Variable-Temperature Variable-Field Magnetic Circular Dichroism

Downloaded by EAST CAROLINA UNIV on November 8, 2016 | http://pubs.acs.org Publication Date: August 19, 2003 | doi: 10.1021/bk-2003-0858.ch018

Chapter 18

Variable-Temperature Variable-Field Magnetic Circular Dichroism Combined with Electron Paramagnetic Resonance: Polarizations of Electronic Transitions in Solution Edward I. Solomon, Mindy I. Davis, Frank Neese, and Monita Y. M. Pau Department of Chemistry, Stanford University, 333 Campus Drive, Stanford,CA94305

The behaviour of saturation magnetization in VTVH MCD is determined by the polarization of an electronic transition relative to the magnetic ground state D tensor and/or g matrix. The latter can be derived from molecular orbital calculations including spin-orbit coupling. Thus the combination of V T V H MCD, EPR and molecular orbital theory allow the determination of polarizations of electronic transitions in a randomly oriented solution, providing new experimental insight into electronic structure and its contribution to reactivity.

328

© 2003 American Chemical Society

Telser; Paramagnetic Resonance of Metallobiomolecules ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

329


Introduction Many active sites in bioinorganic chemistry are highly colored as they exhibit intense low energy ligand-to-metal charge transfer (LMCT) transitions in absorption which reflect highly covalent ligand-metal bonds. These are often paramagnetic and in many cases (particularly Kramers ions) also exhibit rich electron paramagnetic resonance (EPR) spectra. Variable-temperature variablefield (VTVH) magnetic circular dichroism (MCD) provides a method to combine these to obtain new insight into electronic structure that can be important to function. For example, F e active sites play key roles in catalysis in the substrate activating enzymes, intradiol dioxygenases and lipoxygenases which are high spin, and in the peroxide level intermediates present in bleomycin (BLM) (activated BLM, a low-spin Fe -OOH complex) and possibly in the non-heme iron enzymes (which would be high-spin) (1). In contrast to Fe centers, ferric sites are EPR active and exhibit intense LMCT transitions in their absorption spectra. Variable temperature (and possibly variable frequency) EPR provide spin hamiltonian parameters (g values for S = Vi, and zero field splitting (ZFS) parameters (D, E) for S = / ) which, however, are not generally interpreted in terms of geometric and electronic structure. For the LMCT transitions, absorption, CD (circular dichroism), MCD and resonance Raman excitation profiles (the latter analyzed with time domain Heller theory (2, 3)) allow resolution of transitions (based on differences in selection rules) and general assignments to specific ligands (based on excited state distortions) (4). However, there is more information in the temperature and field dependence (VTVH) of the MCD data, particularly in the non-linear region, approaching the saturation limit (i.e., high H and low T). This allows correlation of excited state with ground state properties and the determination of the polarization of electronic transitions in a randomly oriented frozen solution (rather than in a single crystal). However, this is a complex problem because the spin sub-levels of the ground state can cross and mix for specific orientations of the magnetic field which in turn is dependent on the polarization of the electronic transitionfromthe ground to the excited state. m

m

11

5

2

Methodology We have derived expressions (5) which allow the analysis of V T V H MCD saturation magnetization curves. Equation 1 is for an S = Υι system, while eq 2 is the general expression, where the ZFS of the i sub-levels of the ground state is included.


330 A

£

2

2

- = -const f ï t a n h f ^ l — ( / x g , M ^ +/ g Mg y

{{

v ) y

where 7= ^ ( G X + G * + G * ) and G -l g P

ir

.

Δ£ ν

P

P

with p = x,y,z

2/r

const | " | ^ "£ Μ ( / Χ { 5 Χ ) Μ ^ + / ( S y ) . M Î + / » ( 5 . ) Μ ί : ) 8 Ϊ η β Ι β 1 ^ (2)

α


+Ι^Μ%^θάφ

y

2kT

y

(

For both equations, θ and φ are the angles between the incident light and the molecular ζ axis and the xy plane, respectively, l , l and 1 describe the orientation of the magnetic field relative to the molecular coordinate system, and the M f are the products of the polarizations of the electronic transitions. Two perpendicular transition moments i and j are required for MCD intensity. In a low symmetry protein site a transition is uni-directional, so M f Φ 0 is accomplished by spin-orbit mixing with an excited state that has transition moments with perpendicularly polarized components. In eq 1 for the S = Vi case, the g values are input from experimental EPR data. Equation 2 is dependent on Ni the Boltzmann population, and (S )., the spin expectation value in the ρ direction, for the spin sub-levels i of the ground state. These are obtained directly from the energies and wave-functions of the spin hamiltonian, eq 3, solved with values of D, Ε and g obtainedfromEPR data (6). x

y

2

p

P

P

H=D(SÏ

2

~S )

+ E(S * - S Î ) + )ff(g*HxS, +gyHySy +g«H«S«) 2

(3)

Thus the combination of V T V H MCD and EPR data allow the determination of the relative polarization products, Mf , for a transition to a given excited state, which through eq 4 (with cyclic permutations of indices to obtain % y and z) give the polarizations of electronic transitions for a randomly oriented protein solution (5).

%x = ΙΟΟΧτ fatMiJ (MUM t J +tyl*M%J + ( Μ ί Μ ί J

( 4 )

Experimentally, V T V H MCD data require spectroscopy on a strain free optical quality glass. The study of intermediates, an application important to our research, often requires these to be trapped using rapid freeze quench (RFQ) methods. Thus we have also developed a protocol (Figure 1) to obtain good quality V T V H MCD data on a RFQ intermediate. This involves trapping the



331 intermediate in liquid nitrogen and then making a 50% glycerol glass sample at 30° where the intermediates we are studying are stable. This is then transferred to an M C D cell also maintained at -30° for spectroscopy (7). The polarizations of electronic transitions obtained above are in the g matrix or D tensor coordinate system. The g matrix or D tensor must then be correlated to the molecular structure to obtain electronic structure information. We have been generally interested in the information content of spin hamiltonian parameters in terms of electronic structure and have shown that the standard ligand field model used to interpret these in terms of d orbital energy splitting must be modified to include differential orbital covalency (8). Each d orbital has a different covalent interaction with its ligand environment which modifies the spin-orbit interactions between the ground and excited states that give the ZFS and g value deviationsfrom2.00.

Figure 1. Protocol for trapping rapid freeze quench (RFQ) intermediates.

The absolute orientation of the g matrix and the D tensor can be measured with single crystal EPR spectroscopy. However, this depends on the availability of single crystals. Alternatively, the orientation of the g matrix and the D tensor in the molecule can be calculated by quantum chemical methods. In general, the elements of the D tensor are defined as (9): ±l)

D$

1

q

- - ^ ^ ( A S A M S . A | / / , V | B S B M S , B ) X ( B S B M S . B \H \ASAMS.A) S0C

(5)

Β

with I AS A M s , A) being the ground state function of total spin SA and corresponding MS.A value; the|BSBMs,B) are the excited states which appear in the sum over B ; the indizes(/?,g)= x,y,z refer to the cartesian components of


332 the D tensor; H$ is the spin-orbit coupling operator; the superscripts (0; ±1) represent the allowed ^4S = S A - S B following the selection rule of H and ΔΒ =E*-EA } The elements of the Ag matrix which define the shifts of the gvalues relative to the free electron value (2.00) are obtained from the equation (9): oc

soc

/,

"(ASAMS, |L |BSBMS.B)(BSBMS,B | / / V | A S A M S . A ) A

S

1

+(ASAMS,A|H V|BSBMS.B)(BSBMS3|L |ASAMS.A)J

1

9


Β

}

S

1

with U being the orbital angular momentum operator. The elements of the D tensor in eq 5 arise from products of spin-orbit coupling matrix elements whereas the g shifts in eq 6 emerge from products of spin-orbit coupling and Zeeman matrix elements. This is the reason why only excited states of the same total spin as die ground state (SB =SA ) contribute to the Ag matrix whereas the D tensor has additional contributionsfromstates with SB = SA ± 1. These molecular orbital expressions for g and D have been implemented in an I N D O / S - C I semi-empirical M O program to calculate the Du tensor and go matrix for a low symmetry active site and by diagonalization determine the orientation of these principle magnetic directions in the molecular frame (9). This allows the evaluation of possible structural models for an active site (e.g., intermediates), determines specific bonding contributions reflected by the spin Hamiltonian parameters, and, in particular, fixes the polarization of electronic transitions from V T V H M C D to specific acive site structural features. This has, as an example, proved critical in determining the different phenolate-Fe bonding interactions in the intradiol dioxygenases as described below. m

Application: Intradiol dioxygenases - Nature of the Tyr -> F e Bonds and Their Contributions to Reactivity (11) ra

The crystal structures of the intradiol dioxygenases, including protocatechuate 3,4-dioxygenase (3,4-PCD) show a trigonal bipyramidal Fe site with axial Tyr and His, and equatorial Tyr, His and OH" ligands, Figure 2 left (12, 13). Protocatechuate (PCA, 3,4-dihydroxybenzoate) binds as the dianion, replacing the axial Tyr and equatorial OH" forming a square pyramidal structure (14). The catecholate is asymmetrically bound with a long Fe-0 bond trans to the ra

1

By applying the Wigner-Eckhard theorem to the definition of H > Ms.A = SA and Ms.Β =SB are the only components needed in the definition of D$ and ng &, 10). soc

±l)

Pq


333 equatorial Tyr. Resting 3,4-PCD has a deep burgundy red color reflecting low energy intense phenolate-to-Fe charge transfer transitions (15). As we have emphasized, low energy intense charge transfer transitions reflect highly covalent ligand-metal bonds which can activate the metal site for reactivity (16). The absorption spectrum shows a broad band at ~ 22,000 cm" (ε ~ 3,000 M^cm" ) and a second feature at ~ 30,000 cm" (ε ~ 8,000 M^cm" ). The combination of absorption, CD, and MCD data (each with a different selection rule) demonstrates that at least seven transitions, Figure 3, are required to fit the Tyr —> Fe charge transfer region. The VTVH MCD data for these transitions given in Figure 4 reveal different saturation behaviors reflecting different polarizations for the electronic transitions. Fitting these saturation magnetization curves using eq 2 shows that bands 1, 3 and 6 are y polarized and that bands 2, 4 and 7 are dominantly ζ polarized (Table I). ra

1

l

1

1


ffl

Figure 2. Schematic representation of the binding of substrate protocatechuate (PCA) to protocatechuate 3,4-dioxygenase (3,4-PCD). (Structures were generated using crystallographic coordinates from PDB files 2PCD (from reference 13) and 3PCA (from reference 14))

As developed above these polarizations are relative to the D tensor orientation which was mapped onto the trigonal bipyramidal resting site in Figure 2 left using INDO/S-CI calculations. These calculations allow for the second-order spin-orbit coupling of the A i ground state with the complete manifold of quartet and sextet excited states including differential orbital covalency. The calculations give EID = 0.32 and D = -1.3 cm" which are in fortuitously good agreement with experiment {EID = 0.33 and D = 1.2 cm" ). Note that in the rhombic limit, while the sign of D does not affect the EPR or magnetic susceptibility data, it does have a strong effect on the MCD analysis as 6

1

1


334 it determines the specific orientations of the effective g values of the first and third Kramers doublets of the ground state. These calculations show that the zdirection of the D tensor is along the axial Tyr-Fe direction and the y axis is approximately along the equatorial Tyr-Fe direction. Thus we can assign the charge transfer transitions associated with each Tyr-Fe bond. From the above studies we find that there are at least three Tyr —• F e charge transfer transitions associated with each Tyr-Fe bond, and that the two sets of CT transitions hence the axial and equatorial Tyr-Fe bonds are inequivalent. m

ra

ra


m

Figure 3. Abs at 4 °C (A), CD at 4 °C (B) and MCD spectra at 5 Κ and 7 Τ (C) of3,4-PCD. Gaussian resolution (—)is shown along with spectra ( ). (Reprinted from reference 11. Copyright 2002 American Chemical Society.) t



335

Figure 4. Variable-temperature variable-field MCD data for 3,4-PCD. (A) Temperature dependence of the MCD spectrum at 7 Τfor 5 Κ ( ), 15 Κ (—) and 50 Κ (-·-). The arrows indicate energies where VTVH MCD data were taken. VTVH MCD data (·) collected between Ο Τ and 7 Τ and between 1.6 Κ and 50 Κ and the fit ( ) at (B) 32575 cm Band 7. (C) 28820 cm Band 6. (D) 25250 cm Band 4. (E) 23150 cm Band 3. (F) 19300 cm Band 2. (G) 16390 cm' Band 1. (Reprinted from reference 11. Copyright 2002 American Chemical Society.) 1

1

1

1

1

1


336 Table I. Gaussian Resolution and Spectroscopic Parameters for Transitions Observed in MCD data collected at 7T and 5K for 3,4-PCD band

MCD energy (cm )


1

1 2 3 4 6 7

17 600 20 700 22100 25 200 30150 31900

M (M-'cm') -2.33 12.6 9.20 -1.21 15.0 11.5

MCD v (cm ) 3000 3400 3400 3000 3600 3400

m

Polarization

1

y ζ y ζ y ζ

SOURCE: Adapted from reference 1 1 . Copyright 2 0 0 2 American Chemical Society.

Each Tyr (axial (ax) and equatorial (eq)) has two occupied valence orbitals ( π (out-of-plane), n (in-plane), Figure 5) available for donor bonding interactions with the five h occupied Fe d orbitals that are split in energy by the trigonal bipyramidal ligand field. This donor bonding strongly depends on the Fe-O-C angle θ (and to some extent on the Fe-O-CC dihedral angle). For Θ = 180°, both Tyr π orbitals in Figure 5 would be involved in π donor bonding with the Fe d orbitals; as θ decreases one (or both depending on dihedral angle) Tyr π level becomes pseudo-σ interacting with the do orbitals on the Fe, the CT spectrum (which reflects donor/acceptor orbital overlap) acquires more bands, and the Tyr-Fe bond strength increases. For the equatorial Tyr, θ = 133° while for the axial Tyr, θ = 148 . This large difference in structure affects the strengths of the Tyr-Fe bonds where from Table Π the axial Tyr has less donor interaction with the Fe and a weaker bond. From geometry optimization using DFT, the axial Tyr-Fe bond angle decreases and becomes equivalent to the equatorial Tyr in its donor interaction with the Fe . This indicates that the large θ of the axial Tyr-Fe bond is imposed on the active site by the protein and labilizes the axial Tyr for substitution by catecholate substrate. Alternatively, the equatorial Tyr-Fe bond involves a strong donor interaction which is trans to the second oxygen of the catecholate and can contribute to the latter's weak bonding interaction with the Fe which is thought to activate the substrate for attack by 0 . Thus the active site in 3,4-PCD has evolved to use the same residue, Tyr, in two essential but different roles by controlling the coordination geometry of the Tyr-Fe bonds. ορ

ip

x

ra

e

m

ffl

ffl

ffl

m

ra

2

111

Substrate coordination, Figure 2 right, produces new very low energy charge transfer transitions associated with the catecholate-Fe bonds (19, 20). Studies are now underway to use the above methodology to probe the nature of this bond in the intradiol dioxygenases and evaluate the mechanism of substrate activation by this non-heme ferric active site. ffl



337

Figure 5. Molecular orbitals representing the π (out-of-plane) and π (inplane) interactions of both the axial (ax) and equatorial (eq) Tyr. (Reprinted from reference 11. Copyright 2002 American Chemical Society.) ορ

ίρ

Table Π. Bond Order and Charges for DFT and INDO/S-CI Calculations

tyr (ax) tyr(eq)

Charge Mulliken (ADF ") -0.8 -0.5

Charge Mulliken (INDO) -0.7 -0.6

Charge Hirschfeld (ADF ") -0.5 -0.4

Fe-Frag Overlap (INDO) 0.4 0.5

a

Amsterdam density functional package (17,18).

b

Fragment-Fragment overlap, i.e. Fe with tyr (ax) and Fe with tyr (eq).

b

SOURCE: Reprinted from reference 11. Copyright 2002 American Chemical Society.


338

Acknowledgement


This research was supported by NIH grant GM40392 (E.I.S.). F.N. thanks the Deutsche Forschungsgemeinshaft for a post-doctoral fellowship. M.I.D. thanks the Evelyn Laing McBain Fund for a doctoral fellowship. The studies in PCD were done in collaboration with professors John D. Lipscomb and Allen M. Orville.

References

1.

2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Solomon, Ε. I.; Brunold, T.C.;Davis, M. I.; Kemsley, J. N.; Lee, S. K.; Lehnert, N.; Neese, F.; Skulan, A. J.; Yang, Y.-S.; Zhou, J. Chem. Rev. 2000, 100, 235-349. Lee, S. Y.; Heller, E. J. J. Chem. Phys. 1979, 71, 4777-4788. Tannor, D. J.; Heller, E. J. J. Chem. Phys. 1982, 77, 202-218. Solomon, Ε. I.; Hanson, M. A. In Inorganic Electronic Structure and Spectroscopy Vol. 2; Solomon, Ε. I., Lever, A. B. P., Eds.; John Wiley: New York, 1999, pp 1-129. Neese, F.; Solomon, Ε. I. Inorg. Chem. 1999, 38, 1847-1865. Abragam, Α.; Bleaney, B. Electron Paramagnetic Resonance of Transition Ions; Dover: New York, 1986. Lee, S.-K.; George, S. D.; Antholine, W. E.; Hedman, B.; Hodgson, K. O.; Solomon, Ε. I. J. Am. Chem. Soc. 2002, 124, 6180-6193. Deaton, J.C.;Gebhard, M. S.; Solomon, Ε. I. Inorg. Chem. 1989, 28, 877-889. Neese, F.; Solomon, Ε. I. Inorg. Chem. 1998, 37, 6568-6582. McWeeny, R. Methods of Molecular Quantum Mechanics; Academic Press: London, 1992. Davis, M. I.; Orville, A. M.; Neese, F.; Zaleski, J. M.; Lipscomb, J. D.; Solomon, Ε. I. J. Am. Chem. Soc. 2002, 124, 602-614. Ohlendorf, D. H.; Weber, P.C.;Lipscomb, J. D. J. Mol. Bio. 1987, 195, 225-227. Ohlendorf, D. H.; Orville, A. M.; Lipscomb, J. D. J. Mol. Biol. 1994, 244, 586-608. Orville, A. M.; Lipscomb, J. D.; Ohlendorf, D. H. Biochemistry 1997, 36, 10052-10066. Fujisawa, H.; Hayaishi, O. J. Biol. Chem. 1968, 243, 2673-2681. Solomon, Ε. I.; Lowery, M. D. Science 1993, 259, 1575-1581.


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17. Baerends, Ε. J.; Ellis, D. E.; Ros, P. Chem. Phys. 1973, 2, 41-51. 18. te Velde, G.; Baerends, E. J. J. Comp. Phys. 1992, 99, 84-98. 19. Bull,C.;Ballou, D. P.; Otsuka, S. J. Biol. Chem. 1981, 256, 1268112686. 20. Elgren, T. E.; Orville, A. M.; Kelly, Κ. Α.; Lipscomb, J. D.; Ohlendorf, D. H.; Que, L. J. Biochemistry 1997, 36, 11504-11513.


Variable-Temperature Variable-Field Magnetic Circular Dichroism

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