Sulfanyl stabilization of copper-bonded phenoxyls in model complexes and galactose oxidase
Pratik Vermaa, Russell C. Pratta, Tim Storrb, Erik C. Wasingerc, and T. Daniel P. Stacka,1
aDepartment of Chemistry, Stanford University, Stanford, CA 94305; cDepartment of Chemistry and Biochemistry, California State University, Chico, CA 95929; and bDepartment of Chemistry, Simon Fraser University, Burnaby, BC, Canada V5A-1S6
Edited by Judith P. Klinman, University of California, Berkeley, CA, and approved September 15, 2011 (received for review June 20, 2011)
GOGO activeactive Integrating sulfanyl substituents into copper-bonded phenoxyls GOGO inactiveinactive oxyoxy O semisemi O significantly alters their optical and redox properties and provides -1e- 1 e- insight into the influence of cysteine modification of the tyrosine NHiHis IIII O NHiHis IIII O CuCu S CCuu S cofactor in the enzyme galactose oxidase. The model complexes NHiHis H2O2 NHiHis RCHRCH2OHOH ½1SR2 þ are class II mixed-valent CuII-phenoxyl-phenolate species that exhibit intervalence charge transfer bands and intense visible O 2 OH RRCHCHO sulfur-aryl π → π transitions in the energy range, which provides a HHOO N greater spectroscopic fidelity to oxidized galactose oxidase than HiHis CCuuI S N non-sulfur-bearing analogs. The potentials for phenolate-based HiHis oxidations of the sulfanyl-substituted 1SR2 are lower than the alkyl- GGOOredred ca. substituted analogs by up to 150 mV and decrease following Fig. 1. The active site and observable forms of GO. the steric trend: −StBu > −Si Pr > −SMe. Density functional theory calculations suggest that reducing the steric demands of the sul- fanyl substituent accommodates an in-plane conformation of the energetic influence in copper-bonded phenoxyls including GOoxy alkylsulfanyl group with the aromatic ring, which stabilizes the remain unknown (9, 10). phenoxyl hole by ca. 8 kcal mol−1 (1 kcal ¼ 4.18 kJ; 350 mV) Copper complexes of bisimine bisphenolate salen ligands through delocalization onto the sulfur atom. Sulfur K-edge X-ray are known to catalytically oxidize benzylic alcohols with O2 under tBu2 þ absorption spectroscopy clearly indicates a contribution of ca. 8– basic conditions (11, 12). The one-electron oxidized form ½1 13% to the hole from the sulfur atoms in ½1SR2 þ. The electroche- acts as a stoichiometric oxidant of benzyl alcohol and exists in III mical results for the model complexes corroborate the ca. 350 mV an equilibrium between a Cu -phenolate and a ferromagnetic II (density functional theory) contribution of hole delocalization on Cu -phenoxyl radical species (13–15). Previous work suggests to the cysteine–tyrosine cross-link to the stability of the phenoxyl localization of the hole on the ligand rather than the metal cor- radical in the enzyme, while highlighting the importance of the relates with increased rate of alcohol oxidation (14–16). A sulfa- in-plane conformation observed in all crystal structures of the nyl substituent creates a more easily oxidized aromatic ring and II enzyme. biases the equilibrium toward the Cu -phenoxyl radical species. In this work, we examine the redox and spectroscopic influence II Marcus–Hush analysis ∣ density functional theory reduction potentials ∣ of a para-sulfanyl substituent in a series of Cu -phenoxyl radical noninnocent ligands ∣ magnetic coupling complexes (Fig. 2). The variation of the steric demands of the alkylsulfanyl groups (−SMe, −SiPr, −StBu) modulates their alactose oxidase (GO) is an enzyme that selectively oxidizes orientation relative to the aromatic ring, which in turn modulates Gprimary alcohols to aldehydes via a unique CuII-tyrosyl the delocalization of the phenoxyl hole. For structural fidelity, radical with concomitant reduction of dioxygen to hydrogen per- sulfanyl substituents in GO model complexes are generally posi- ortho 2SMe2 – para oxide (1, 2). The active site of GO contains a Cu center ligated by tioned to the oxygen (e.g., ; refs. 12, 17 26). The 1R2 two histidines, one unmodified tyrosine (axial) and one tyrosine positioning of sulfanyl substituents in is designed purposefully residue (equatorial) that is covalently cross-linked to a cysteine to assure minimal copper coordination changes by preserving the ortho residue in a posttranslational oxidative modification step (3–5) sterically abutting -t-butyl substituents (R3, Fig. 2). (Fig. 1). The consensus mechanism involves two catalytically re- EPR characterization of the sulfur contribution to the hole sta- I bilization is not feasible for GOoxy as antiferromagnetic coupling levant forms: the reduced (GOred), which contains a Cu -tyrosine −1 II (J > 200 cm ) between the CuII center and the tyrosyl radical unit, and the oxidized (GOoxy), which contains a Cu center and a cysteine-modified tyrosyl radical. One-electron reduction of leads to a diamagnetic ground state (9, 27). We have evaluated sulfur K-edge X-ray absorption spectroscopy (XAS) as a compli- GOoxy generates the inactive semireduced (GOsemi) form, which contains a CuII-tyrosinate unit. mentary technique for experimental quantification of the sulfur Cysteine-modification of the redox-active tyrosine significantly contribution in complexes with complicated or silent EPR spectra ½1SR2 þ influences the redox properties of the active oxidant: The poten- such as and GOoxy. The delocalization of hole onto the • ∕ sulfur atom and the resulting stabilization of the phenoxyl radical tial of the Tyr-Cys/Tyr -Cys couple estimated from the GOsemi ca. is modeled by DFT computations and correlated to trends in GOoxy interconversion is 400 mV [vs. normal hydrogen electrode (NHE), pH 7.5], which is significantly lower than free experimental reduction potentials. tyrosine in solution (ca. 900 mV) and unmodified redox-active ca. – tyrosines in other enzymes ( 660 1,000 mV) (1, 6). Delocaliza- Author contributions: P.V., R.C.P., and T.D.P.S. designed research; P.V., R.C.P., T.S., and E.C.W. tion of the tyrosyl radical onto the thioether bridge, which is pre- performed research; P.V., R.C.P., and E.C.W. contributed new reagents/analytic tools; P.V., dicted by density functional theory (DFT) computations (7, 8) R.C.P., T.S., E.C.W., and T.D.P.S. analyzed data; and P.V., T.S., and T.D.P.S. wrote the paper. and EPR studies of copper-free GO (9), is postulated to contri- The authors declare no conflict of interest. bute to the energetic stabilization of GOoxy. This postulate is con- This article is a PNAS Direct Submission. sistent with the in-plane conformation of the cysteine–tyrosine 1To whom correspondence should be addressed. E-mail: [email protected]. – cross-link observed in all known crystal structures of GO (3 5), This article contains supporting information online at www.pnas.org/lookup/suppl/ yet the extent of hole delocalization on sulfur and consequent doi:10.1073/pnas.1109931108/-/DCSupplemental.
18600–18605 ∣ PNAS ∣ November 15, 2011 ∣ vol. 108 ∣ no. 46 www.pnas.org/cgi/doi/10.1073/pnas.1109931108 Downloaded by guest on September 23, 2021 R R 3 5 Table 1. Key electrochemical parameters racrac 1tBu2tBu2 tBu tBu E E ΔEK SMe2SMe2 t 1 2 c* N N 1 Bu SMeSMe tBu2 Cu SiPr2SiPr2 t i 1 450 650 200 2,450 1 Bu S Pr 2 R5 O O R5 1StBu 450 550 100 50 1StBu2StBu2 tBuBu StBu SiPr2 R3 R3 1 340 440 100 50 SMe2 — 60† 10 2SMe2SMe2 t 1 300 < < steric interactiointeractionn SMeSMe Bu Units in millivolt vs. Fc0∕þ. Fig. 2. Symmetric copper-salen complexes. *Kc ¼ expðΔE∕59 mVÞ at RT. †Upper limit. GO and ½1SR2 þ are mixed-valent species in which the π oxy ½1SR2 þ ½1tBu2 þ SI Appendix orbitals of the phenolate and phenoxyl radical rings comprise dependence for than for (see , redox-active centers that are bridged by the d orbitals of the CuII Table S11). Owing to the lower energy and higher intensity of center. Complexes ½1SR2 þ are assigned as class II mixed-valent the NIR absorptions relative to d-d bands and the mixed-valent ½1SR2 þ species based on a Marcus–Hush analysis of the intervalence oxidation state of , these features are attributed to pheno- charge transfer (IVCT) transitions (28–32). The correspondence late-phenoxyl IVCT. with the energies of the IVCTand sulfur-aryl π → π transitions in Both one- and two-electron oxidized forms are colored due to ½1SR2 þ supports the assignment of both these types of transitions intense bands in the visible region. The origins of these absorp- ½1tBu2 þ∕2þ ½1SR2 þ∕2þ for the broad optical band in GO . tions in vs. differ distinctly; upon oxidation, oxy the ca. 18;000 cm−1 feature of ½1tBu2 þ disappears, whereas the Results ca. 17;000 cm−1 features of ½1SR2 þ nearly double in intensity. The ½1SR2 þ π → π Electrochemistry. Room temperature (RT) differential pulse vol- features in can be assigned to intraring sulfur-aryl tammograms of 1SR2 show two overlapping oxidation peaks that transitions (see below), though the energies and intensities are ortho were fit with Gaussian functions to estimate the potentials for higher than those observed for -alkylsulfanyl substituted the two oxidations (Table 1 and SI Appendix, Fig. S3). In general, phenoxyl radicals (18, 20, 26). The intensity of these features 2 ½1SR þ ½1SR 2þ − t − i the redox processes of 1SR occur at lower potentials than those of in and increases in the order S Bu > S Pr > tBu2 − 1 .E1 increases with increasing steric demands of the alkylsul- SMe, which correlates with increased delocalization of the
StBu2 tBu2 − CHEMISTRY fanyl substituent; whereas E1 for 1 is close to 1 , values for hole on the sulfur atom with SMe. 1SiPr2 1SMe2 ca. and are 110 and 150 mV lower, respectively. All 2 SR2 XAS Spectroscopy. 1SR of the sulfanyl-substituted complexes, 1 , have E2 values well Sulfur K-edge XAS spectra of the neutral 2 − − i below that of 1tBu . ( SMe and S Pr) and the corresponding one-electron oxidized ca. The AgI salts generally used for oxidation of sulfur-free ana- complexes are dominated by intense edge features at 2,474 eV 2 A SI Appendix logs cause immediate precipitation with 1SR , presumably due (Fig. 5 and , Fig. S11). The edge features occur to coordination of AgI to the thioethers as demonstrated by at higher energy for the oxidized forms with a larger difference − − i Halcrow and coworkers (33). Instead, thianthrenyl radical hexa- for SMe in comparison with S Pr. The oxidized forms exhibit •þ − fluoroantimonate (Th SbF6 ) was used as a readily prepared, an additional preedge feature (2,470.9 eV, integration 0.35, 0∕þ •þ ½1SMe2 þ ½1SiPr2 þ titratable oxidant [MeCN, 860 mV vs. Fc (ferrocene), Th : ; 2,471.2 eV, integration 0.21, ); analysis of these ϵ ¼ 8 500 −1 −1 features using methods reported for metal-bonded sulfur com- 546 nm ; M cm , Th: colorless] (34). Diacetylferroce- þ − nium hexafluoroantimonate (Ac2Fc SbF6 ) was used as a sulfur- plexes affords sulfur contributions to phenoxyl hole of 13% SMe2 þ SiPr2 þ free oxidant for S K-edge XAS studies. and 8% for ½1 and ½1 , respectively (Table 2 and SI Appendix). The Cu L-edge XAS spectra of the neutral and ca. UV-Visible-Near-Infrared (UV-vis-NIR) Spectroscopy. In CH2Cl2, the one-electron oxidized complexes contain L3-edge features at UV-vis absorbance spectra of 1R2 exhibit low-intensity features 931.5 eV, consistent with a CuII oxidation state (Fig. 5B and at ca. 17;500 cm−1, attributed to CuII d-d transitions, and more SI Appendix, Fig. S1). intense features near 25;000 cm−1, assigned to Schiff base π-π and/or phenolate-Cu ligand-to-metal charge transfer transitions. EPR Spectroscopy. Frozen solution (77 K) X-band EPR spectra of R2 Oxidation of 1R2 using Th•þ is accompanied by development 1 exhibit nearly identical rhombic signals with ligand hyperfine 1R2 structure resolved in toluene, which indicates that all neutral of intense UV-vis-NIR features (Fig. 3). Complexes can be 2 oxidized completely to the one- and two-electron oxidized forms, 1R complexes exist as monomeric, nearly planar complexes with ½1tBu2 2þ ½1StBu2 2þ the intended homology of coordination around the copper center with the sole exception of .* Except for , the oxi- 2 þ dized complexes can be back-titrated with decamethylferrocene (35) (see SI Appendix, Fig. S9). The EPR spectra of ½1SR con- as the reductant (Fc , −530 mV vs. Fc0∕þ) to form solutions with tain features in a broad range (2,800–3,600 G) that are similar in the optical features of the neutral complexes and ½Fc þ, demon- shape to the neutral complexes (see SI Appendix, Fig. S2). Spin strating chemical reversibility of the oxidations. quantitation was carried out by double integration of derivative The most notable features for ½1R2 þ are the NIR absorptions at 5;000–11;000 cm−1 that are absent in 1SR2 and ½1SR2 2þ (Fig. 4 ABSiPr2 SI Appendix ½1SR2 þ ) 1 -1 and , Table S2). Complexes absorbs at ener- 2 ) −1 2 þ -1 gies 4;000–5;000 cm higher than ½1tBu , but with less than half 0.1 SR2 þ the intensity. For ½1 , a small shift in the energy of the NIR Cu cm -1 ½1SMe2 þ 1 bands is observed with having the lowest energy and M 4 ½1StBu2 þ having the highest (9,400 and 10;500 cm−1, respectively). (10 abs (10,000 cm In CH3CN, a more polar solvent, the absorbance maxima for the 0 0 NIR transitions occur at higher energies, with a greater solvent 25,000 15,000 5,000 0 0.5 1 1.5 2 2.5 energy (cm-1) equiv Th
SiPr2 •þ •þ ½1tBu2 þ Fig. 3. Spectrophotometric titration of 1 with Th in CH2Cl2 at RT. (A) *The spectral changes accompanying addition of Th to solutions of are −1 •þ •þ 10;000 – consistent with incomplete oxidation and accumulation of Th . This incomplete UV-Vis-NIR spectra (B) absorbance at cm vs. Th eq. From 0 1eq oxidation may be due to the high potential for the second oxidation (þ650 mV)or added, the NIR intensity increases; from 1–2 eq, the intensity decreases; past •þ low polarity of CH2Cl2 in absence of electrolyte. 2 eq, the intensity increases due to excess Th .
Verma et al. PNAS ∣ November 15, 2011 ∣ vol. 108 ∣ no. 46 ∣ 18601 Downloaded by guest on September 23, 2021 tBu2 Table 2. Phenoxyl hole delocalization parameters 2 1 H λ† r‡ § ¶ AB* [spin] %S x5 ½ StBu2 þ — 1 1 2,060 (2,000) 10,500 2.6 0.55 ½1SiPr2 þ 2,030 (2,200) 10,200 2.9 0.95 8 ½1SMe2 þ 1,510∥ (2,300) 9,400 4.1** 1.25 13 −1 *Units in cm , from Kc (from IVCT band assuming charge 1StBu2 – r ¼ 2 7 2 transfer distance is the OA OB distance, i.e., . Å). †Units in cm−1, from IVCT band. ) -1 ‡Å, effective CT distance. 1 x5 §EPR spin concentration.
Cu cm ¶
-1 XAS S contribution to phenoxyl hole. ∥ M
4 Upper limit. **Lower limit. (10 SiPr2 2 1
bond lengths are elongated and the C–O and C–S bond lengths 1 x5 are contracted in the ring with the −SMe group in plane (ring B). DFT computed spin density distribution correlates with a predo- minant localization of the hole on ring B in the (90°, 0°) confor- SMe2 SI Appendix 2 1 mation (see , Table S12). The energetic and structural distortions within the analogous ½2SMe2 þ complexes containing ortho-methylsulfanyl substituents 1 x5 follow the same trends as ½1SR2 þ. However, in the nonoxidized 2SMe2 form, the lower-energy conformation is (0°, 0°), presumably because of unfavorable steric or lone-pair/lone-pair interaction 35,000 30,000 25,000 20,000 15,000 10,000 5,000 ortho− energy (cm-1) between the oxygen and SMe groups in the (90°, 90°) con- formation. R2 R2 þ SMe2 SMe2 þ Fig. 4. RT CH2Cl2 solution UV-Vis-NIR spectra of 1 (dotted), ½1 (solid), The ΔE computed for 1 and ½1 structures with 2 2þ rxn and ½1R (dashed). both −SMe groups constrained 90° out of plane is −7.4 kcal mol−1 or þ320 mV, which indicates that ½1SMe2 þ is a stronger 2 EPR spectra and normalized to 1R intensity (Table 2). Whereas oxidant in this conformation (Table 3). With both −SMe groups 2 þ 2 þ ½1tBu is effectively EPR silent, the spectra of ½1SR contain constrained in plane, ½1SMe2 þ becomes a weaker oxidant ½1StBu2 þ Δ ¼þ4 4 −1 −190 − appreciable intensity, which increases in the order < ( Erxn . kcal mol or mV). Constraining one SMe 2 þ 2 þ ½1SiPr < ½1SMe . group in an out-of-plane orientation and one in plane yields a DFTestimated ½1SMe2 0∕þ redox potential of −155 mV, matching DFT Calculations. A dihedral angle defines the orientation of the that observed experimentally at −150 mV (vs. ½1tBu2 0∕þ). The alkylsulfanyl substituent relative to the plane of each aromatic decrease in oxidizing power with increasing orientation of the θ θ − θ θ ring ( A, B; Scheme 1): Maximal out-of-plane orientation corre- SMe group toward an in-plane conformation ( A and B) fol- SMe2 sponds to 90°. The DFToptimized 1 structure is C2 symmetric lows a cosine-torsional dependence (Fig. 6 and SI Appendix, with both −SMe groups oriented out of plane and with equal Fig. S5). For ½1SMe2 þ, the energetic stabilization and the increase Cu–O, C–O, and C–S bond lengths for both rings (see SI in sulfur spin density show a similar correspondence. Appendix, Table S4). Structures optimized with one or both −SMe These DFT calculations suggest that orientations of the groups constrained in plane are slightly less stable by 1.7 and methylsulfanyl substituent can alter the redox potential of a phe- −1 3.5 kcal mol , respectively, without significant structural distor- noxyl radical by nearly 510 mV in the para position and 350 mV in tions in the remaining portion of the complex. the ortho position (Table 3). In a spectroscopically calibrated 2 þ By contrast, the one-electron oxidized form, ½1SMe , opti- computational model of GO reported by Szilagyi and coworkers mizes to a C2 symmetric structure with both −SMe groups in (model 6 in refs. 7 and 8), a second-sphere tryptophan residue −1 plane, which is 8.5 kcal mol more stable than the conformation (W290) constrains the cross-linked cysteine (C228) side chain in with both −SMe groups 90° out of plane; this energetic difference the plane of the modified tyrosine (Y272) ring and the orienta- II 3 quantifies the maximal resonance stabilizing influence of a tion of the aromatic plane perpendicular to the Cu dx2−y2 plane. sulfanyl substituent in para-substituted phenoxyl radicals. As the For structures derived from this model with the tryptophan 2 þ −1 (90°, 0°) conformation of ½1SMe is 7.9 kcal mol more stable removed, DFT estimated Y272/Y272• reduction potential for than the (90°, 90°) conformation, the majority of the stabilization the structure with the sulfanyl group oriented 90° out of the plane is achieved by placing only a single sulfanyl substituent in plane. of the Y272 ring is nearly 350 mV more oxidizing than with the 2 þ In this conformation of ½1SMe , significant metrical differences sulfanyl group oriented in plane, on par with simple synthetic are found between the two rings in DFT calculations: the Cu–O models. 3 6 A B NA NB 2 Cu 4 SA A OA OB B SB
R t R
Intensity t 1 2 Bu Bu 1SR2 & [1SR2]+ Normalized intensity 0 0 2,470 2,474 2,478 930 934 938 E SMe2SMe2 + tBu2tBu2 rxnrxn SMe2SMe2 tBu2tBu2 + energy (eV) energy (eV) [1 ] + 1 1 + [1 ] (S(S = 1)1) ((SS = 1/21/2)) (S(S = 1/2)1/2) (S(S = 11)) Fig. 5. XAS spectra of 1SMe2 (dotted) and ½1SMe2 þ (solid): sulfur K-edge (A) and copper L-edge (B). Scheme 1.
18602 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1109931108 Verma et al. Downloaded by guest on September 23, 2021 Table 3. DFT estimated redox potentials with −SMe Table 4. TD-DFT analysis of the visible feature in ½1SR2 þ conformation constrained Exp TD-DFT ΔE ν ϵ f † θ θ ‡ ν f rxn* max* ( ) A, B max* SMe2 þ E1 (exp) 90° out of plane In plane Difference ½1 16,850 12,400 (0.15) 90°, 0° 17,500 0.26 SMe2 − 0°, 0° 17,600 0.23 1 150 +320 -190 -510 SiPr2 þ † † ½1 16,800 9,050 (0.11) 18°, 18° 17,100 0.22 (-155) (-475) ½1StBu2 þ 16,900 7,450 (0.09) 37°, 37° 15,900 0.16 2SMe2 -20 +190 -165 -355 GO -450 +415 +70 -345 *Units in cm−1. †Units in M−1 cm−1, exp oscillator strength in parentheses. Millivolt vs. ½1tBu2 0∕þ, experimental from refs. 7, 14, and 35. ‡−SR conformation (Scheme 1). *Scheme 1. Positive values imply forward reaction is favored. †Only one −SMe in plane. ½1tBu2 þ exists as an equilibrium mixture of CuIII-phenolate and II † Time-Dependent DFT Calculations. TD-DFT calculations predict Cu -phenoxyl radical isomers at RT (13). Cu L-edge XAS II ½1SR2 þ transitions with significant intensity (oscillator strengths >0.1) measurements confirm the Cu oxidation state in all SI Appendix II in the 10;000–25;000 cm−1 range for ½1SMe2 þ only if at least one (see , Fig. S1). The Cu -phenoxyl-phenolate species π −SMe group is oriented in plane (Table 4). With only one −SMe are mixed-valent, in which the orbitals of the phenolate and in plane, the donor and acceptor orbitals are localized on the ring phenoxyl radical rings comprise the redox-active centers that are II with the in-plane group (B) and the transition is best described coupled through the d orbitals of the Cu center (Fig. 7). The 10 000 −1 ½1SR2 þ as an intraring transition between aryl π orbitals that contain IVCT transitions observed near ; cm in are consis- significant S p character (Fig. 7 B and D). With both −SMe groups tent with a class II mixed-valent description with solvent-dependent in plane, the nature of the donor and acceptor orbitals remains energies, broad bandwidths exceeding the high-temperature limit, 2 ∕λ 0 7 – and Hab ratios within the class II limit (< . )(28 32). The elec- the same but contains equal contributions from both rings (see 2 SI Appendix, Fig. S4). TD-DFT calculations predict similar types trochemically determined comproportionation constants for 1SR of transitions for ½1SiPr2 þ (18°, 18°) and ½1StBu2 þ (37°, 37°), but are also within the range expected for a class II assignment 1 000 with lesser oscillator strengths due to reduced sulfur p overlap (Kc < ; at 298 K). with the aromatic ring. This trend correlates with a decrease in Although the effective charge transfer distance is well defined CHEMISTRY intensity of the 17;000 cm−1 feature in the order ½1SMe2 þ > for metal-based redox centers, it is ambiguous for a phenolate- ½1SiPr2 þ > ½1StBu2 þ observed experimentally. A similar π–π tran- phenoxyl IVCT; whereas the negative charge of the phenolate sition is not predicted for 1R2. is expected to be predominantly localized on the oxygen atoms, the hole on a phenoxyl radical is expected to be delocalized over Discussion the oxygen atom and the aromatic ring. For ½1StBu2 þ, the effective Models of GO generally use ortho-sulfanylphenols to provide charge transfer distance obtained from Marcus–Hush analysis faithful structural correspondence to the covalent bond between agrees well with the OA-OB distance (2.7 Å) (13, 16); this suggests Cys228 and the ortho position of Tyr272 (12, 17–26). The results that the locus of oxidation in the phenoxyl ring lies predominantly para herein show that -sulfanyl substituents have electrochemical near the oxygen atom. The effective charge transfer distance in- ortho 2 þ 2 þ 2 þ and spectroscopic effects comparable to those at position, creases in the order ½1StBu < ½1SiPr < ½1SMe (Table 2). while allowing variation in the steric demands of the alkylsulfanyl This increase indicates a shift in the locus of oxidation from the group with minimal potential changes in the copper coordination oxygen atom toward the aromatic ring and the sulfur atom for less geometry. The nearly identical EPR spectra of all 1SR2 indicate a II sterically demanding alkylsulfanyl substituents, which can better similar distorted planar geometry around the Cu center. More- accommodate an in-plane orientation with the aromatic ring to over, nearly identical distorted planar coordination is found in all delocalize the hole onto the sulfur atom. DFT optimized geometries of 1R2 and ½1R2 þ (see SI Appendix). para GOoxy is EPR silent due to antiferromagnetic coupling of In the position, the preferred orientation of the methyl- the phenoxyl radical to the copper(II) center (1, 9, 27). In small- sulfanyl substituent is in plane with respect to the aromatic molecule CuII-phenoxyl radical complexes, the difference be- ring for a phenoxyl radical and is 90° out of plane for a phenolate tween antiferromagnetic and ferromagnetic coupling arises from (see SI Appendix). The larger steric demands of the −SiPr and differences in the angular overlap of the unpaired spin containing −StBu substituents preclude an in-plane orientation. Whereas 2 þ orbitals (27, 12, 20, 24, 36–38). The orientation of the aromatic DFT geometry optimizations of ½1SMe orient the −SMe groups 2 þ 2 þ ring relative to the Cu coordination basal plane observed in crys- in plane, the alkylsulfanyl substituents of ½1SiPr and ½1StBu tal structures of Cu(II)–salen complexes precludes the orbital are rotated out of plane by 18° and 37°, respectively. DFT geo- overlap required for antiferromagnetic coupling (13, 16). metry optimizations orient the alkylsulfanyl substituent signifi- ½1SR2 þ 1SR2 θ θ ¼ 90 The X-band EPR spectra of contain broad features cantly out of plane for all nonoxidized ( A and B °). ¼ 2 II ½1SR2 þ II near the g region similar to a Cu center and do not contain The one-electron oxidized forms exist as Cu -pheno- Δ ¼ 2 late-phenoxyl radical complexes, whereas the non-sulfur-bearing half-field signals ( ms ), which would be characteristic of a triplet species (27).‡ In contrast to the triplet EPR response 6 observed in non-sulfur-bearing analogs (16), the larger interspin A B ) 0.6 distance resulting from significant delocalization of the hole
-1 3 onto the para-sulfur atoms in ½1SR2 þ lead to weaker spin–spin 0 0.4 coupling and a more diradical EPR response (39). The integrated
(kcal mol -3
rxn 0.2 E -6 †The one-electron oxidized form ½1tBu2 þ exists in a valence-tautomeric equilibrium Mulliken Spin Density Mulliken 0 involving a diamagnetic CuIII-bisphenolate and a paramagnetic CuII-phenolate-phenoxyl radical species in solution at RT. The two isomers are iso-energetic (ca. 1∶1 mixture at RT), & & which implies that both isomers have identical redox potentials determined from A B A B electrochemical measurements. Fig. 6. DFT computed scan along Cm-Cp-S-Calkyl dihedral angle (Scheme 1). ‡The possibility of decomposition of ½1SR2 þ to 1SR2 as the source of the ambiguous EPR (A) ΔErxn.(B) Mulliken atomic spin density for S (solid circles), O (open circles), spectra is countered by the full chemical reversibility of redox processes confirmed 2 þ and the aromatic ring in ½1SMe (open squares). through optical titrations.
Verma et al. PNAS ∣ November 15, 2011 ∣ vol. 108 ∣ no. 46 ∣ 18603 Downloaded by guest on September 23, 2021 such as ½1OMe2 þ and ½1tBu2 þ (13, 16, 43), highlighting the stron- ½1SR2 þ ger spectroscopic fidelity of to GOoxy. The electrochemical influence of the alkylsulfanyl substituents in 1SR2 highlights the importance of the in-plane conformation in enhancing the stability of phenoxyl radicals, including in para GOoxy. Relative to an alkyl group, a -sulfanyl substituent has a moderate electron-donating influence: The potential for first phenolate oxidation of 1SMe2 is ca. 150 mV lower than 1tBu2.Anortho-methylsulfanyl group lowers this potential by a lesser amount in the range of 10–50 mV (20 mV for 2SMe2) (24, 33, 36, 44, 45). For para substituted 1R2, the potential for the first oxidation varies linearly with the σþ Hammett constant (see SI Appendix, Fig. S8); a similar correlation is observed for free phenolates (46, 47). Based on a linear free energy relation- ship (LFER) analysis, the net electron-donating influence of a para−SMe substituent, relative to an alkyl group, is attributed to a large electron-donating resonance effect attenuated by a 2 þ Fig. 7. Selected molecular orbitals of ½1SMe . smaller electron-withdrawing field effect (SI Appendix, Table S7). The resonance stabilization energy estimated by a LFER analysis using experimental redox potentials is ca. 8 kcal mol−1 for StBu2 þ SiPr2 þ EPR signal intensity increases in the order ½1 < ½1 < ½1SMe2 þ, which corresponds well with the 8.5 kcal mol−1 pre- SMe2 þ ½1 (Table 2); the corresponding increase in distance be- dicted by DFT.¶ II tween the phenoxyl and Cu unpaired spins is supported by the At parity of the field effect, increasing out-of-plane rotation increase in effective charge transfer distance determined from to accommodate the steric demands of −SiPr and −StBu groups Marcus–Hush analysis. The shift in spin density distribution attenuates the electron-donating resonance effect; indeed, the based on the alkylsulfanyl orientation is reproduced in DFT 1StBu2 1tBu2 2 þ potential for first phenolate oxidation of and are iden- calculations: the phenoxyl radical spin density in ½1SMe shifts tical. DFTcalculations also support that the increase in energetic from the oxygen atom with both −SMe groups oriented 90° out stabilization of ½1SMe2 þ is due to increased in-plane orientation of of plane toward the sulfur atom with both groups oriented in the −SMe group, and the concomitant increase in the sulfur spin plane (Fig. 6). density results in a decrease in the ½1SMe2 0∕þ redox potential. The Because of coupling between the spins in copper(II)-phenoxyl maximal potential influence of the orientation of the methylsul- 2 species such as GOoxy, quantitative characterization by EPR spec- fanyl substituent on the redox potential of 1SMe is nearly 510 mV troscopy is not possible. We have used sulfur K-edge XAS as a and nearly 350 mV in ortho-sulfanyl-substituted analog 2SMe2 complimentary technique to quantify the sulfur contribution to 2 þ (Table 3). the phenoxyl hole in ½1SR . Although this technique has been In crystal structures of GO, an in-plane conformation of the used recently to detect sulfur-centered free radicals not bonded cysteine–tyrosine cofactor is observed exclusively. Based on the to a metal center (40), we demonstrate its utility for quantitative comparison with 1SMe2, this conformation enhances the stability 2 analysis in such species. The energy of the edge feature in 1SR of the phenoxyl radical in GO by up to 350 mV (DFT). Given the ca. ¼ 175 ( 2,474 eV) is consistent with other thioether compounds and a weak substrate binding interactions (Km mM for galac- ½1SR2 þ small shift to higher energy in is consistent with an in- tose) and slow activation of GOsemi under aerobic conditions, the crease in positive charge (40) (Fig. 5). The additional preedge remarkable stability of the phenoxyl radical (t1∕2 ¼ 7.2 d) is es- 2 þ 2 features observed in ½1SR , but not 1SR , are attributed to a S sential to its function: reduction of dioxygen to peroxide coupled 1s to phenoxyl singly unoccupied molecular orbital transition with oxidation of primary alcohols (1). To put this observation (Fig. 7 B); the preedge energies are similar to sulfur-centered into perspective, the phenoxyl radical in another copper-radical radicals reported by Martin-Diaconescu and Kennepohl (40). In oxidase that generates hydrogen peroxide, glyoxal oxidase, is ca. metal-bonded sulfur complexes, the preedge features intensity 40-fold less stable (t1∕2 ¼ 4 h) (48, 49). Although the oxidized reflects S p mixing due to covalency of the metal-sulfur bonds (41). form of glyoxal oxidase is a stronger oxidant than galactose oxi- A similar analysis reveals significant S p contribution due to delo- dase by 240 mV (experimental), it is able to efficiently oxidize 2 þ calization of the phenoxyl hole for ½1SR (Table 2). This deloca- only substrates with much weaker C–H bonds (acetals generated lization should be maximal for a methylsulfanyl substituent from aldehydes). oriented in the plane of the aromatic ring and ca. 13% S contribu- In conclusion, the CuII-phenolate-phenoxyl radical complexes tion to the phenoxyl hole is observed experimentally. The lesser S bearing sulfanyl substituents reported here provide insight into 2 þ contribution in ½1SiPr (8%) is consistent with the larger steric the extent of influence of the cysteine-modification of tyrosine demands of the −SiPr group precluding an in-plane orientation. in the enzyme GO. One-electron oxidation of symmetric sulfa- The electronic spectrum of the fully oxidized GO exhibits nyl-containing CuII–bisphenolate complexes 1SR2 yields relatively multiple overlapping features in the vis-NIR region (8;000– stable paramagnetic CuII-phenolate-phenoxyl radical complexes −1 −1 −1 20;000 cm , 3;200 M cm ) (1) that are attributed alterna- ½1SR2 þ, which are assigned as class II mixed-valent species based tively to either sulfur-aryl π → π (7, 8) or phenolate-phenoxyl on a quantitative Marcus–Hush analysis of the IVCT transition 2 þ IVCT (42) transitions. The electronic spectra of ½1SR exhibit and electrochemical measurements. aryl π → π transitions [Fig. 7 B (acceptor) and D (donor)] in Relative to non-sulfur-bearing analogs, the CuII-phenolate- −1 the visible near 17;000 cm and IVCT transition in the NIR near phenoxyl radical complexes bearing sulfanyl substituents exhibit −1 10;000 cm .§ These features are either not observed in the IVCT and sulfur-aryl π → π features with a high fidelity to the appropriate energy range or at all for non-sulfur-bearing analogs broad feature in the vis-NIR region observed for GOoxy. The nature of the IVCT transition in ½1SR2 þ lends credence to the contributions of similar transitions from the tyrosinate to the §The IVCT of class III mixed-valent species are akin to π-π transitions and their energies are well reproduced by single point TD-DFT calculations. The lack of vibronic-electronic coupling in such calculations limits their applicability for modeling IVCT transitions of ¶DFT computed resonance stabilization energy is the difference in energy between the class II mixed-valent species. (90°, 90°) and (0°, 0°) conformations of ½1SMe2 þ.
18604 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1109931108 Verma et al. Downloaded by guest on September 23, 2021 cysteine-modified tyrosyl radical in GOoxy, a pointed disputed in cross-link, the structure of the enzyme is optimized for formation the literature (7, 42). of a stable tyrosyl radical, which is critical for its function. The coupling between the phenoxyl radical and the CuII unpaired spins in oxidized GO precludes its characterization Materials and Methods 2 by EPR spectroscopy. Sulfur K-edge XAS spectral features Details of synthesis of 1SR (R ¼ Me, iPr, tBu), spectrophotometric titrations, associated with sulfur-bearing phenoxyl radicals have been iden- EPR sample preparation and spectra, XAS data collection and analysis, tified and the extent of sulfur contribution to the phenoxyl hole Marcus–Hush analysis, LFER analysis, DFT calculations, and the complete in CuII-phenoxyl species is quantified: If a planar conformation Gaussian 09 reference are included in the SI Appendix. can be achieved, the hole is significantly delocalized (ca. 0.13 e−) from the oxygen and aromatic ring onto the sulfanyl group. ACKNOWLEDGMENTS. We thank Prof. E. I. Solomon for instrument use and The corresponding resonance stabilization is ca. 8 kcal mol−1 Prof. C. E. D. Chidsey and Dr. M. D. Newton for helpful discussions. This work and leads to a significant lowering of the redox potential. was supported by an Urbanek Family Stanford Graduate Fellowship (R.C.P.) and funding from the National Institutes of Health (GM50730 to T.D.P.S.) and The influence of the sulfanyl substitution in copper-bonded from California State University Chico (E.C.W.). Portions of this research were phenoxyl radicals in model complexes is maximized with the carried out at Stanford Synchrotron Radiation Lightsource, a national user orientation of the alkylsulfanyl group in the plane of the aromatic facility operated by Stanford University on behalf of the US Department ring. With the in-plane conformation of the cysteine–tyrosine of Energy, Office of Basic Energy Sciences.
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