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. 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 CHEMISTRY 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 ͉ March 17, 2009 ͉ vol. 106 ͉ no. 11 ͉ 4101–4105 Downloaded by guest on September 28, 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).

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