Assignment of the spectra of protein radicals in cytochrome c

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J. Phys. Chem. 1993,97, 831-836

831

Assignment of the Spectra of Protein Radicals in Cytochrome c Peroxidase M. Krauss’ Center of Advanced Research in Biotechnology, National Institute of Standards and Technology, Gaithersburg, Maryland 20899

D.R. Gamer Department of Physiology and Biophysics, Mt. Sinai Medical Center, New York.New York 10029 Received: August 27, 1992; In Final Form: October 30, 1992

In oxidized cytochrome c peroxidase a peak a t 570 nm has been attributed to an intermediate tryptophan free radical of indeterminate protonation state. Ab initio calculations of the spectra of the neutral and cationic free radicals of the indole side chain of tryptophan are used to assign the absorption spectra to the neutral radical. Earlier assignment attempts were confused by the large blue shift in water of the in vacuo transition. The relevant calculated transitions of both the neutral and cationic indole radicals, in fact, shift substantially to the blue in aqueous solution. The theoretical cation spectra do not agree with the experimental spectra and suggest a tautomeric form is observed. Calculations of the valence singlet and triplet excited states of benzene and triplet states of indole are also presented to support the ab initio calculations of the indole radical. Spin densities are calculated for both neutral and cation radicals. The small spin density on nitrogen found for the cation is more in agreement with experimental ENDOR data. The visible and ENDOR data suggest different states of protonation and cannot be reconciled to the electronic properties of the ground-state radical tautomers.

1. htroduction The oxidation of cytochromec peroxidase (CcP) with hydrogen peroxide yields an intermediate, called compound I or ES, in which an oxidation equivalent is reversibly stored as an amino acid radica1.l-2 Recent experiments have identified T r p 191 as the radical site34 but the electronic characteristics of the radical are still to be determined, although assignmentto a radical cation is favored.1*6An absorption peak at about 570 nm has been assigned to this radical int~rmediate.~ An enhancement of the shoulder between 580 and 640 nm was observed by Mauro et al. and is assumed here to be the same tran~ition.~ Assigning either the neutral or cation free radical has not been possible from a simple comparison with transient absorption spectra of indoleor tryptophan-containingmolecules.s-10 Kinetic and pH dependence analyses have concluded that the neutral radical in water has an absorption peak near 510 nm, while the cation has one near 580 nm. Ho et al.7 note that the protein spectra resemble a red-shifted version of the neutral radical spectra in solution. The aqueous environment is distinct from the protein and the experimental protocols are very different. Therefore, the identification of the various species would be aided by a theoretical analysis of the radical spectroscopy and electronic structure relating the aqueous spectra to the in vacuo excitation energies. Valence excitation spectra can be calculated with accuracy only from a multireferenceconfigurationinteraction calculation, since the electronic description of the excited states is not well represented by a single configuration and it must be balanced with the ground state. First-order configuration interaction (FOCI) calculations are the simplest multireferencecalculation that can provide an accurate representation of the excited states. All possiblecouplingsamong the active orbitals span the electronic configurationspace, providing a qualitatively correct description of the excited states similar to the Hartrec-Fock (HF) description of the ground state. Extra valence orbitals are included in the active space to provide antibonding character to account for the correlation involved in breaking of bonds associated with the excited states. Correlationdue to degeneracy is considered, while the dynamical correlation between electron pairs is assumed to be comparable between the states under considerationand could therefore be neglected. The valence orbitals of the excited states 0022-3654/58/2097-083 1$04.00/0

of the radicals should span the same spaceas the dominantorbitals for the ground radical state. Rydberg or diffuse electronic states are not relevant for the spectroscopic regions considered here. Single excitations into the virtual space from these reference configurationsrelax the originally chosen molecular orbital basis by allowing for all orbital polarizations in each state. The molecular orbital basis can then be improved by iterating with the natural orbitals (INO) obtained from the CI wave function. This procedure has been shown to be accurate for analyzing the valence spectra of the SS bond in both neutral and anionic mo1ecules.l’ The I N 0 procedure was shown to produce appropriate valence orbitals even when the initial H F orbitals were poor for the excited states. FOCI calculationsare presented here for the neutral and cationic radicals of indole. The calculated excitation energy of the valence triplet states of indole can be compared with experimental absorption spcctra.9J0 The well-known valence singlet and triplet states in benzene’*were also calculated to support the excitation energies presented for the indole radicals. The influence of the media on the spectroscopic transitions was estimated from the reaction field theory but including only the lowest order dipole moment term in both classical13and quantum calc~lations.l~-’~ The T r p 191 in CCP is hydrogen-bonded to Asp235, which is also hydrogen bonded by its other carboxyl oxygen to the proximal iron-heme ligand His-175.2v3 However, the electronic structure of the iron coordination spherecan not be reliably determined for the purposes of modeling environmentaleffects on T r p 191 and our efforts in this direction have concentrated solely on the hydrogen-bonded Asp235. This paper is primarily concerned with the visible spectra of the indole radicals. However, the spectral evidence mostly used to date regarding the identity of the residue radicals is from the hyperfine splittings obtained from ENDOR spectroscopy6 with proton- and deuterium-substituted spectra. The lack of observation of spin density on the nitrogen atom6 suggests that the radical speciesof T r p 191is a u radical cation. Since the electronic properties of the radicals will lead to different assignments regarding the state of protonation, the spin densities will also be reported. The conflicting conclusions cannot easily be reconciled but the more complete description of the radical electronic Q 1993 American Chemical Society

Krauss and Garmer

832 The Journal of Physical Chemistry, Vol. 97, No. 4, 1993

1.391

Indole

1

1.409

11.421

)

-N/1.430 1.419

Indole x radical

1.415

1.430

-N/1.330 1.424

Indole radical cation Figure 1. Optimized bond distances of the indole radicals and the atomic numbering scheme. Hydrogen atoms are deleted. They are bound to N1, C2, C3, C4, CS. C6, and C7 in indole and the cation with no H atom bound to N1 in the neutral T radical.

properties presented here underlines the difficulty in assigning the species involved in this oxidative intermediate. 2. Method

Self-consistent-field and FOCI calculations were done with the GAMESS system of ~ 0 d e s . lAll ~ results were obtained with a double tlevel basis set with K-shell orbitals replaced by compact effective core potentials (CEP).I* Since we are interested in the absorption spectra, the geometries were obtained by restricted open-shell Hartree-Fock (ROHF) gradient optimization of the radical ground states of indole, the triplet state of indole, and the ground singlet and triplet states of benzene. The complete geometries are provided in tables in the supplementary material. For the symmetrical singlet ground state of benzene the calculated CC bond distance is 1.41 1 A compared with the experimental value of 1.396 A. The indole radical bond distances are given in Figure 1 with the atomic numbering of the atoms indicated. All FOCI calculations were started with appropriate ROHF orbitals. The ground-state doublet ROHF orbitals were used to generate a singles CI wave function. Natural orbitals from this wave function started the I N 0 procedure with an FOCI active space described below by using the lowest state natural orbitals at each iteration." The singlet- and triplet-state natural orbital iterations were started. by using triplet-ground-state ROHF orbitals. Occasionally state energies drift upward during the I N 0 procedure and therefore excitation energies are reported as the difference between the lowest energies obtained for the ground and excited states during the natural orbital iterations. The valence states are defined as those without significant contribution from diffuse or Rydberg orbitals. This is a particular consideration for the neutral benzene molecule where the ionization potential of 9.25 eV19J0suggests that Rydberg states

or significant diffuse contributions to the excited wave functions are expected above 5.5 eV. The valence excitation from the eIg orbital to the excited ezu orbital results in three singlet states, lBzurlBlu,and IElU,and three triplet states, 3Blu,3Elu,and 3B2u. However, we see from a catalogue of benzene state energies2I that only the first excited singlet and the two lowest triplet states are likely to be substantially free of diffuse components. Both components of the doubly degenerate valence excited orbital, ezur are included in the active space of the FOCI. In order to reduce the size of the CI matrix and the associated computational requirements, only five doubly occupied orbitals were included in the FOCI active space. The triplet states are calculated with the same number of active orbitals used for the singlets but are coupled to a triplet spin. Feasible CI calculations for indole also require that the active valence space is restricted by freezing the most tightly bound doubly occupied orbitals. Since there is no orbital degeneracy only one valence orbital was included in an active space with seven doubly and one singly occupied orbital. This extra valence orbital is antibonding within the r space of the benzene ring contributing to the correlation for a single r bond that is broken upon excitation into this orbital. For the radicals two natural orbital iterations decreased the state energies and converged to about 0.001 hartreea20 Within the limited CI that is possible, the active valence space of the triplet state of indole has five doubly occupied and two singly occupied orbitals, both with spin a,and one valence orbital. An additional valence orbital is required because the excited states relevant to the triplet absorption spectra are sufficiently high in energy that correlation of another r bond is also required. In solution the solute dipole moment for state i, pi,will orient the solvent dipoles to produce a reaction field, Ri= pia-32f(D), at the center of a sphere of radius a that represents the solute molecule, where D is the static dielectric constant due to the permanent dipole of the solvent. The reaction field perturbation in the quantum hamiltonian is H I = -pRi. The quantum calculation assumes this perturbation is present for all states and cannot distinguish different coupling time scales. However, the classical calculation can select terms that represent different couplings to different states. Assuming that the solvent dipoles do not rearrange during the electronic transition and the reaction field is frozen but the induced dipoles of the solvent do relax, the classical excitation energy can be expressed as13

+

+

wheref(D) = (D - 1)/(2D 1) and g(n) = (n2 - 1)/(2n2 1 ) with n the refractive index. The scalar product is taken between the ground-state dipole moment and the differenceof the excitedand ground-state dipole moments. The critical parameter is the effective reaction field radius. By fitting the absorption shift for the neutral indole22in comparable FOCI calculati~ns,~~ we obtain a value of 6.5 bohr. For the radicals it is assumed that the radius is smaller because hydration binding is probably stronger for both neutral and cationic radicals and a value of 6.0 bohr was chosen. The reaction field for the cation is calculated with respect to the center of charge of the ground state. The absorption shift of the radicals will be calculated from both the quantum reaction field FOCI energies and the classical formula and related to the observed spectrum. The dipole moments of the radicals required in eq 1 are calculated from the FOCI wave functions in the solvent reaction field self-consistentlydetermined from the ground-state dipole m ~ m e n t s . ' ~The ? ~ moments ~ are presented in Table I1 for all states with and without the reaction field showing that these differences can be substantial in many cases due to the large polarizability of radicals, T systems, and excited states. Various explicit models of the protein environment were included in the FOCI calculations as described below. The

Spectra of Protein Radicals

The Journal of Physical Chemistry, Vol. 97, No. 4, 1993 833

TABLE I: Benzene Excitation Energies excitation energy, eV/nm final state, neutral 'BIv

3B~, 3E~u

calcd 4.96 eV 250 nm

exptl 3.90 eVo 253 nm

3.83 324 4.87 255

3.950 314 4.750 26 1

Reference 12.

accuracy of the representations are restricted by affordability considerations. Only Asp235 in various protonation states was considered becauwe this is clearly the most strongly interacting residue but is also the only oneof the heme-His-Asp(-Trp)network for which the electronicstructure can now be reliably calculated. 3. Results Trial calculations of the valence states of benzene and the triplet states of indole validate the FOCI method for calculating excited-state energies. Experimental and FOCI values of state energy differencesfor benzene are given in Table I. Four natural orbital iterations converged the excitation energy for the IB2" state to 4.96 eV, which compares favorably to the experimental value of 4.90 eV.I2 Convergence behavior of the natural orbitals provides a rough test of the sufficiency of thechosen active space. The natural orbital occupancy of the most importantvirtual orbital not included in the active space is substantially lower than the degenerate pair of valence orbitals for both the ground and first excited states. For the ground state the open valence orbitals each have an occupancy of 0.1023, while the largest virtual occupancy is 0.0232. In the excited singlet state the occupancies are 0.5647 and 0.0693, respectively. The theoretical excitation energy for the jBlu state represents the adiabatic excitation energy, since the triplet geometry rather than the singlet ground state is used. The threshold behavior for the triplet states is complicated by the distortion of the lowest energy conformation to a lower ~ymmetry.2~ Theexcitationenergy between the triplet states is 0.2 eV larger than the peakseparations of the experimental electron impact spectrum but the excitation energy at the triplet geometry cannot easily be discerned from the threshold behavior of the electron impact spectrum. The restricted active space, however, could lead to errors of this order, which is still comparable to the more extensive CI calculations in the literature.21 The excitation energies, oscillator strengths, and dipoles of the valence triplet states of indole are summarized in Table 11. The corresponding triplet spectra have been assigned by flash photolysis of tryptophan at 465 nmS9Two transitions are predicted is this region with their wavelength in agreement with the experiment. However, again we note that two transitions were not expected and this could be of interest for substituted indoles where the wavelength separation could be larger. These transitions are to states with dipole moments very comparable to the ground state and there is essentially noshift from thein vacuospectra. Another transition is also predicted around 390 nm in water and a bump in the triplet curve is observed near this wavelength. The ground state of the neutral indole radical is A type with A" symmetry as seen in Table 11. The first, third, and fourth excited states are also A type but the second excited state is u type with A' symmetry. The latter is the upper state for the spectroscopicallyrelevant transition and is particularly interesting because the dipole moment is much reduced from the ground state and there is no longer a lone pair on the nitrogen because of the nu T transition. In this transition charge is transferred from the nitrogen lone pair to alternating carbon atoms in both the pyrrole and benzene rings. This decreasesthe dipole moment by equalizing the charge around the periphery of the fused rings

-

TABLE II: Indole Excitation Energies: In Vacuo and Reaction Field final state (nm;f; state 1 neutral 2.97

shift, c shift, q 4.01

d

3

5

4 ~~

958.5 0.007 3.68 999.0 986.4 4.72

580.9 0.002 0.30 530.9 537.1 1.02

400.4 0.039 5.07 421.2 413.0 6.05

311.3 0.008 4.16 318.3 315.8 5.21

11664 1.77 957.2 95 1.8 0.0001 2.73

502.2 0.073 1.86 469.4 473.0 0.058 3.21

318.9 0.007 2.02 301.7 301.6 0.009 2.93

288.4 0.185 1.94 279.0 280.5 0.156 3.45

773.5 0.002 2.22 778.9 2.80

475.3 0.0010 1.85 475.7 2.37

458.8 O.OOO5 1.41 456.7 1.93

384.8 0.013 5.37 394.3 5.89

cation 0.0002 3.12

shift, c shift, q 5.00

triplet 1.81

shift, q 2.24

The transition wavelength is given in nanometers,fis the oscillator strength, and fi is the dipole for each state in debyes.

as well as reducing the lone-pair dipole. This is reflected in the electrostatic potentials shown in Figures 2 and 3. Figure 2 represents the potential for the ground A radical, and Figure 3 for the u radical draws attention to the small values of the electrostatic potential in the lone-pair region in the excited state. A similar reduction in the dipole moment of the u radical is calculated for the pyrrole radical alone, suggestingthat the fused rings play a small role in equalizing charge on the ring. Because there is a hydrogen atom bonded to the nitrogen, there is no comparable n A transition in the cation but the dipole moment of the ground state is also larger than for the excited states leading to a noticeableblue solvent shift for the spectroscopically relevant second excited state. This transition in the cation transfers charge from the benzene ring to the two carbon atoms and the nitrogen in the pyrrole ring segment. The FOCI dipole moment of the ground and excited states coupled to the reaction field include the polarization of the solute to the extent that the basis set allows. The reaction field increases the dipole moment substantially, but the differencein the ground and excited dipole moments remains about the same in most cases. The solvent shifts are enhanced by the increase in the solute dipole moment in the ground state. The solvent-shifted spectra are also summarized in Table I1 for the quantum and classical reaction field calculations. The oscillator strengths were also calculated for the cation in vacuo and with the reaction field with little change. The effect of local hydrogen bonding in the protein environment was considered by inserting a point charge model of the Asp235 anion into the hamiltonian for an FOCI run on the radical cation. Only the effective charge of the oxygen is included at the H-bonded distance. This field perturbation induces a blue shift in the important transition from 505.2 nm in vacuo to 493.5 nm. Thus, both the aqueous continuum and the protein Asp anion preferentially stabilize the ground state of the cation and cause noticeable blue shifts. Addition of an all-electron model of Asp235 makes the FOCI calculations impractical but excited states can occasionally be examined at low resolution when the HF calculations converge to a state other than the ground state from poor initial guess orbitals. A calculationintended to model the indole radical cation in contact with the Asp235 anion with an 0-N distance of 2.5 A instead converged to an excited state having the electron hole localized on the Asp235 oxygens. This state is estimated to be lower in energy than the important spectral regions in vacuo but

-

Krauss and Garmer

834 The Journal of Physical Chemisfry, Vol. 97, No. 4, 1993

/" \

'\

./

I

\

,

\

J , (/ -I \

i

/

( / \

-

-

\

Figure 2. Electrostatic potential for the ground neutral r radical: (a, top) in the plane of the molecule; (b, middle) 1.5 A above the plane of the molecule; (c, bottom) perpendicular to the plane of the molecule along the axis indicated in (a).

could be less well solvated than the ground state because the cation-anion dipole is eliminated. In this case an excimer coupling reduces the ionic coupling. This indicates that a deprotonated Asp may add bands to the spectroscopy of the indole radical cation but a quantitative analysis requires a more complete environment and a larger CI treatment that is not possible at present. Analogous excimer couplings could occur with the heme or the histidine imidazole ring but would be weak for the overlap in theresting stateand include toomany electronstovary geometry at the FOCI level at this time. Spin populations were determined for the radical ground and tautomeric states at the ROHF level and are reported in Table 111. The H F configuration dominates the ground state of both the neutral and cation radicals according to the configuration coefficients in the FOCI wave functions, so these spin populations should be qualitatively correct. The current code we are using does not calculate spin-related properties from the CI wave function but the electrostatic potentials from the ROHF and CI are nearly identical, which would not be possible if there are substantial shifts in the spin populations. In T radicals there is

Figure 3. Electrostatic potential for the excited neutral u radical; (a, top) in the plane of the molecule; (b, middle) 1.5 A above the plane of the molecule; (c, bottom) perpendicular to the plane of the molecule along the axis indicated in (a).

TABLE III: Indole Radical ROHF Spin Powlrtioss spin population (a- B ) neutral ~

atom

N1 c2 c3 c4

c5 C6

c7 C8 c9

0.77 -0.004 0.13 0.01, 0.02 0.005 0.04

-0.001 0.03

H3

cation

0.006 0.006

0.08 0.24 0.40

H2 0.02 0.09 0.77 0.04 -0.000

0.97 -0.002 0.OOO

0.04

0.OoO

0.08

0.002 0.04 -0.012

0.00, 0.015 -0.ooo

0.04 0.04

0.09

0.001

0.017

nos-orbital contribution to the Fermi contact term in the hyperfine interaction for the ROHF or the present FOCI wave function but atomic exchange polarization is expected to develop spin density at the heavy nuclei and magnetic interaction at bonded protons, leading to observable EPR and ENDOR signals. The ROHF spin populations for the ground states are qualitatively similar for the ring atoms to the values obtained for the tryptophanyl radical by Hoffman et and although the ab initio population at nitrogen in the cation is twice as large as the semiempirical

Spectra of Protein Radicals

TABLE I V R o t a Aflinities of Species Relevant to the Qoertioa of Rotolrrtion states im oxidized CCP protonating species indole radical imidazole imidazole anion methyl formate anion indole anion water

proton affinity, kJ/mol DZ/SCF DZd/SCF DZ/FOCI 1029 1018 1034 1021 1548 1460 1553 129

value it is still small. For the cation the spin density is mostly on C2 and C3 in the pyrrole ring with the density largest on C3. In addition to nitrogen only the C3 atom has any appreciable spin density in the neutral radical. The spin populations of two tautomericneutral radicals where the hydrogen shifts fromeither C2 (-H2) or C3 (-H3) to N1 are also given to show the spin density is almost totally on the C3 atom. Both tautomers are calculated in vacuo at the ROHF level to be 93 and 57 kJ higher in energy relative to the ground state for the H2 and H3 tautomers, respectively. The r tautomers are the lowest in energy but for the H2 tautomer the u tautomer is only about 48 W,higher in energy at the FOCI level. For this tautomer the spin density will undoubtedly be localized on the C2 atom. Shifting the hydrogen from carbon atoms on the benzene ring results in u radicals as the ground state with the spin population on the carbon that has lost its hydrogen atom. The presence of low-lying excited states would indicate that radical species could be unusually polarizable. In order to test this possibility, an ab initio model of the Asp235 as formic acid was inserted in the configurationrelative to the indole taken from the crystal structure but with varying N-O distances. For the indole radical the oxygen closest to the indole nitrogen was protonated to give a good interaction with the lone pair evident in the electrostaticpotential plots. Another model added a formate anion representingthe deprotonated Asp to interact stronglywith the radical cation leading to an energy minimum at an N-O distance of approximately 2.5 A. Neither of these models produced a significant shift in spin populations in the radicals, with changes only in the second decimal place. The population of the nitrogen atom in the radical cation remained at about 0.1 electron at all N-O distances. In vacuo estimates of proton affinities of the indole radicals and other relevant species are given in Table IV. These provide a means of assessing the ability of the indole radical cation to lose a proton in solution and in the protein where Asp235 is available as a proton acceptor. Values obtained from self-consistent-field (ROHF) energies are usually found to be accurate for neutral singlet systems.26 For an open-shell radical, electron correlation may be more significant, so we have obtained the radical proton affinity using both the ROHF and FOCI. Since the zero-point energies will mostly cancel for the proton transfers considered here, only the electronic contribution to the proton affinity is given. Both the DZ and DZd basis sets were used for the ROHF calculation. The DZd basis adds a d set of functions to all the heavy atoms. The FOCI calculations in both the radical neutral and cation used the same active space so the correlation contributions are comparable. 4. Mseuarion

The radical excitation energies are applied first to the assignment of the radical spectra observed in CCP. We will then return to a discussion of the transient spectra observed in pulse radiolysis of indole-containing species. The Trp- 191 residue in CCP is embedded within the protein and H-bonded to Asp-235. The indole plane is approximately parallel to the proximal His175,whichi~alsoH-bondcdtotheAsp235.~~ Inmostperoxidases the two oxidizing equivalents are both stored in the iron-heme

The Journal of Physical Chemistry, Vol. 97, No. 4, 1993 835 complex with 1 equiv as the *-cation radical of the porphyrin. When Trp-191 is replaced by a phenylanaline, the spectrum characteristic of a *-cation appc~rs,3,~ suggesting that the porphyrin is initially oxidized but the Trp radical is produced by electron transfer in CCP. The calculated cation spectra have no transitions that can be assigned to the 570-nm peak attributed to the radicaL7 The in vacuo transition is calculated at 502 nm and would shift to the blue slightly assuming a low dielectric media of the protein interior. Modeling of the Asp235 carboxylateasanelectrostaticfieldshifts thecalculatedcationspectra to the blue from 502.2 to 493.5 nm. The calculated spectrum of the radical cation is inconsistent with that spectrum attributed to a protein radical intermediate. On the other hand the neutral radical transition is calculated in vacuo at 582 nm and a modest shift from the environment either to the red or the blue would still leave it in better agreement with the experimental spectrum. The calculated transition at 400 nm is of interest because a perturbation of the optical spectra is observed in this region upon oxidation of CCP.3.27These shifts may also be attributed to Fc-heme transitions in the Soret region of the spectra but the calculated radical spectra show that these speciesalso produce spectra in unsuspected regions, and a complete catalogue of all transitions is needed and can, at present, only be obtained from calculations. Since the indole side chain is H-bonded to the Asp, proton transfer from the indole cation to the Asp is certainly possible but competitivewith a similar proton transfer from His to Asp. This competition was decided in favor of the His for the resting state involving the neutral residues and the driving potential of the Fe+3 cation.28 However, after oxidation the proton is transfered from a neutral indole radical to an Asp anion, which should increase the driving force for this proton transfer since the calculated proton affinity of the neutral radical is over 500 kJ less than that for the imidazole anion. The proton shift from the radical cation would leave the neutral radical hydrogen bonded to the neutral side chain of the Asp. A blue shift is expected because the lone-pair orbital on the indole nitrogen, which is H-bonded to the protonated Asp, loses one electron in then r transition, so the excited state does not have as strong a H-bond in the molecular plane as the ground state. However, the electronic transition shifts a large negative electrostatic potential in the plane of the nitrogen atom in the T ground state to a substantially increased potential perpendicular to the plane over both rings as seen in the electrostatic potentials for both the T and u radicals in Figures 2 and 3. Although the largest H-bond energy is for water donating a proton to nitrogen in the plane, the H-bond for water to the *-electron cloud of indole is also substantial, as suggested by the 17 kJ/mol bond energy calculated for a single water binding to ben~ene.2~ Therefore, there is an unquantifiable first shell compensating stabilization of the u radical excited state in CCP, which is ignored in the simple continuum model being used to interpret aqueous data. The simplest interpretation of the ENDOR spectra6 and, in particular, the lack of a nitrogen spectra points to an indole radical cation. However, the majority EPR spectra attributed to the residue radical have features that make it difficult to assign to a molecule such as indole with first-row a t o m ~ . ~ JThere ~ J ~ has been a strong tendency to attribute the spectra to nearby sulfurcontaining rcsidues25.30and the difficulty in modeling the observed ENDOR and EPR spectra with indole parameters suggests that another residue is involved either as part of a kinetic path or strongly spin coupled to the indole radical. The pulse radiolysis spectra of indole can be analyzed with the continuum reaction field model. The FOCI reaction field transition in water is calculated to be 537 nm and the classical reaction field value is 531 nm. The experimental spectrum attributed to the neutral indole radical has a quite broad peak

-

836 The Journal of Physical Chemistry, Vol. 97, No. 4, 1993 at about 530 nm.10 There is a shift to the blue to 510 nm for L-tryptophan and 3-methylind01e.l~ This comparison suggests that small changes in solvent accessibility, which may change preferred hydrogen bonding behavior only modestly, perturb the spectrum with the continuum model generally valid for semiquantitative estimates of shifts. The absorption spectrum also shows a bump at 410 nm and a larger peak with a maximum at about 305 nm. The calculated spectrum predicts two electronic transitions at about 417 and 317 nm but with a larger oscillator strength in the first transition. The cation valence spectra in water are calculated to shift to the blue for all transitions. Because there is a hydrogen atom bonded to the nitrogen, there is no comparable n r transition in the cation and the dipole moment of the ground state is larger than the excited-state dipole moments. The FOCI reaction field yields three transitions at about 952, 473, and 302 nm with comparable classical values as seen in Table 11. None of these correspond to the broad transition peaking near 580 nm that is ascribed to the cation from its pH dependence.* However, the cation spectra have been studied at microsecond time scales and the proton may now be binding to a carbon site.1° The lack of any correspondencewith the theoretical spectra suggests that the observed spectra are due to a tautomer or subsequent kinetic product. The cation that is produced initially probably transfers its proton from the NH bond at very short times and the tautomer is produced by a subsequent protonation reaction.

-

5. Conclusion The visible spectral assignment determines the protein radical in CCP is the neutral indole side chain of the Trp residue. This residue is H-bonded to the Asp235 in the resting state of the enzyme. The proton transfers after oxidation to the cation because the proton affinity of the Asp side chain far exceeds that for a radical cation. The relevant neutral transition in solution is substantially blue-shifted by both the classical and FOCI calculations, in good agreement with pulse radiolysis spectra attributed to the neutral indole radical. The calculated radical cation spectra are not found in the relevant region and are blueshifted away by local fields that stabilize the ground state. The calculated radical cation spectra are found to differ from assignments from pulse radiolysis experiments. This suggests that the radical cation spectra observed in pulse radiolysis experiments are not due to the cation protonated at the nitrogen but to that protonated at another site produced by chemical reactions subsequent to the initial ionization. Support for the calculations of the indole radicals is found in the accurate predictions for the triplet absorption spectra of indole and the valence states of benzene. Ab initio spin densities for the indole radicals superficially lead to a contrary conclusion and support earlier suggestions that

Krauss and Garmer the lack of spin density on the nitrogen atom points to the radical cation. However, there are sufficient problems in interpreting the EPR and ENDOR spectra that the assignment of the EPR spectra to the tryptophanyl species is in question, even though the radical population must certainly correlate with production of this radical. The present study emphasizes the apparent contradiction between the visible and ENDOR spectra.

Supplementary Material Availabk Tables of internal distances

(A) for indoleneutral radical, indole radical cation, indole triplet,

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