Correlating the para-Substituent Effects on Ru(II)-Polypyridine

May 24, 2017 - [Ru(4,4′-dimethoxy-2,2′-bipyridine)2(2,2′-bipyridine)]·2PF6 (6) ..... Better data fitting using the Brown-Okamoto Σσp+ rather ...
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Correlating the para-Substituent Effects on Ru(II)-Polypyridine Photophysical Properties and on the Corresponding Hybrid P450 BM3 Enzymes Photocatalytic Activity Hadil Shalan, Alexander Colbert, Thanh Truc Nguyen, Mallory Kato, and Lionel Cheruzel* Department of Chemistry, San Jose State University, San Jose, California 95192-0101, United States S Supporting Information *

ABSTRACT: Ru(II)-diimine complexes covalently attached near the heme active site of P450 BM3 enzymes have been used to rapidly inject electrons and drive selective C−H functionalization upon visible light irradiation. Herein, we have generated a series of hybrid P450 BM3 enzymes containing a photosensitizer of general formula [Ru(4,4′-X2bpy)2(PhenA)]2+ where X = Cl, H, tBu, Me OPhe, OMe, or NMe2, bpy = 2,2′-bipyridine, and PhenA = 5acetamido-1,10-phenanthroline. We then probed the effect of electronwithdrawing and -donating groups at the para position of the 4,4′-X2bpy ligands on the corresponding hybrid enzymes photocatalytic activity. A 3-fold improvement in initial reaction rate was noted when varying the substituent from Cl to tBu, however, the reaction rates decrease thereafter with the more electron donating groups. In order to rationalize those effects, we investigated the variation of the substituent on the photophysical properties of the corresponding [Ru(4,4′-X2bpy)2(bpy)]2+ model complexes. Several linear correlations were established between the E(III/II) potential, the MLCT emission, and absorption energies as well as the logarithm of the luminescence quenching rate vs the summative Brown-Okamoto parameter (Σσp+). Moreover, a downward curved Hammett plot is observed with the hybrid enzyme initial reaction rate revealing mechanistic details about the overall lightdriven enzymatic process.

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INTRODUCTION Because of the long-lived nature of their excited states, Ru(II) polypyridyl complexes have been extensively studied for their unique light-harvesting and redox properties.1 Several applications have capitalized on their photochemical properties in dyesensitized solar cells2 and enzymatic reactions requiring timely electron delivery.3 We previously established that the strategic covalent attachment of a Ru(II)-polypyridyl unit to non-native single cysteine mutants of P450 enzyme heme domains enables the activation of molecular dioxygen at the heme center and thus the selective light driven hydroxylation of various substrate C− H bonds often with high photocatalytic activity.4 Using sodium diethyldithiocarbamate (DTC) as the reductive quencher under established flash-quench conditions (Figure 1), a highly reductive species is photogenerated capable of rapidly delivering electrons to the heme active site to initiate the enzymatic process.5 In an attempt to optimize their photocatalytic activity, we generated a series of hybrid P450 enzymes (P1−P7) where a [Ru(4,4′-X2bpy)2(PhenA)]2+ (X = Cl, H, tBu, Me OPhe, OMe, or NMe2; bpy = 2,2′-bipyridine; PhenA = 5-acetamido-1,10phenanthroline) complex is covalently attached to the P450 BM3 L407C heme domain mutant. Herein, we report on the effect of the X substituent on the photocatalytic activity of the hybrid enzyme as well as on the photophysical properties of © 2017 American Chemical Society

Ru(II)-diimine model complexes of general formula [Ru(4,4′X2bpy)2(bpy)]2+. The substituents investigated include electron withdrawing (Cl) and electron donating (tBu, Me, OPhe, OMe, NMe2) groups at the para positions of the ancillary bipyridine ligands. The seven model complexes (1−7) were characterized using 1 H NMR, mass spectrometry, cyclic voltammetry as well as UV−vis and luminescence spectroscopy. Several linear correlations are observed in the model complexes relating the E(III/II) redox potential, the absorption and emission energies as well as the chemical shift of the adjacent proton and quantum yield with the summative Brown-Okamoto values (Σσp+). Across the series, the luminescence of the complexes is effectively quenched by DTC, and a linear free energy relationship was obtained with the logarithm of the quenching rates and the Σσp+ values. The photocatalytic activity and initial reaction rate (k0) of the hybrid enzyme series was determined using a colorimetric assay involving the O-dealkylation of nitrophenoxy based substrates (Figure 1).6 A 3-fold increase in initial reaction rate resulted within the series with the lowest rate observed with the Cl substituent and the highest with the tBu. Moreover, the Received: March 15, 2017 Published: May 24, 2017 6558

DOI: 10.1021/acs.inorgchem.7b00685 Inorg. Chem. 2017, 56, 6558−6564

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Inorganic Chemistry

Figure 1. Schematic representation of the hybrid P450 BM3 enzymes investigated where the X substituents on the ancillary ligands of the photosensitizer covalently attached to the heme domain have been systematically varied to include electron donating and withdrawing groups. The Ru(II) excited state is quenched by DTC (step 2) to generate the highly reductive ruthenium species that is able to inject electrons to the heme domain (step 3) and initiate the P450 mechanism. A colorimetric assay involving the O-dealkylation of nitrophenoxy analogues was utilized to compare enzymatic activity across the series.

Hammett plot for the log(k0X/k0H) reveals a downward curvature indicative of a change in the rate limiting step during the photocatalytic process.

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added. Upon dissolution of the ligand, reducing equivalents were sequentially added at varied concentrations and allowed to react for an additional 30 min. After cooling of the mixture to room temperature, saturated NaCl (10 mL) was added, and the resulting solution was further cooled at 0 °C for 1 h. Filtering the mixture yielded a colored filtrate and a violet to black microcrystalline product. The solid was washed with several portions of brine and dried under a vacuum. Yields were typically 70−75%. This powder was then added to a MeOH/water solution with 1.1 equiv of 2,2′-bipyridine and heated to reflux for 3 h to generate the desired heteroleptic complexes (1−7). The product was precipitated by the addition of a saturated KPF6 solution. The orange powder was vacuum filtered, washed with water, dried with excess ether, and finally recrystallized using warm methanol and hexane/ether. Yields ranged from 60 to 90%. The complexes were characterized by 1H NMR and high-resolution mass spectrometry (Figure S1) confirming the integrity of the compounds. [Ru(4,4′-dichloro-2,2′-bipyridine)2(2,2′-bipyridine)]·2PF6 (1). Isolated as an orange powder (0.044 g, 90%). 1H NMR (400 MHz, DMSO-d6): 9.09 (s, 4H), 8.76 (t, 2H), 8.13 (d, 2H), 7.73 (d, 4H), 7.59 (m, 6H), 7.46 (d, 2H) HR-ESI-MS: m/z = 353.9768 exp. (theoretical 353.9759) [M]2+ [Ru(2,2′-bipyridine)3]·2PF6 (2). Isolated as a red-orange powder (0.051 g, 86%). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.76 (d, 6H), 8.11 (d, 6H), 7.65 (d, 6H), 7.47 (d, 6H). HR-ESI-MS m/z = 285.0553 exp. (theoretical 285.0559) [M]2+. [Ru(4,4′-di-tbutyl-2,2′-bipyridine)2(2,2′-bipyridine)]·2PF6 (3). Isolated as a light orange powder (0.037g, 90%). 1H NMR (400 MHz, DMSO-d6): 8.78 (s, 4H), 8.74 (d, 2H), 8.11 (t, 2H), 7.62 (d, 2H), 7.49 (m, 10H), 1.31 (s, 36H). HR-ESI-MS m/z = 307.1805 exp. (theoretical 397.1811) [M]2+. [Ru(4,4′-dimethyl-2,2′-bipyridine)2(2,2′-bipyridine)]·2PF6 (4). Isolated as an orange powder (0.46 g, 76%). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.75 (d, 2H), 8.63 (s, 4H), 8.08 (t, 2H), 7.65 (d,

EXPERIMENTAL SECTION

Materials. All reagents used in this work were of analytical grade and purchased from Thermo Fisher Scientific and Sigma-Aldrich except for the 4,4′-bis(dimethylamino)-2,2′-bipyridine ligand, which was purchased from Hetcat, Switzerland. Measurements. 1H NMR was recorded on a Varian 400 MHz NMR spectrometer. Electrospray ionization mass spectrometry (ESIMS) data were obtained from an Agilent 6520 quadrupole time-offlight LC/MS instrument. UV−visible and luminescence spectra were recorded on a Cary 60 UV−vis and a Varian Cary Eclipse fluorescence spectrophotometers, respectively. Quantum yields in acetonitrile at 25 °C were determined using Ru(bpy)32+ as a reference (ϕ = 0.044, λex = 455 nm) as previously reported.7 Cyclic voltammograms were obtained on a Pine Wavenow potentiostat instrument in a single-compartment three-electrode cell with a glassy carbon working electrode, Ag/AgCl reference electrode, and platinum wire auxiliary electrode. The supporting electrolyte was 0.1 M tetrabutylammonium hexafluorophosphate (TBA·PF6), and dry acetonitrile was used as the solvent. Solutions were degassed with N2 prior to measurements, and the voltage was swept at 100 mV/s. Potentials were calibrated using the ferrocene/ferrocenium (Fc/Fc+) couple with a measured E1/2(Fc/Fc+) of 0.45 V vs Ag/AgCl as a reference and are reported vs NHE. Synthesis of Model Complexes 1−7. All the Ru(4,4′X2bpy)2Cl2 precursors could be synthesized by expanding on a reported procedure using saccharides as reducing agents (see Supporting Information for detailed procedures).8 In a typical reaction, RuCl3·3H2O (0.25 g, 1 mmol) and LiCl (1.7 g, 39 mmol) were dissolved in ethylene glycol/water (25 mL, 4:1) with stirring at 110 °C for 15 min. At this point, the desired 4,4′-X2bpy ligand was 6559

DOI: 10.1021/acs.inorgchem.7b00685 Inorg. Chem. 2017, 56, 6558−6564

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Inorganic Chemistry Table 1. Summary of Photophysical and Electrochemical Properties for the Complexes 1−7 complexes

E(III/II)a

E(II/I)a

E(I/0)a

1 2 3 4 5 6 7

1.629 1.532 1.429 1.427 1.400 1.298 0.905

−0.855 −1.093 −1.140 −1.169 −1.101 −1.190 −1.182

−1.048 −1.295 −1.370 −1.376 −1.268 −1.393 −1.648

a

E(0/−I)a λmax (nm)/Eabs (cm−1) −1.435 −1.553 −1.633 −1.678 −1.506 −1.616 −1.843

465/21505 456/21929 463/21598 464/21551 469/21321 472/21186 488/20491

λem (nm)/Eem (cm−1)

quantum yield (%)

tau (ns)

650/15385 620/16129 633/15798 638/15674 640/15625 661/15129 702/14245

3.90 4.20 3.06 3.11 3.08 2.08 0.80

450 377 395 362 345 398 180

quenching rate (s−1)b 8.72 7.24 4.21 4.31 3.34 1.13 5.39

× × × × × × ×

108 108 108 108 108 108 107

Potential reported vs NHE. bSodium diethyldithiocarbamate (DTC) as reductive quencher.

2H), 7.47 (m, 6H), 7.28 (m, 4H), 3.23 (s, 12H). HR-ESI-MS m/z = 313.0866 exp. (theoretical 313.0870) [M]2+. [Ru(4,4′-diphenoxy-2,2′-bipyridine)2(2,2′-bipyridine)]·2PF6 (5). Isolated as an orange powder (0.032g, 78%). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.75 (d, 2H), 8.61 (dd, 4H), 8.11 (t, 2H), 7.89 (d, 2H), 7.62 (d, 2H), 7.52 (m, 10H), 7.45 (m, 10H), 7.23 (d, 4H) 6.84 (dd, 2H). HR-ESI-MS m/z = 469.1077 exp. (theoretical 469.1046) [M]2+. [Ru(4,4′-dimethoxy-2,2′-bipyridine)2(2,2′-bipyridine)]·2PF6 (6). Isolated as a dark red powder (0.047g, 93%). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.76 (d, 2H), 8.41 (s, 4H), 8.08 (t, 2H), 7.78 (d, 2H), 7.77 (d, 2H), 7.48 (m, 2H), 7.06 (dd, 4H), 4.05 (s, 6H), 3.95 (s, 6H). HR-ESI-MS m/z = 345.0764 exp. (theoretical 345.0770) [M]2+. [Ru(4,4′-bis(dimethylamino)-2,2′-bipyridine)2(2,2′-bipyridine)]· 2PF6 (7). Isolated as a brown powder (0.022g, 60%). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.64 (d, 2H), 7.93 (d, 2H), 7.80 (d, 2H), 7.64 (d, 4H), 7.42 (t, 2H), 7.06 (d, 2H), 6.87 (d, 2H), 6.69 (d, 2H), 6.56 (d, 2H), 2.42 (s, 24H). HR-ESI-MS m/z = 371.1397 exp. (theoretical 371.1399) [M]2+. Hybrid Enzymes P1−P7. The desired hybrid P450 BM3 enzymes (P1−P7) were generated by reacting the Ru(4,4′-X2bpy)2(PhenIA) (PhenIA = 5-iodoacetamido-1,10-phenanthroline) (see Supporting Information for characterization) with the non-native single cysteine mutant (L407C) of the P450 BM3 heme domain mutant.6 The hybrid enzymes were then purified and characterized as previously reported.3,6 The ESI mass spectrometry (Figure S2), UV−vis and fluorescence spectra of the labeled proteins (Figure 5) confirmed the covalent attachment of the various complexes to the heme domain.3 The photocatalytic activity was recorded using the chromogenic substrates, 16-nitrophenoxyhexadecanoic acid and benzoxy-4-nitrobenzene, as previously reported.6

Figure 2. 1H NMR spectra of the aromatic region for the complexes 1−7.

complexes 1, 3, and 4. However, loss of symmetries is observed in the remainder complexes 5−7 where splitting of peaks is observed for the substituted ligands. As expected, the proton resonances for these ligands vary significantly throughout the series, with the more electron-poor Cl ligand having resonances shifted downfield relative to those of the electron-rich NMe2 and OMe substituted ligands. Most dramatic is the singlet peak pertaining to the Hc proton adjacent to the X substituent, which is shifted by 2 ppm between the complexes 1 and 7. Cyclic Voltammetry. The electrochemical properties of each complex were investigated by cyclic voltammetry in dry and degassed acetonitrile with 0.1 M tetrabutylammonium as the supporting electrolyte. All complexes exhibit quasireversible Ru3+/2+ redox couples, as well as three reduction waves (Figure 3). The measured redox potentials are summarized in Table 1. The redox potentials for the Ru3+/2+ couple, E(III/II), span more than 700 mV from 0.91 V for compound 7 with the

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RESULTS Synthesis and Characterization of Model Complexes. All of the model complexes (1−7) reported herein have the same general structure: [Ru(4,4′-X2bpy)2(bpy)]2+, where X = Cl, H, tBu, Me, OPhe, OMe, NMe2, respectively. The heteroleptic complexes were easily synthesized in good yields using the same overall procedure,8 in contrast with other synthetic approaches.9 The characterization of cis-Ru(4,4′X2bpy)2Cl2 complexes was rendered difficult due to their limited solubility; however, the final model complex chromophores, isolated as their PF6 salts, were characterized using a combination of techniques. The determined parameters summarized in Table 1 are in agreement and complement previously reported data.1a,9 1 H NMR. The aromatic region of the 1H NMR spectrum of each complex in DMSO-d6 is shown in Figure 2. The complexes 1−4 exhibit C2 symmetry with a single 2-fold axis bisecting the bpy ligand as apparent in each spectrum. There are four distinct proton resonances for the 2,2′-bpy ligands, and their chemical shifts remain relatively unaffected across the series. In addition, as a result of the C2 symmetry, the 4,4′X2bpy ligands show three unique proton resonances in the

Figure 3. Cyclic voltammograms for the complexes 1−7 in dry degassed acetonitrile with 0.1 M TBA·PF6 as supporting electrolyte and ferrocene (E0(Fc/Fc+) = 0.45 V vs Ag/AgCl) as reference. 6560

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Inorganic Chemistry NMe2 substituent to 1.63 V for the Cl substituent (1). The oxidation potential of the heteroleptic complexes are consistently higher than their homoleptic counterpart, Ru(4,4′-X2bpy)32+.10 All complexes show three reduction waves within the potential window of the experiments with scans extended to −2.3 V. As shown in Figure 3, three ligand-based reduction waves are observed arising from reduction at the bipyridine ligand for 2−7, followed by reduction at each of the substituted ligands.1a The first reduction potential of the complexes ranges from −0.86 V (vs NHE) for the Cl substituent (1) to −1.18 V for the NMe2 derivative (7). UV−Vis Absorption and Steady-State Emission Spectra. The absorption spectra of all of the complexes in a mixture of acetonitrile/water (20/80) feature the characteristic prominent bands11 including the intense π → π* absorptions below 300 nm (ε ≈ 5.1−104 M−1 cm−1) as well as the characteristic lower-energy metal-to-ligand charge-transfer (MLCT) absorptions from 400 to 500 nm (Figure 4 and Table 1). A change of 3000 cm−1 is observed in the absorbance energies for the MLCT transition across the series of complexes 1−7.

Figure 5. Absorption and emission spectra for the hybrid enzymes P1−P7.

model complexes (Figures 4 and S3). Subtle changes in intensity and blue-shifted emission are noticed, which can be attributed to changes in the local environment of the photosensitizers covalently attached to the protein. On the basis of the recent crystal structure of P2,5 the photosensitizer is located in a bowl-shaped cavity with the ancillary bipyridine ligands pointing toward the bulk solvent away from any protein residues.5 We thus infer that introduction of the X substituent does not perturb the position and orientation of the photosensitizer across the series of functionalized hybrid enzymes.

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DISCUSSION Linear Correlations with Brown-Okamoto Parameters. Several relationships have been established to correlate the intrinsic properties of Ru(II)-polypyridyl complexes, such as redox potentials or energies of the absorption and emission, with various parameters (e.g., the redox potential of the free ligand,12 the ΔE difference between the oxidation and the first reduction potentials).1 We confirmed the linear correlation between the absorption and emission energies and the ΔE difference as seen in Figure S4. Noteworthy is the recent work by Connick et al. correlating the E(III/II) potential of a hundred complexes with the total Brown-Okamoto parameter (Σσp+) quantifying the electronic effect of the substitution on the diimine ligands.10a As shown in Figure 6A, similar linear correlation is observed in the current heteroleptic series of compounds 1−7 with a slope of 0.093 (R2 = 0.985). A better fit is obtained using the Σσp+ parameters rather than the Hammett Σσp (R2 = 0.959, Figure S5), taking into account the resonance contribution of the substituents. Such correlation with the Σσp+ parameters extends to the absorption and emission energies (Eabs and Eem), the 1H NMR shift for the singlet proton (Hc, Figure 2) adjacent to the X substituent and the quantum yield13 as shown in Figure 6. Taken altogether, these data are consistent with the X substituent affecting mainly the metal-based dπ6 orbital energy levels due to a decrease in π back bonding14 with smaller variations in the low-lying π* energy level of the ancillary ligands (see partial MO diagram in Figure S6). The greater changes in the oxidation potential versus the first reduction potential across the series of substituted complexes 1−7 are also consistent with this picture. Decreased back-bonding destabilizes the dπ6 core, resulting in lower Ru(III/II) redox potentials (Table 1), while the E(II/I) reduction potentials are mainly ligand based involving the unsubstituted bipyridine and are less affected by the substitution (see Figure S7). The

Figure 4. UV−visible absorption and emission spectra of complexes 1−7 (1.5 uM) in aerated acetonitrile/water (20/80) mixture at 23 °C following excitation at 470 nm.

The emission spectra for each complex are shown in Figures 4 and S3, and the emission maxima, energies, and quantum yields are listed in Table 1. The emission energies vary over ∼2000 cm−1 with λmax = 620 nm (Eem = 16129 cm−1) for 2 to λmax = 702 nm (Eem = 14245 cm−1) for 7 (Table 1). The bands are broad with a low energy shoulder whose intensity is sensitive to the substituent. As mentioned previously for heteroleptic complexes, the emission originates in most cases from a manifold of 3MLCT excited states involving the ligand that is easier to reduce, likely the bpy in 2−7 as indicated by the first reduction potential values reported in Table 1. In addition, the excited state lifetimes fall in the 180−450 ns range, which is consistent with the spin-forbidden character of the excited state. Hybrid P450 Enzymes P1−P7. The hybrid enzymes were generated as previously described by reacting the Ru(4,4′X2bpy)2(PhenIA) (PhenIA = 5-iodoacetamido-1,10-phenanthroline)3 compounds with the P450 BM3 L407C heme domain mutant. The covalent attachment was confirmed by mass spectrometry (Figure S2) where the expected shift in the overall mass is observed. In Figure 5, the UV−vis spectra of the hybrid enzymes is dominated by the strong Soret band associated with the low spin six-coordinate ferric-aquo heme species.19 Shoulders in the 450 nm region are due to the MLCT band of the corresponding photosensitizer covalently attached to the enzyme.3 The emission maxima in the hybrid enzymes P1−P7 are consistent with those observed with the 6561

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Figure 6. Linear correlations between (A) the E(III/II) potential, (B) the absorption and emission energies, (C) the 1H chemical shift of the Hc atom, and (D) quantum yield vs the summative Brown-Okamoto values (Σσp+) in the series of complexes 1−7.

changes in the visible absorption (MLCT and π → π* transitions) and excitation maxima also point to the X substitution mainly affecting the metal-based dπ6 orbital energies. Better data fitting using the Brown-Okamoto Σσp+ rather than Σσ p parameters suggests that the charge delocalization in the ligands and resonance contribution of the substituent play a larger role in the photophysical properties of the photosensitizer. In order to successfully deliver electrons to the heme domain and sustain photocatalytic activity, we have utilized the flash quench reductive technique3 with DTC as the electron source. Among several reductive quenchers investigated,6 we have previously identified that DTC was a suitable quencher to sustain photocatalytic activity under aerobic conditions.4b The DTC anion is able to efficiently quench the Ru(II) excited state via electron transfer (Figure 1, Step 2) and generate a highly reductive species.5,15 We investigated the luminescence quenching of the complexes 1−7 excited states with DTC. The Stern−Volmer plots are linear as shown in Figure S8 over a wide range of quencher concentration. The quenching rates listed in Table 1 decrease across the series of Ru(II) complexes as the electron donating ability of the substituent is increased. A linear free energy relationship was established between the logarithm of the ratio of quenching rates of the substituted complexes over the unsubstituted variant vs the Σσp+ values (Figures 7 and S9). The positive slope is consistent with electron transfer from the quencher to the Ru(II) excited state and the addition of a negative charge to the overall complex. Similar trends have been established in other systems involving excited state quenching via electron transfer. The excited state of the homoleptic [Ru(COOH-bpy)3]2+ complex is quenched by a series of organic sulfides,16 while the excited states of quinoxaline and C-60 fullerene are quenched by various electron-poor alkene17 or aniline18 derivatives, respectively,

Figure 7. Linear free energy relationship between the logarithm of the ratio of quenching rates vs the Σσp+ parameters.

for organic examples. In each case, the slope of the Hammett plot is positive consistent with the increase of negative charge in the transition state, which would be disfavored by the presence of electron-donating substituent. Photocatalytic Activity of the Hybrid Enzyme P1−P7. The motivation for synthesizing and characterizing the series of hybrid P450 BM3 enzymes (P1−P7) was to explore the effect of the X substitution on their photocatalytic activity. The photocatalytic activity and initial reaction rates of the series of hybrid enzymes were measured as previously described using a colorimetric assay based on chromogenic substrates, 16nitrophenoxyhexadecanoic acid and benzoxy-4-nitrobenzene (Figure 1). The determined reaction rates are summarized in Figures 8A and S10A. It is apparent that the substituents effect the overall initial reaction rate (k0) of the hybrid enzyme across the series. A 3-fold rate increase is noticed between the Cl and tBu derivatives. However, the reaction rates decrease thereafter with the more electron donating ligands. The substituents fall into two groups and do not follow a typical Hammett plot as 6562

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Figure 8. (A) Initial reaction rates in the selective hydroxylation of the chromogenic nitro-phenoxy substrate, 16-nitrophenoxyhexadecanoic acid, by the hybrid enzymes P1−P7; (B) linear free energy relationship between the logarithm of the ratio of initial reaction rates vs the mixed Σσp+ and Σσp parameters.

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shown in Figure 8B and Figure S10B where the log(k0X/k0H) is plotted against a combination of Σσp+ and Σσp values.20 The Hammett plots show downward curvature centered at the tBu substituent, indicative of a change in the rate-limiting step in the overall photocatalytic reaction.20 For the Cl, OPhe, Me, and tBu substituents, the negative slope (−0.24) supports an electron transfer step between the photogenerated ruthenium species and the heme center (Figure 1, Step 3) as the rate-limiting step. As noted previously, enhancing ET resulted in increased photocatalytic activity of the hybrid enzymes.5 Accordingly, this step is favored by the increasing inductive effect of the substituents and is then governed by the Σσp parameter. A better fit is obtained using the Σσp values as opposed to the Brown-Okamoto parameters (Figure S11). This is particularly evident in the OPhe derivative, where the reaction rate is identical to the rate obtained for the unsubstituted hybrid enzyme (Figure 8A). The OPhe effect is thus better described using the Σσp value of −0.12 (vs Σσp+ = −2), closer to the Σσp = Σσp+ = 0 for the H substituent. A similar argument is used to rationalize the rate observed for the Me substituent (Σσp = −0.28 vs Σσp+ = −1.24). The downward curvature of the Hammett plots and the observed positive slope on the left side of the plot for the more electron-donating groups (OMe and NMe2) suggest a change in the rate limiting step to one that no longer involves electron transfer from the photosensitizer unit to the heme domain (Step 3). With these derivatives, the rate of DTC quenching (Figure 1, Step 2) is likely accountable for the rate-determining step. The positive slope is consistent with the buildup of a negative charge on the photosensitizer as previously mentioned in the luminescence quenching of the model complexes (1−7).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b00685. The following files are available free of charge. Detailed synthetic procedures for the model complex precursors. Theoretical and experimental mass spectra for the photosensitizers and hybrid enzymes; table of Hammett and Brown-Okamoto substituent parameters used in this study; correlation using Hammett substituent parameters; partial MO diagram; luminescence decay with fit and Stern−Volmer plots (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Lionel Cheruzel: 0000-0003-0283-6816 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by the National Institute of Health through Grant Number SC3 GM095415 and by the National Science Foundation through Grant Number 1509924.

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REFERENCES

(1) (a) Campagna, S.; Puntoriero, F.; Nastasi, F.; Bergamini, G.; Balzani, V. Photochemistry and photophysics of coordination compounds: Ruthenium. Top. Curr. Chem. 2007, 280, 117−214. (b) Kalyanasundaram, K. Photophysics, Photochemistry and SolarEnergy Conversion with Tris(Bipyridyl)Ruthenium(II) and Its Analogs. Coord. Chem. Rev. 1982, 46, 159−244. (2) Gratzel, M. Recent advances in sensitized mesoscopic solar cells. Acc. Chem. Res. 2009, 42, 1788−98. (3) Lam, Q.; Kato, M.; Cheruzel, L. Ru(II)-diimine functionalized metalloproteins: From electron transfer studies to light-driven biocatalysis. Biochim. Biophys. Acta, Bioenerg. 2016, 1857, 589−97. (4) (a) Kato, M.; Nguyen, D.; Gonzalez, M.; Cortez, A.; Mullen, S. E.; Cheruzel, L. E. Regio- and stereoselective hydroxylation of 10undecenoic acid with a light-driven P450 BM3 biocatalyst yielding a valuable synthon for natural product synthesis. Bioorg. Med. Chem. 2014, 22, 5687−91. (b) Tran, N. H.; Nguyen, D.; Dwaraknath, S.; Mahadevan, S.; Chavez, G.; Nguyen, A.; Dao, T.; Mullen, S.; Nguyen, T. A.; Cheruzel, L. E. An efficient light-driven P450 BM3 biocatalyst. J. Am. Chem. Soc. 2013, 135, 14484−7.

CONCLUSIONS

We described herein the study of a series of Ru(II)-polypyridyl complexes in which substitutions on the ancillary ligands were used to modify the photophysical properties of the complexes. In the corresponding hybrid enzymes, the modifications resulted in a 3-fold increase in photocatalytic activity. The substituent effects could be rationalized using a combination of Hammett and Brown-Okamoto parameters and shed light on the rate limiting steps in the overall photocatalytic activity of the hybrid enzymes. These correlations will help improve the design of future light-driven systems involving Ru(II)-diimine complexes. 6563

DOI: 10.1021/acs.inorgchem.7b00685 Inorg. Chem. 2017, 56, 6558−6564

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DOI: 10.1021/acs.inorgchem.7b00685 Inorg. Chem. 2017, 56, 6558−6564