Role of Thiolate Ligand in Spin State and Redox Switching in the

Mar 28, 2017 - Synopsis. The spin state (high-spin) of a five-coordinated heme thiolate model complex of cytochrome P450 was readily changed to low-sp...
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Role of Thiolate Ligand in Spin State and Redox Switching in the Cytochrome P450 Catalytic Cycle Hiroshi Suzuki, Kanako Inabe, Yoshinori Shirakawa, Naoki Umezawa, Nobuki Kato, and Tsunehiko Higuchi* Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan S Supporting Information *

ABSTRACT: The catalytic cycle of cytochrome P450 involves a change from the resting-state, water-bound, sixcoordinated form (1, low-spin state) to a five-coordinated form (2, high-spin state) upon binding of a hydrophobic substrate. Here, we used a heme−thiolate model complex (SR complex) with THF as a model of nonionic H2O to address the question of whether or not coordination of nonionic water is sufficient to induce the low-spin state. Measurements of electronic absorption spectra and magnetic properties confirmed that five-coordinated SR complex has a high-spin state, and THF-bound, sixcoordinated SR has a low-spin state in dichloromethane at ambient temperature. The redox potential E1/2 (FeII/FeIII) of THF-bound SR was 80−90 mV more negative than that of five-coordinated SR. These properties indicate SR is a good model of P450. Our results suggest that thiolate coordination plays a key role in setting the low energy barrier between the high-spin and low-spin states.

Figure 1. Catalytic cycle of cytochrome P450.

(named SR complex) which retains thiolate coordination during catalytic oxidation, and we observed several remarkable axial thiolate ligand effects (Figure 2).5 Since there has been no

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ytochrome P450 (P450) is a family of heme-containing enzymes that catalyze various types of oxidative processes in steroid biosynthesis, fatty acid metabolism, and detoxification of xenobiotic compounds, mainly via monooxygenase-type NADPH/NADH- and O2-dependent reactions.1 Another heme-containing enzyme with strong oxidizing ability, nitric oxide synthase (NOS), shares an unusual structure with P450; i.e., the heme iron has alkanethiolate coordination in both enzymes. Consequently, the role of the axial thiolate ligand in P450 chemistry has attracted much interest. The catalytic cycle of P450 generally involves a change from a water-bound sixcoordinated form (1, Fe(III), low-spin state) to a fivecoordinated form (2, Fe(III), high-spin state) upon entry of a hydrophobic substrate into the binding pocket of P450 (Figure 1).2,3 The redox potential of 1 is almost 130 mV lower than that of 2 in the case of P450cam.4 Formation of electrochemically more positive 2 enables P450 to interact with reductase.4 This process is of great significance in the catalytic cycle, because it means that uneconomical electron uncoupling is avoided during P450 reaction. In the absence of a substrate, electrochemically negative 1 stays in this form without reduction by reductase. Thus, the system can be considered as a redox switch that is turned on by substrate binding. It is of interest to know what role the heme thiolate coordination structure plays in this redox switching. We previously succeeded in synthesizing the first synthetic heme thiolate © 2017 American Chemical Society

Figure 2. Structure of SR complex.

report of a model complex that reproduces the reversible redox switching of P450, we set out to investigate the coordination chemistry and electrochemistry of the synthetic heme thiolate complex. In most cases, water coordinates to heme iron in form 1 as the resting state in the catalytic cycle of P450. The neutral form of H2O has only weak field strength as a ligand, and it remains Received: October 21, 2016 Published: March 28, 2017 4245

DOI: 10.1021/acs.inorgchem.6b02499 Inorg. Chem. 2017, 56, 4245−4248

Communication

Inorganic Chemistry

ligand. As shown in Figure 3b, the spectrum of the SR complex (Fe(III)) changed greatly with an increase of HO− concentration in CH2Cl2. The altered spectrum can be assigned to HO−-coordinated SR; this is the first reported example of the spectrum of HO−-coordinated heme thiolate. The λmax (444 nm) of the Soret band in the spectrum of HO−-coordinated SR was very different from those of P450 in the resting state (λmax around 420 nm) and THF-coordinated SR in the low-spin state (λmax: 418 nm). This result indicates that hydroxide is unlikely to have a significant role as a major sixth ligand of cytochrome P450 in the resting state, judging from the large difference in the spectra. The values of effective magnetic moment (μeff) of SR and the SR−THF complex were determined using the Evans method in order to confirm the spin state.12 The μeff of the SR complex (Fe(III)) in CD2Cl2 was 4.92 at 298 K, showing mainly a highspin state, and this value is fairly close to that of P450cam (μeff = 5.2).13 The μeff value of five-coordinated SR at 298 K is considered to be the average resulting from the high spin state−low spin state equilibrium, but we cannot rule out the possibility of an intermediate spin state. On the other hand, the value of the SR−THF complex was 1.64 at 298 K, indicating a low-spin state (S = 1/2). These results clearly indicate that coordination of a weak, neutral oxy ligand such as THF changes the spin state of heme thiolate from high to low spin. We previously reported that the SR complex in toluene solution showed EPR spectra indicating a low-spin state at 77 K.5a Therefore, we examined the temperature dependency of the spin state of SR. As shown in Figure 4, the μeff of SR in

controversial whether or not coordination of a weak ligand such as water is sufficient to change the spin state of heme thiolate. Previous calculations suggested that the assistance of an electrostatic field due to the protein would be needed to alter the spin state.7 On the other hand, another computational study indicated that thiolate coordination to heme would enable the spin state change.8 An experimental study using various anionic ligands reached a similar conclusion.9 Water may actually coordinate to iron as a hydroxo anion form with the assistance of some proximal amino acid residue in the binding pocket, or another water molecule, but this is also controversial.10 Therefore, we were interested in the spin statechanging ability of tetrahydrofuran (THF), which cannot be converted to an oxy anion species and has a field strength as weak as that of H2O.11 To address this question, we investigated how the properties of the SR complex are influenced by coordination of THF as a nonionic H2O ligand model. First, we tried to evaluate the coordination ability of H2O to the SR complex by using a H2O-saturated nonpolar solvent, such as dichloromethane (CH2Cl2), because polar solvent molecules themselves generally coordinate to iron of the SR complex. However, only a slight spectral change was observed (Figure S1). This is probably due to both the low coordination ability of H2O and the low H2O concentration in CH2Cl2 (0.17% at maximum). We examined the suitability of watermiscible solvents, such as acetonitrile and acetone, as cosolvents for evaluation of the spin-switching ability of water. However, the SR complex was already in the low-spin state in abs. acetonitrile and abs. acetone, due to coordination of the solvent molecule to iron (Figure S2), so these solvents are unsuitable for the purpose. On the other hand, THF and CH2Cl2 are freely miscible, and the spectra of the SR complex (Fe(III)) changed greatly with an increase of THF concentration in CH2Cl2 (Figure 3a).

Figure 3. (a) Electronic absorption spectral changes of SR (10−5 M in CH2Cl2 at 298 K) with increasing THF concentration. [THF] (mM): 0, 50, 100, 500, 4000. (b) Electronic absorption spectral changes of SR (10−5 M in CH2Cl2 at 298 K) with increasing TBAOH concentration. [TBAOH] (mM): 0, 0.1, 0.5, 1.0, 2.5, 5.0, 7.5.

Figure 4. Plot of μeff versus temperature (K) of the SR complex in CD2Cl2. The values of μeff were corrected for the cubical expansion coefficient of CH2Cl2 (1.39 × 10−3 °C−1 at 298 K).

Tetrahydropyran also similarly changed the spectra of SR in CH2Cl2 (Figure S3). SR in a noncoordinative solvent such as toluene, benzene, or CH2Cl2 is in a high-spin state. On the other hand, the spectra due to the six-coordinated SR complex increased in proportion to the increase of THF concentration. The coordination equilibrium constant (K) of THF to the SR complex was calculated as 0.11 M−1, which is a rather low value. We also examined the spectrum of the HO−-coordinated SR complex to see whether or not HO− can serve as the sixth

CD2Cl2 considerably decreased as the temperature was lowered, whereas the μeff of SR was constant above 273 K. A previous study showed almost the same temperature-dependent change in the spin state in camphor-bound cytochrome P450cam.14 This agreement indicates that SR in CD2Cl2 or other aprotic nonpolar solvent is an appropriate model for the noncoordinating substrate-bound P450 form (2 in Figure 1) without a change of pH accompanied by a change of temperature in the case of aqueous buffer solution. 4246

DOI: 10.1021/acs.inorgchem.6b02499 Inorg. Chem. 2017, 56, 4245−4248

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Inorganic Chemistry The ligand-field strength of THF is weak.11 Therefore, it is usually expected to have little ability to change the properties of a metal complex, such as the spin state, by coordination. Indeed, Holm et al. reported that a 2-MeTHF solution of FeIII(protoporphyrin IX dimethyl ester) p-nitrothiophenolate showed absorption spectra indicating a high-spin state at 298 K, even though this is a heme thiolate complex.13b In contrast, we observed direct conversion of the five-coordinated SR complex in the high-spin state to a six-coordinated THF−SR complex in the low-spin state, and each spin state was well-defined by determining its effective magnetic moment in this study. It is known that water ligation to heme coordinated by an imidazole does not change the spin state (high-spin state).15 Therefore, we conclude that the axial alkanethiolate ligation plays a critical role in the spin state-switching of heme. Next, we investigated the electrochemistry of SR in both high-spin and low-spin states by means of cyclic voltammetry. The former was measured in abs. CH2Cl2 and the latter in 2.5 M THF/CH2Cl2. A reversible cyclic voltammogram of SR in each spin state was obtained (Figure 5 and Figure S4). The

Thus, the synthetic heme thiolate complex could reproduce the redox switching and spin state change of P450 upon coordination of the weak ligand THF as a model of nonionic water. These results indicate that the redox switching in P450 chemistry is an intrinsic property of the heme thiolate coordination structure itself. The influence of thiolate ligation to heme on the coordination field is so great that even a weak sixth ligand such as water or THF can change the spin state of the complex to low spin. These results support the conclusions drawn from Green’s calculation8 and Dawson’s experimental study9 and indicate that thiolate ligation to the heme in P450 serves to avoid electron uncoupling in the absence of substrate.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b02499. Listing of electronic absorption spectra and magnetic data (spectra in various solvents; spectral changes with increasing THP conc.; determination of μeff and magnetic susceptibility); CV of SR complex at 293 K (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Tsunehiko Higuchi: 0000-0002-3586-4680 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Dr. Takuya Kurahashi (Institute for Molecular Science) for advice on the determination of μeff. We also thank Prof. Junpei Yamanaka and Dr. Tohru Okuzono for helpful discussions. This work was supported by the Platform Project for Supporting in Drug Discovery and Life Science Research from MEXT, and Japan Agency for Medical Research and Development (AMED).

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Figure 5. Cyclic voltammogram (CV) of the SR complex. The CV of the SR complex in the high-spin state (in CH2Cl2) is shown in blue. The CV of the SR complex in the low-spin state (in 2.5 M THF/ CH2Cl2) is shown in red. Complex, 0.3 mM; Bu4NClO4, 100 mM; solvent, CH2Cl2 or 2.5 M THF/CH2Cl2; working electrode, Pt; reference electrode, Ag/AgCl; sweep rate, 0.2 V/s. These measurements were carried out at 258 K under an Ar atmosphere.

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E1/2(FeII/FeIII) of low-spin SR (−0.81 V at 258 K and −0.78 V at 293 K vs Ag/AgCl electrode) was 80−90 mV lower than that of high-spin SR (−0.90 V at 258 K and −0.86 V at 293 K). This is the first report of the redox potential of a neutral oxy ligandcoordinated synthetic heme thiolate in a low-spin state. The difference between the two states is comparable to that of the case of P450cam (130 mV).4 The E 1/2 (Fe III/FeIV or isoelectronic form) of SR in the low-spin state (0.64 V) also showed a slight negative shift from that of SR in the high-spin state (0.68 V) at 258 K. It seems likely that the difference in E1/2(FeII/FeIII) between the high-spin state and low-spin state directly corresponds that in P450. 4247

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