Sample concentrations were 800 M in Fe. The quently slowing down the rate of O binding relative to that seen samples were either run at room temperature, or cooled to 77 K in a quartz 2 liquid nitrogen finger dewar (Wilmad) and hand spun to minimize sample in CcO. When O2 binding studies with 3 were carried out in a pH decomposition during scan collection. 7 buffered-acetonitrile mixture, a 5-fold increase in O2 binding ϭ Ϫ1⅐ Ϫ1 rates was found [kon(O2) 30 M s ], which suggests that Water Titration Experiments. In the glove box, 10.5 mg of porphyrin 3a (7.6 ϫ unlike dichloromethane this solvent mixture may mimic the 10Ϫ6 mol) in solution in anhydrous toluene (SPS, stored over activated sieves, hydrophilic character of water channels of CcO. In addition to 20 mL) was placed in a 100 mL-capacity flame-dried Young flask. The flask was solvating the displaced water cluster, it is also expected that the sealed, transferred outside the box and connected to the one end of a hydrogen bonding network inside the bound water cluster can be T-shaped glass, whereas the other end was connected to another empty extended to the external aqueous environment, resulting in a Young flask. The T and empty Young glasses were flame dried under vacuum. decreasedfree energy barrier to removal of the iron-bound water The porphyrin solution in Flask A was frozen and put under vacuum. Then flask cluster. These results are consistent with other reports of oxygen A was closed, the solution was melted and plunged in an oil bath at 120 °C for 30 min. Flask B was plunged in liquid N2 and the main vacuum tap was closed. binding with water soluble porphyrins (47, 48). They also high- The tap in Flask A was carefully opened and distillation of the toluene light the possible relevance of water channels in CcO, which occurred. When all of the solvent in Flask A was distilled and collected in Flask allow the removal of water before oxygen binds. The O2 binding B, the tap in both flasks was closed. The solution in flask B was slowly melted rate that could not be measured with dry samples may be related by plunging the vessel in water. Both flasks were transferred in the glove box. to that found in CcO. An earlier report suggested that O2 binding The titration of water from the content of Flask B was carried on a Metler 7 Toledo DL39 Karl Fischer Coulometer, using the coulometric method (39). A in free CcO is in the 10 range (20), whereas O2 binding to flash photolyzed CO bound CcO is in the 108 range (21, 49). This quality check of the toluene used for the extraction showed it was anhydrous Ͻ difference may account for bound water in the free CcO, that is ( 1 ppm water). Flask A was refilled with dry toluene in the box and the operation was repeated to ensure that no water was present. The first not present in the CO bound CcO. Upon photolysis of the CO distillate contained 36 ppm of water, which corresponds to Ϸ5 molecules of bound species, a competition may occur between O2 and water water per tris-imidazole porphyrin, the second distillate 11 ppm (1.3 mole- binding. Nevertheless these fast rates are consistent with the high cules), the third distillate 2 ppm (no water was detected). IR spectra were spin state found in ferrous deoxy heme a3 in CcO (31, 50, 51). recorded on a Mattson Galaxy 4030 FT-IR spectrometer on 1-cm-thick KBr The present results imply that efficient water pumps must pellets by evaporation of a solution of porphyrin in THF. operate in CcO to remove tightly bound molecules of water and to prevent the formation of a low spin state in CcO. In the ACKNOWLEDGMENTS. We thank Professor Edward I. Solomon for the reso- Fe(III)/Cu(II) resting state the water pumping should be even nance Raman, Todd Eberspacher and Ali Hosseini for helpful discussions, and the Stanford Mass Spectrometry Facility for access to the Karl Fisher Coulom- stronger because (i) the ionic diameter of iron in the ferric state eter. This work was supported by National Institute of Health Grant GM- is 1.2 times smaller than that in the ferrous state, which will leave 17880-35 and by a Lavoisier fellowship (to R.A.D.).

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