Thermodynamic and Kinetic Studies on the Binding of Nitric Oxide to a

Published on Web 09/22/2006

Thermodynamic and Kinetic Studies on the Binding of Nitric Oxide to a New Enzyme Mimic of Cytochrome P450 Alicja Franke,† Natalya Hessenauer-Ilicheva,† Dominik Meyer,‡ Graz˘ yna Stochel,§ Wolf-D. Woggon,*,‡ and Rudi van Eldik*,† Contribution from the Institute for Inorganic Chemistry, UniVersity of Erlangen-Nu¨rnberg, Egerlandstrasse 1, 91058 Erlangen, Germany, Department of Chemistry, UniVersity of Basel, St. Johanns-Ring 19, 4056 Basel, Switzerland, and Faculty of Chemistry, Jagiellonian UniVersity, Ingardena 3, 30 060 Krako´ w, Poland. Received February 4, 2006; E-mail: [email protected]

Abstract: A new model for the P450 enzyme carrying a SO3- ligand coordinated to iron(III) (complex 2) reversibly binds NO to yield the nitrosyl adduct. The rate constant for NO binding to 2 in toluene is of the same order of magnitude as that found for the nitrosylation of the native, substrate-bound form of P450cam (E‚S-P450cam). Large and negative activation entropy and activation volume values for the binding of NO to complex 2 support a mechanism that is dominated by bond formation with concomitant iron spin change from S ) 5/2 to S ) 0, as proposed for the reaction between NO and E‚S-P450cam. In contrast, the dissociation of NO from 2(NO) was found to be several orders of magnitude faster than the corresponding reaction for the E‚S-P450cam/NO system. In a coordinating solvent such as methanol, the alcohol coordinates to iron(III) of 2 at the distal position, generating a six-coordinate, high-spin species 5. The reaction of NO with 5 in methanol was found to be much slower in comparison to the nitrosylation reaction of 2 in toluene. This behavior can be explained in terms of a mechanism in which methanol must be displaced during Fe-NO bond formation. The thermodynamic and kinetic data for NO binding to the new model complexes of P450 (2 and 5) are discussed in reference to earlier results obtained for closely related nitrosylation reactions of cytochrome P450cam (in the presence and in the absence of the substrate) and a thiolate-ligated iron(III) model complex.

Introduction

Synthetic iron porphyrins have received considerable attention from many groups, in particular regarding their use as chemical models in a variety of enzymatic oxidations such as those catalyzed by cytochromes P450, catalases, and peroxidases.1 It was shown that the nature of the anionic proximal ligand coordinating to iron plays a crucial role in controlling the properties and reactivity of the heme model complexes in such catalytic processes.2 In this context, heme-thiolate proteins like cytochromes P450 are of particular interest due to the significance of the quite unusual thiolate ligand with respect to spin†

University of Erlangen-Nu¨rnberg. University of Basel. § Jagiellonian University. ‡

(1) (a) Watanabe, Y. In The Porphyrin Handbook; Kadish, K. M., Smith, K. M., Guilard, R., Eds.; Academic: New York, 2000; Vol. 4, Chapter 30, pp 97-117. (b) McLain, J. L.; Lee, J.; Groves, J. T. In Biomimetic Oxidations Catalyzed by Transition Metal Complexes; Meunier, B., Ed.; Imperial College Press: London, 2000, pp 91-169. (c) Montanari, F.; Cassela, L. Metalloporphyrins Catalyzed Oxidations; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1994. (d) Traylor, T. G.; Traylor, P. S. In ActiVe Oxygen in Biochemistry; Valentine, J. S., Foote, C. S., Greenberg, A., Liebman, J. F., Eds.; Blackie Academic and Professional, Chapman and Hall: London, 1995; pp 84-187. (e) Ingold, K. U.; MacFaul, P. A. In Biomimetic Oxidations Catalyzed by Transition Metal Complexes; Meunier, B., Ed.; Imperial College Press: London, 2000; pp 45-89. (f) Meunier, B. Chem. ReV. 1992, 92, 1411. (2) (a) Dawson, J. H. Science 1988, 240, 433. (b) Poulus, T. L. J. Biol. Inorg. Chem. 1996, 1, 356. (c) Goodin, D. B. J. Biol. Inorg. Chem. 1996, 1, 360. (d) Franzen, S.; Roach, M. P.; Chen, Y.-P.; Dyer, R. B.; Woodruff, W. H.; Dawson, J. H. J. Am. Chem. Soc. 1998, 120, 4658. 10.1021/ja060650o CCC: $33.50 © 2006 American Chemical Society

state equilibria of the resting state, redox potential of the iron porphyrin, and the electronic nature of the high-valent iron(IV) oxo intermediate. Consequently, over the past three decades, many groups have focused their efforts on the synthesis of thiolate-ligated iron porphyrins in order to understand the function of the thiolate ligand and to mimic P450 reactivity.3-5 Although a number of sophisticated iron(III) porphyrin-thiolate complexes were synthesized, most of them remained structural models of the active site, displaying significant differences from (3) (a) Aissaoui, H.; Ghirlanda, S.; Gmu¨r, C.; Woggon, W.-D. J. Mol. Catal. A: Chem. 1996, 113, 393. (b) Aissaoui, H.; Bachmann, R.; Schweiger, A.; Woggon, W.-D. Angew. Chem., Int. Ed. 1998, 37, 2998. (c) Woggon, W.-D.; Wagenknecht, H.-A.; Claude, C. J. Inorg. Biochem. 2001, 83, 289. (d) Lochner, M.; Meuwly, M.; Woggon, W.-D. Chem. Commun. 2003, 1330. (e) Lochner, M.; Mu, L.; Woggon, W.-D. AdV. Synth. Catal. 2003, 345, 743. (4) (a) Higuchi, T.; Uzu, S.; Hirobe, M. J. Am. Chem. Soc. 1990, 112, 7051. (b) Higuchi, T.; Shimada, K.; Maruyama, N.; Hirobe, M. J. Am. Chem. Soc. 1993, 115, 7551. (c) Higuchi, T.; Hirobe, M. J. Mol. Catal. A: Chem. 1996, 113, 403. (d) Urano, Y.; Higuchi, T.; Hirobe, M.; Nagano, T. J. Am. Chem. Soc. 1997, 119, 12008. (e) Ohno, T.; Suzuki, N.; Dokoh, T.; Urano, Y.; Kikuchi, K.; Hirobe, M.; Higuchi, T.; Nagano, T. J. Inorg. Biochem. 2000, 82, 123. (f) Dokoh, T.; Suzuki, N.; Higuchi, T.; Urano, Y.; Kikuchi, K.; Nagano, T. J. Inorg. Biochem. 2000, 82, 127. (g) Suzuki, N.; Higuchi, T.; Nagano, T. J. Am. Chem. Soc. 2002, 124, 9622. (h) Suzuki, N.; Higuchi, T.; Urano, Y.; Kikuchi, K.; Uekusa, H.; Ohashi, Y.; Uchida, T.; Kitagawa, T.; Nagano, T. J. Am. Chem. Soc. 1999, 121, 11571. (5) (a) Matsu-ura, M.; Tani, F.; Nakayama, S.; Nakamura, N.; Naruta, Y. Angew. Chem., Int. Ed. 1989, 2000, 39. (b) Tani, F.; Matsu-ura, M.; Nakayama, S.; Ichimura, M.; Nakamura, N.; Naruta, Y. J. Am. Chem. Soc. 2001, 123, 1133. (c) Tani, F.; Matsu-ura, M.; Nakayama, S.; Naruta, Y. Coord. Chem. ReV. 2002, 226, 219. (d) Matsu-ura, M.; Tani, F.; Naruta, Y. J. Am. Chem. Soc. 1941, 2002, 124. J. AM. CHEM. SOC. 2006, 128, 13611-13624

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Figure 1. P450 enzyme models (1 and 27, 44) and coordination sphere of P450cam (3).

P450 enzymes regarding redox potentials and spin-state equilibria of the iron(III) resting state.6 Furthermore, most of these approaches failed to provide oxidation catalysts with decent turnover, which could be attributed to the instability of both the porphyrin chromophore and the thiolate ligand in the presence of strong oxidants such as PhIO, mCPBA, and H2O2 which are usually employed in the “shunt” pathway.7 To find more suitable enzyme mimics, the Woggon group has synthesized two new complexes, 1 and 2 (Figure 1), in which the RS- ligand is replaced by a RSO3- group.8 The SO3group has been introduced because DFT calculations revealed that ca. 40% of a negative charge is localized at the oxygen that coordinates to iron.8d Thus, it seemed reasonable that such a system would mimic the coordination sphere of several native P450’s, such as P450cam, P450terp, P450eryF, and P450BM-3, which display hydrogen bonding of the thiolate ligand to two amino acids of the protein backbone (see 3, Figure 1). This concept was confirmed by several experiments. For example, 1 and 2 show redox potentials Eo ) -210 and -290 mV, respectively (vs SCE measured in 0.1 M LiClO4, LiBr saturated DMF with ferrocene as an internal standard),8b indicating that exchanging the thiolate for SO3- ligand causes an anodic shift of ca. 300 mV.3 The Eo value of 2 is close to that obtained with a model compound that has two hydrogen bonds to a (6) Woggon, W.-D. Chimia 2001, 55 (4), 366. (7) (a) Tang, S. C.; Koch, S.; Papaefthymiou, G. C.; Foner, S.; Frankel, R. B.; Ibers, J. A.; Holm, R. H. J. Am. Chem. Soc. 1976, 98, 2414. (b) Caron, C.; Mitschler, A.; Riviere, G.; Ricard, L.; Schapacher, M.; Weiss, R. J. Am. Chem. Soc. 1979, 101, 7401. (c) Ogoshi, H.; Sugimoto, H.; Yoshida, Z. Tetrahedron Lett. 1975, 27, 2289. (8) (a) Woggon, W.-D.; Leifels, T.; Sbaragli, L. Cytochromes P450: Biochemistry, Biophysics and Drug Metabolism; Presented at the 13th International Conference on Cytochromes P450, Prague, Czech Republic, June 29-July 3, 2005. (b) Meyer, D. N.; Woggon, W. -D. Chimia 2005, 59 (3), 85. (c) Woggon, W.-D. Acc. Chem. Res. 2005, 38, 127. (d) Kozuch, S.; Leifels, T.; Meyer, D.; Sbaragli, L.; Shaik, S.; Woggon, W.-D. Synlett. 2005, 4, 675. (e) Sbaragli, L.; Woggon, W.-D. Synthesis 2005, 9, 1538. (f) Meyer, D.; Leifels, T.; Sbaragli, L.; Woggon, W.-D. Biochem. Biophys. Res. Commun. 2005, 338, 372. 13612 J. AM. CHEM. SOC.

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thiolate coordinating to iron (Eo ) -350 mV vs SCE in CH2Cl2)9 and is also similar to the redox potential determined for the E‚S complex of P450cam (Eo ) -170 mV vs NHE, which is equivalent to -411 mV vs SCE). However, it should be kept in mind that Eo depends very much on the selected solvent and counterion. Hence, it is not possible to interpret a difference of 20-50 mV between model and native systems in more detail. Further DFT calculation predicted, and X-ray structures confirmed, the coordination of one oxygen of the SO3- group to Fe(III).8d,f Complexes 1 and 2 were fully characterized by UV-vis, EPR, and ESI-MS spectroscopy, and in particular the more reactive, electron-deficient meso-dichlorophenyl-substituted porphyrin 1 displayed a rather broad spectrum of P450type reactions such as epoxidation, N-dealkylation, cleavage of diols, and hydroxylation of nonactivated C-H bonds.8e,f Preliminary UV-vis experiments also revealed that the oxo iron(IV) porphyrin radical cation can be produced from 2.8f The main practical advantage of 1 and 2, as compared to corresponding model complexes with a thiolate ligand, is the inertness of the SO3- ligand toward oxidation and S-nitrosation. In efforts to improve the mechanistic understanding of the catalytic action of cytochrome P450 and the essence of its biological functions, we have focused on the dynamics of NO interaction with the iron(III) heme-thiolate centers. It is known that NO binding to the catalytic site of enzymes such as nitric oxide reductase (P450nor),10a,b nitric oxide synthase (NOS),10c-f and nitrophorins from Cimex lectularius (NO transport heme(9) Ueyama, N.; Nishikawa, N.; Yamada, Y.; Okamura, T.; Nakamura, A. J. Am. Chem. Soc. 1996, 118, 12826-12827. (10) (a) Shimizu, H.; Obayashi, E.; Gomi, Y.; Arakawa, H.; Park, S.-Y.; Nakamura, H.; Adachi, S.; Shoun, H.; Shiro, Y. J. Biol. Chem. 2000, 275, 4816. (b) Obayashi, E.; Takahashi, S.; Shiro, Y. J. Am. Chem. Soc. 1998, 120, 12964. (c) Hurshman, A. R.; Marletta, A. Biochemistry 1995, 34, 5627. (d) Abu-Soud, H. M.; Wang, J.; Rousseau, D. L.; Fukuto, J. M.; Ignarro, L. J.; Stuehr, D. J. J. Biol. Chem. 1995, 270, 22997. (e) Couture, M.; Adak, S.; Stuehr, D. J.; Rousseau, L. J. Biol. Chem. 2001, 276, 38280.

Binding NO to a New Enzyme Mimic of Cytochrome P450

thiolate proteins from the bedbug)11 is essential for their biological function. In this context, an earlier report from the Erlangen/Krakow laboratories focused on the reaction of NO with native cytochrome P450cam and revealed mechanistic details on the reversible binding of NO to P450cam in the absence and in the presence of the substrate (+)-camphor.12 Very recently, these groups studied NO binding to the synthetic heme-thiolate complex 4,4 which was considered as a reasonable model for the kinetics of the analogous reactions with Fe(III) hemethiolate proteins.13 However, under conditions of even a small excess of NO, the initially formed NO complex of 4 proved to be very unstable; i.e., subsequent attack of a second NO on the sulfur atom of the RS- group was clearly observed.13 It was anticipated that these problems would not be encountered when employing complex 2, since the RSO3- group replacing the thiolate ligand (i) is less accessible due to steric hindrance and (ii) is “forced” into coordination to iron because the ligand belongs to a bridge spanning the porphyrin. The present work was undertaken to evaluate thermodynamic and kinetic data for NO binding to iron(III) of the high-spin complex 2 (g ) 5.67, 2.03), which is insoluble in water but soluble in organic solvents such as CH2Cl2, CHCl3, BuCN, toluene, and methanol. To investigate the influence of the iron(III) coordination sphere on the dynamics of the reaction with NO, the studies were carried out in non-coordinating toluene and in coordinating methanol. In toluene the five-coordinate complex 2, reminiscent of the E‚S complex of P450cam, is produced, whereas in methanol the six-coordinate complex 5 should be formed, resembling the resting state of P450cam. Employing lowtemperature stopped-flow and laser flash photolysis techniques at ambient and high pressure, suitable rate and activation parameters can be derived that reveal insight into the mechanisms for the “on” and “off” processes for NO binding and allow a comparison of the model systems with the corresponding states of cytochrome P450. Experimental Section Materials. All solutions were prepared in toluene (puriss from Aldrich or Acros Organics) or methanol (g99.9% from Roth). Complex 2 was prepared as described in the literature and characterized by UVvis, EPR, and ESI-MS spectroscopy.8 All other chemicals used throughout this study were of analytical reagent grade. NO gas, purchased from Riessner Gase or Linde 93 in a purity of at least 99.5 vol %, was cleaned to remove trace amounts of higher nitrogen oxides such as N2O3 and NO2 by passing it through an Ascarite II column (NaOH on silica gel, Sigma-Aldrich) and then bubbling through a gas scrubbing bottle containing 5 M NaOH (Aldrich). Solution Preparation. All solutions were prepared under strict exclusion of air. Toluene was distilled from a purple solution of benzophenone over sodium under Ar directly before use. Methanol was deaerated for extended periods with pure Ar before it was brought in contact with complex 2 or NO. The concentration of NO in NOsaturated methanol was determined with the NO electrode to be 0.014 M at 23 °C. The concentration of NO at saturation in toluene was (11) (a) Walker, F. A. J. Inorg. Biochem. 2005, 99, 216. (b) Ding, X. D.; Weichsel, A.; Andersen, J. F.; Shokhireva, T. Th.; Balfour, C.; Pierik, A. J.; Averill, B. A.; Mantfort, W. R.; Walker, F. A. J. Am. Chem. Soc. 1999, 121, 128. (c) Weichsel, A.; Maes, E. M.; Andersen, J. F.; Valenzuela, J. G.; Shokhireva, T. Kh.; Walker, F. A.; Montfort, W. R. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 594. (12) Franke, A.; Stochel, G.; Jung, C.; van Eldik, R. J. Am. Chem. Soc. 2004, 126, 4181. (13) Franke, A.; Stochel, G.; Suzuki, N.; Higuchi, T.; Okuzono, K.; van Eldik, R. J. Am. Chem. Soc. 2005, 127, 5360.

ARTICLES assumed to be 0.011 M at 25 °C.14 Dilutions of known concentration were prepared from these saturated solutions by a syringe technique. NO Electrode Measurements. The concentration of free NO in methanol was determined with an ISO-NOP electrode connected to an ISO-NO Mark II NO sensor from World Precision Instruments.15 To determine the concentration of NO in methanol solutions, the following procedure was followed. A known volume of the NO-saturated methanol solution (at 23 °C) was dropped into a known volume of degassed water. The concentration of NO in the resulting solution was measured with the NO electrode. The NO electrode was calibrated daily with fresh solutions of sodium nitrite and potassium iodide according to the method suggested by the manufacturer. The calibration factor nA/µM was determined with a linear fit program. UV-Vis Measurements. UV-vis spectra at appropriate temperatures were recorded in gastight cuvettes on a Shimadzu UV-2100 spectrophotometer equipped with a thermostated cell compartment CPS260. A pill-box cell combined with high-pressure equipment16 was used to record UV-vis spectra under high pressure (up to 150 MPa). EPR Measurements. To investigate the spin state of 2 in the presence of an excess of nitric oxide (nitrosyl complex of 2) and methanol (complex 5), cw-EPR experiments were carried out using a Bruker ESP-300 X-band spectrometer equipped with a T102 cell and an ER 4111 VT liquid nitrogen cryostat. EPR measurements of the nitrosyl complex of 2 were performed under strict exclusion of air oxygen. FTIR Measurements. FTIR measurements on complex 2 in the presence of a large excess of NO were performed in toluene under exclusion of air oxygen at ∼5 °C. The spectra were recorded with Mattson Infinity FT/IR-60 AR spectrophotometer. Laser Flash Photolysis. Laser flash photolysis kinetic studies were carried out with an LKS.60 spectrometer from Applied Photophysics for detection and a Nd:YAG laser (SURLITE I-10 Continuum) pump source operating in the second harmonics (λexc ) 532 nm, 245 mJ pulses with ∼7 ns pulse widths). Spectral changes at appropriate wavelength were monitored using a 100 W xenon arc lamp, monochromator, and PMT-1P22 photomultiplier tube. The absorbance reading was balanced to zero before the flash, and data were recorded on a DSO HP 54522A digital storage oscilloscope and then transferred to a personal computer for subsequent analysis. Gastight quartz cuvettes and a pill-box cell combined with high-pressure equipment16 were used at ambient and high pressure (up to 150 MPa), respectively. At least 10 kinetic runs were recorded under all conditions, and the reported rate constants represent the mean value of these. Low-Temperature Stopped-Flow Instrument and Software. Time-resolved UV-vis spectra were recorded with a Hi-Tech SF-3L low-temperature stopped-flow unit (Hi-Tech Scientific, Salisbury, UK) equipped with a J&M TIDAS 16/300-1100 diode array spectrophotometer (J&M, Aalen, Germany). The optical cell had a light path of 1.0 cm and was connected to the spectrophotometer unit with flexible light guides. Five-milliliter driving syringes were used. The mixing chamber was immersed in an ethanol bath which was placed in a Dewar flask containing liquid nitrogen. The ethanol bath was cooled by liquid nitrogen evaporation, and its temperature was measured by use of a Pt resistance thermometer and maintained to (0.1 °C by using a PID temperature-controlled thyristor heating unit (both Hi-Tech). Complete spectra were collected between 400 and 760 nm with the integrated J&M software Kinspec 2.30. All kinetic experiments were performed under pseudo-first-order conditions, i.e., at least a 10-fold excess of NO. The studied reactions exhibited pseudo-first-order behavior for at least three half-lives. In all stopped-flow experiments, at least five kinetic runs were recorded (14) Shaw, A. W.; Vosper, A. J. J. Chem. Soc., Faraday Trans. 1 1977, 73, 1239. (15) Kudo, S.; Bourassa, J. S. E.; Sato, Y.; Ford, P. C. Anal. Biochem. 1997, 247, 193. (16) Spitzer, M.; Gartig, F.; van Eldik, R. ReV. Sci. Instrum. 1988, 59, 2092. J. AM. CHEM. SOC.

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Figure 2. Electronic absorption spectra of 2 before (solid line) and after saturation with NO (dashed line) in toluene solution. Experimental conditions: [2] ) 4.5 × 10-6 M, [NO] ) 1.1 × 10-2 M, 20 °C.

under all conditions, and the reported rate constants represent the mean values. The activation parameters and corresponding error limits were calculated from a weighted linear least-squares fit of the data.

Results

I. Reaction of Complex 2 with Nitric Oxide in Toluene. A. Effect of Temperature and Pressure on the Equilibrium Constant, KNO. The UV-vis spectrum of 2 in toluene solution exhibits a Soret band maximum at 416 nm and a Q-band at 512 nm (Figure 2). Exposure of a degassed solution of 2 to a large excess of NO gas leads to spectral shifts of the Soret and Q-bands to 432 and 542 nm, respectively. The uptake of NO appears to be completely reversible, as judged from experiments in which a stream of Ar gas was used to remove NO from solution, consistent with the equilibrium described by eq 1. It kon

2 + NO {\ } 2(NO) k off

KNO

(1)

should be noted that the same reaction carried out with a NOsaturated toluene solution ([NO] ) 0.0055 M after mixing) at 20 °C resulted in the formation of only a very small fraction of the nitrosyl complex (only a weak shoulder at 432 nm). This result suggests that the equilibrium of the nitrosylation reaction (eq 1) is strongly shifted to the left side at room temperature. The effect of temperature on the equilibrium described by eq 1 was studied over the temperature range from 5 to 38 °C (Figure 3a). The values of the equilibrium constants for eq 1 (KNOtol), calculated from the spectral changes over the temperature range 15-38 °C, are summarized in Table 1. The temperature dependence of ln(KNOtol) (Figure 3a, inset) was found to be linear and gave significantly negative values for the thermodynamic parameters ∆H° and ∆S° (Table 1). The effect of pressure on the equilibrium constant KNOtol was examined in the pressure range 5-150 MPa (Figure 3b). The pressure dependence of ln(KNOtol) gave a straight line (Figure 3b, inset) and resulted in ∆V°NO ) -39 ( 2 cm3 mol-1. B. FTIR and EPR Characterization of the Nitrosyl Product, 2(NO). FTIR measurements on a toluene solution of 2 under a large excess of NO revealed formation of a new species (Figure 4a). The difference FTIR spectrum of 2(NO) and 2 (Figure 4b) shows a sharp band at 1833 cm-1, which is 13614 J. AM. CHEM. SOC.

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Figure 3. Spectral changes recorded for the reaction between 2 and NO in toluene. (a) As a function of temperature, from 5 (line 1) to 38 °C (line 8); inset, temperature dependence of ln(KNO). Experimental conditions: [2] ) 4 × 10-6 M, [NO] ) 0.0045 M. (b) As a function of pressure, from 5 (line 1) to 150 MPa (line 6); inset, ln(KNO) as a function of pressure. Experimental conditions: [2] ) 4.5 × 10-6 M, [NO] ) 0.011 M, 25 °C. Table 1. Temperature and Pressure Dependence of KNOtol for the Reaction of 2 with NO in Toluene T (°C)

15 20 25 30 35 38 25

∆H°NO (kJ mol-1) ∆S°NO (J mol-1 K-1) ∆G°NO (kJ mol-1) at 25 °C ∆V°NO (cm3 mol-1)

P (MPa)

0.1

5 30 60 90 120

KNO (M-1)

360 ( 17 197 ( 14 122 ( 10 79 ( 9 54 ( 6 41 ( 4 123 ( 9 175 ( 11 270 ( 13 450 ( 16 752 ( 15 -71 ( 3 -197 ( 10 -12 ( 3 -39 ( 2

very close to the N-O stretching modes found for nitrosyl complexes of natural heme-thiolate-containing enzymes such as P450nor17a and P450cam17a,b (νN-O ) 1853 and 1806 cm-1, respectively) and synthetic heme-thiolate-ligated models such (17) (a) Obayashi, E.; Tsukamoto, K.; Adachi, S.; Takahashi, S.; Nomura, M.; Iizuka, T.; Shoun, H.; Shiro, Y. J. Am. Chem. Soc. 1997, 119, 7807. (b) Hu, S.; Kincaid, J. R. J. Am. Chem. Soc. 1991, 113, 2843.

Binding NO to a New Enzyme Mimic of Cytochrome P450

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Figure 4. (a) FTIR spectra of a toluene solution of complex 2 before (solid line) and after saturation with NO gas (dashed line), measured at 5 °C. (b) The difference FTIR spectrum of toluene solutions of 2 and 2(NO). (c,d) EPR spectra of a toluene solution of 2 before and after saturation with NO gas, respectively.

as SR4a and SR-HB4h (νN-O ) 1828 and 1837 cm-1, respectively18). To investigate the Fe(III) spin state of the complex 2(NO), the EPR spectrum of a degassed toluene solution of 2 was recorded before and after NO saturation (Figure 4c,d). The spectrum of the nitrosyl complex shows a complete disappearance of the signal at g ) 5.78 (characteristic for the high-spin complex 2) and appearance of a weaker new signal at g ) 1.98. The nitrosylation reaction appears to be completely reversible as judged from the EPR spectrum recorded after the removal of NO from the solution by bubbling through a stream of Ar (the spectrum displays complete recovery of the signal at g ) 5.78; spectrum not shown). To investigate the nature of the signal at g ) 1.98, obtained by treatment of a toluene solution of 2 with an excess of NO, an experiment was performed wherein degassed toluene was saturated with NO and an EPR spectrum was recorded for the so-prepared solution (Figure S1, Supporting Information). As judged from this spectrum and a comparison with the spectrum obtained in the EPR experiments for the 2(NO) complex (Figure 4d), it is persuasive to interpret the signal at g ) 1.98 in the latter as stemming from an excess of NO in the complex solution. C. Low-Temperature Stopped-Flow Study of the “On” Reaction. At room temperature, the “on” reaction described by eq 1 was found to be too fast for stopped-flow studies. However, the use of a low-temperature stopped-flow instrument enabled us to measure the rate of this reaction in the NO concentration range (1.4-4.1) × 10-5 M over the temperature range from -70 to -40 °C. Typical spectral changes for nitric oxide binding to complex 2 (Q band), recorded with the rapid-scan, low(18) Suzuki, N.; Higuchi, T.; Urano, Y.; Kikuchi, K.; Uchida, T.; Mukai, M.; Kitagawa, T.; Nagano, T. J. Am. Chem. Soc. 2000, 122, 12059.

Figure 5. Spectral changes of the Q-band region recorded during NO binding to 2 in toluene at -70 °C. Experimental conditions: [2] ) 1.5 × 10-6 M, [NO] ) 1.4 × 10-5 M, spectra recorded within 0-1 s after mixing. Inset: Typical absorbance-time plot for NO binding to 2, recorded at 542 nm under these conditions.

temperature stopped-flow instrument, are shown in Figure 5. A typical kinetic trace, recorded at 542 nm at -70 °C for NO binding to 2 in toluene, is shown in the inset of Figure 5. The rate of reaction (1) with NO in excess is expected to follow pseudo-first-order kinetics, for which the observed rate constant, kobs, can be expressed by eq 2. Plots of kobs versus

kobs ) kon[NO] + koff

(2)

[NO] were linear at all temperatures (Figure S2, Supporting Information), and the resulting kon values are summarized in J. AM. CHEM. SOC.

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ARTICLES Table 2. kon and koff Values and Activation Parameters for the Reversible Binding of NO to Complex 2 in Toluene As Measured with the Low-Temperature Stopped-Flow Technique T (°C)

-70 -60 -50 -45 -40 ∆Hq (kJ mol-1) ∆Sq (J mol-1 K-1) ∆Gq (kJ mol-1) at 25 °C

kon × 10-6 (M-1 s-1)

koff (s-1)

0.79 ( 0.09 0.89 ( 0.06 0.99 ( 0.09 1.04 ( 0.03 1.08 ( 0.05

-3 ( 3 1(2 5(3 9.2 ( 0.8 14 ( 1

2.3 ( 1.0 -118 ( 5 37 ( 1

43 ( 10 -38 ( 42 54 ( 10

Table 2. They appeared not to be very temperature sensitive (the relatively large change in temperature from -70 to -40 °C resulted in only a small change in the kon values). A linear Eyring plot of these data gave a very small value for the activation enthalpy and a significantly large and negative value for the activation entropy (Table 2). D. Kinetics of the Dissociation of NO from the 2(NO) Complex. The reaction described by eq 1 appears to be completely reversible. At room temperature, the equilibrium is strongly shifted to the left side. In principle, koff could be directly determined from the experiment in which the 2/2(NO) equilibrium is disturbed via dilution. Indeed, mixing of the 2/2(NO) equilibrium solution with deoxygenated toluene resulted in the rapid formation of complex 2. However, attempts to determine koff in the temperature range 5-20 °C using the stopped-flow technique failed. The back reaction (binding of NO) appeared to be too fast between 5 and 20 °C, and only the end of this reaction could be observed using the stopped-flow technique. At lower temperatures (