Calculation of the electronic structure and spectra of model cytochrome P450 compound I

Journal of Inorganic Biochemistry 83 (2001) 309–318www.elsevier.nl / locate / jinorgbio

Calculation of the electronic structure and spectra of model cytochromeP450 compound Ia , a ,1 b*Dan Harris , Gilda Loew , Lucy Waskell

aMolecular Research Institute, 2495 Old Middlefield Way, Mountain View, CA 94043, USAbUniversity of Michigan and VA Medical Research Center, 2215 Fuller Road, Ann Arbor, MI 48105, USA

Received 31 March 2000; received in revised form 18 September 2000; accepted 25 September 2000

Abstract

The electronic structure and spectra of the oxyferryl (Fe=O) compound I P450 heme species, the transient putative active intermediateof cytochrome P450s, have been calculated employing a full protoporphyrin IX heme model representation. The principal aim of thiswork was to compare the computed spectra of this species with the observed transient spectra attributed to it. Computations were madeusing both nonlocal density functional theory (DFT) and semiempirical INDO/CI methods to characterize the electronic structure of thecompound I P450 species. Both methods resulted in a similar antiferromagnetic doublet as the ground state with a ferromagnetic quartetexcited state partner, slightly higher in energy. The INDO/ROHF/CI semiempirical method was used to calculate the spectrum of theprotoporphyrin IX P450 compound I heme species in its lowest energy antiferromagnetic doublet state at the DFT optimized geometry. Asa reference, the spectrum of the ferric resting form of the protoporphyrin IX P450 heme species was also calculated. The computed shiftsin the Soret and Q bands of compound I relative to the resting state were both in good agreement with the corresponding experimentallyobserved shifts in the transient spectra of cytochrome P450cam (Biochem. Biophys. Res. Commun. 201 (1994) 1464) andchloroperoxidase (Biochem. Biophys. Res. Commun. 94 (1980) 1123) both ascribed to their common compound I heme site. Thisconsistency provides additional, independent support for the assignment of compound I as the origin of the reported observed transientspectra. 2001 Elsevier Science B.V. All rights reserved.

Keywords: P450 Compound I; Transient species spectra; Density functional theory; Configuration interaction; Multiconfigurational ground states

1. Introduction difficult to isolate and characterize. Recent radical clockexperiments have raised the question whether the oxyferryl

The cytochrome P450s (CYP450s) are a ubiquitous species is indeed the exclusive active species making itsfamily of metabolizing heme proteins with more than 470 spectral identification in CYP450s even more importantcloned and sequenced members from a variety of species [1].including plants, fungi, bacteria, insects, and mammals. All Many of the inferences about the structure and prop-CYP450s have long been believed to have a common erties of a CYP450 compound I (P450-I) species havefive-step enzymatic cycle that transforms them from an been made indirectly from comparisons with the per-inactive ferric heme species to a putative catalytically oxidase compound I species, a closely related metabolizingactive oxyferryl Fe=O species, called compound I. This heme protein family. Particularly useful in this regard areputative active species is consistent with the known comparisons with chloroperoxidase with a proximal cys-monooxygenase function of the CYP450 superfamily of teine ligand in common with the CYP450s. Thus, the hemeenzymes that involves transfer of such an active oxygen sites of the chloroperoxidase compound I (CPO-I) speciesatom to substrates. However, the rapidity of both the and the CYP450 compound I (P450-I) species are identi-formation of this species in the CYP450s and its oxidation cal, consisting of a protoporphyrin IX heme unit with theof substrates render this putative transient active form heme iron axially coordinated to an oxygen atom and a

sulfur atom of a proximal cysteine residue. The greaterstability of (CPO-I) and its close resemblance to that of

*Corresponding author. Tel.: 11-650-210-0310, ext. 105; fax: 11-650-(P450-I) make experimental studies of (CPO-I) particu-210-0318.larly useful to study for additional insight into the nature ofE-mail address: [email protected] (D. Harris).

1Deceased January 5, 2001. the corresponding CYP450 compound I species.

0162-0134/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0162-0134( 00 )00177-X

310 D. Harris et al. / Journal of Inorganic Biochemistry 83 (2001) 309 –318

Over two decades ago, the existence of a Fe=O ferryl While the rapid scan spectroscopic studies with chloro-18oxygen bond was established in O chloroperoxidase peroxidase and P450cam provide evidence for the exist-

compound I by Hager et al. [2]. An EXAFS study in 1983 ence of a common transient species, there is no indepen-of compound I of horseradish peroxidase (HRP-I) estab- dent evidence in these studies that the species that led tolished for the first time the presence of a short Fe–O bond these spectra is indeed the suggested putative oxyferryl

˚of 1.6 A, while a second set of Fe-ligand atom distances form. It is, in fact, very difficult to obtain such directwere deduced to be those of the four Fe-pyrrole N bonds at evidence from experimental studies alone since the tran-

˚|2.0 A [3]. A recent cryogenic crystal structure reported sient nature of these species has thus far precludedby Schlicting and co-workers is quite consistent with the independent experimental structural verification. The tech-previous EXAFS studies [4]. From the energies of the niques of computational chemistry are ideally suited to fill

¨X-ray absorption edge [5,6], and Mossbauer spectra [7,8], this gap by providing a one to one correspondence betweenit was also concluded that the oxidation state of the iron in a calculated spectrum and the species from which itHRP-I was Fe(IV). ESR [9] and ENDOR [10] spec- originates. The finding of such a correspondence wouldtroscopy characterized the porphyrin radical cation nature provide additional independent evidence for the speciesof HRP-I. Oxygen atom ligation was demonstrated in the postulated to be the origin of the spectrum.18O-chloroperoxidase compound I [2]. The vis–UV spec- To examine this possibility, in the work reported heretral of compound I in a variety of peroxidases [11–16] the electronic structure and spectra of the full protopor-provided additional features of peroxidase compound I phyrin IX oxyferryl methyl mercaptide complex model forspecies. The nature of the antiferromagnetic doublet elec- P450-I have been calculated. Specifically, spin unrestrictedtronic state in CPO compound I was reported in 1984 nonlocal density functional theory (DFT) has been used for[17,18]. the first time to calculate the optimized geometry and

The spectrum of CPO-I has been obtained by Palcic et electronic structure of this heme model for P450-I in itsal. as a transient species formed during the reaction of lowest energy quartet and doublet electronic states. Theperacetic acid with the ferric resting form of chloro- visible–UV spectrum of the doublet ground state was thenperoxidase [11] that leads to the direct formation of calculated using the semiempirical INDO/ROHF/CI meth-compound I. Under the conditions of this experiment, od at the DFT optimized geometry. As a reference, thecompound I was stable for about 30 ms before it under- spectrum of the ferric resting form of the protoporphyrinwent a one-electron reduction resulting in a species known IX heme species was also calculated. This is the sameas compound II. A subsequent one-electron reduction species used as reference in the experimental studies. Theresulted in the ferric state of enzyme. The accumulation of INDO/ROHF/CI procedure used here has, in the past,repetitive spectra during this reaction led to the acquisition proven capable of reliably calculating spectra shifts be-at 22 ms in the stopped flow spectrophotometer of the tween different CYP P450 species [21,22]. The calculatedspectrum of a species assigned to compound I. In this shifts in the visible–UV spectra of the ferric resting formexperiment, the initial Soret peak decreased and shifted to and the compound I were then compared to the same shifts367 nm with isosbestic points at 368 and 444 nm with experimentally observed between the ferric resting formrespect to the ferric resting form. In addition to this and the transient spectra of CYP450cam and chloro-

¨transient electronic spectrum, both Mossbauer resonance peroxidase reported to be due to compound I. Finally, theand electron spin resonance spectra have been reported for Soret maximum of CPO-I is known to be 33 nm bluecompound I of chloroperoxidase [7,18]. shifted with respect to HRP-I. Thus, given the near identity

More recently, Egawa et al. [19] studied the spectra of of the CPO-I and P450cam I spectra, we also compute theintermediates in a reaction of m-chloroperbenzoic acid spectra of a model of HRP compound I and compare it towith the ferric form of cytochrome P450cam. This reaction the computed spectra of compound I in P450cam. Theseshould also lead directly to the formation of a compound I comparisons provided an additional, independent assess-species. In that work, the formation of at least four ment for this assumed origin of the experimentally ob-transient intermediates was noted using rapid scan absorp- served transient spectra for a more realistic model thantion spectrophotometry. The first intermediate, noted 10 ms previously reported [23–26].after mixing, gave a spectrum indistinguishable from theputative compound I spectrum reported for chloroperoxid-ase. The B(Soret) and Q(a,b) band absorption maxima of 2. Methodsthis species, at 367 and 694 nm, were very similar to thosereported for chloroperoxidase at 367 and 688 nm. In 2.1. Model used and choice of initial geometriesaddition, these investigators reported the Fe=O stretchingvibration of this putative compound I intermediate [20]. The model of the heme unit of CYP450 compound IThe spectra of compound I in two peroxidases, horseradish used in all calculations reported here is shown in Fig. 1. Itperoxidase (HRP) and CPO have been measured and is an iron-protoporphyrin IX with a methyl mercaptide

2compared [11]. (SCH3 ) axial ligand for the heme iron. An initial

D. Harris et al. / Journal of Inorganic Biochemistry 83 (2001) 309 –318 311

Fig. 1. Model of CYP450 compound I used in this work. A methyl mercapto oxyferryl protoporphyrin IX representation. The structure shown is the initialgeometry chosen for the study.

protoporphyrin IX compound I geometry CYP450 was functional (Becke 1999 exchange functional1Perdew-generated using the CYP450cam ferric low spin state Wang-’91 gradient-corrected correlation functionals). Acrystal structure [27] and adding a ferryl oxygen adduct to double zeta valence polarization DGauss basis (DZVP) set

˚the heme iron at 1.65 A in accord with EXAFS results for employing atomic centered Gaussian basis functions opti-peroxidase compound I. The cysteine linkage was trun- mized for use with DFT was used [29,30] in all calcula-cated at the C carbon and converted to a methyl group. tions. While hybrid functionals give more accurate ex-b

Since CPO and CYP450s share a common heme prosthetic change energy contributions, the use of the BPW91group this species represents the heme complex in both functional, within the context of DGauss, gives accurateCYP450-I and CPO-I. This initial heme structure was then structures of transition metal complexes and makes geome-used as input for unconstrained DFT geometry optimi- try optimization computationally feasible with the use of azation. fitting basis. Convergence criteria and detailed description

The model of the heme unit of the ferric resting form of of methods were as described in detail in previous publi-CYP450 used was an iron-protoporphyrin IX with a cations [22,24].

2methyl mercaptide (SCH3 ) and water as axial ligands forthe heme iron. 2.3. INDO calculations

The geometry used was taken directly from theCYP450cam ferric low spin state crystal structure [27]. The electronic spectra of the lowest energy electron state

The model of HRP-I used to compute the spectra of of the P450-1 heme species and of the low spin groundHRP-I was similarly prepared from initial crystal structure state of the ferric resting form were calculated using thegeometries and geometry optimized using density func- INDO/ROHF/CI method [31,32]. This method has beentional theory in a full protoporphyrin IX representation as successfully used in our past investigations to calculate thedescribed in another publication [28]. electronic structure and spectra of many model heme

proteins, including myoglobin, peroxidases, catalases, and2.2. Density functional theory (DFT) calculations the CYP450s in a variety of spin, oxidation, and ligand

bound states [33–36].Density functional calculations were performed using Computation of INDO spectra requires at least a ROHF-

DGauss version 4.0 in conjunction with the UNICHEM CI-singles calculation. The spectra were calculated frominterface (DGauss 4.0 /Unichem Oxford Molecular, single excitations CI employing the Tamm-Dancoff ap-Beaverton, Oregon). Given the goal of obtaining reason- proximation, in which each excited state is approximatedable model geometries for the full heme compound I as a linear combination of single excitations, i.e., therepresentation of compound I, all DFT calculations were energy of the system with a promoted electron is approxi-computed using a BPW91 nonlocal exchange correlation mated by the energy for such a promotion with orbital


energies given by the solution for the ground state of the for a given spectral line. Each of the calculated frequenciesmolecule. Such an approximation tends to overestimate the has intensity contributions from many single excitations.actual transition energy, because it does not includerelaxation of the orbital into which an electron is excited.To include this effect electron correlation for the excited 3. Results and discussionstates must be added to the spectral calculation. Such alevel of calculation is presently unfeasible for systems the 3.1. DFT results for oxyferryl protoporphyrin IXsize of the heme prosthetic group. The level of calculation CYP450 model compound Ireported here is, however, adequate for the prediction ofspectral shifts among heme species differing in ligand, Table 1 presents the main structural features of theoxidation state, spin-state, and net charge. lowest doublet and quartet electronic states of the oxyferryl

The excitations included in the CI to obtain spectra were protoporphyrin IX (heme) CYP450 compound I species.chosen in accord with the Rumer procedure. For the These results were obtained by unconstrained nonlocalantiferromagnetic doublet compound I state, the CI was DFT geometry optimization performed separately for thedone for doublet states arising from three unpaired spins. two spin states. As seen from this table, the optimized

˚To calculate spectra, it is necessary to specify the number Fe=O distances are 1.66 and 1.67 A for the doublet andof filled and unfilled orbitals to be explored in the CI. All quartet states. The corresponding bond orders of 1.31 andthe calculated spectra in this study included excitations 1.35 for the two states are also very similar. The calculated

˚from the filled 1e 2e , 3e , 1a , 2a , 3a porphine / Fe=O bond length of 1.67 A compares well with theg, g g 1u 2u 2u˚porphyrin p orbitals into the unfilled 4e (p*) and 2b (p*) observed values in EXAFS studies of HRP-I of 1.64 Ag 1u

˚and iron d-orbitals. The excitation space also included all [3,6,37] and of 1.67 A in CCP I (cytochrome C peroxidase)orbitals containing S and O character admixed with [38], as well as the recent report of a cryogenic crystalorbitals of porphine /porphyrin p and p* character. Where- structure of P450cam with an Fe=O bond length of ca.

˚ver possible in this paper, we designate the symmetry 1.65 A [4].labels used for the orbitals on the heme/porphine as In contrast to the similarity in the Fe=O distances in theidealized for D symmetry analogous to the porphine core quartet and doublet state, there is a more pronounced4h

of the porphyrin without substituents, although the difference in the other axial ligand distance, the Fe–S˚ ˚protoporphyrin IX definitely deviates from this idealized bond. It is 2.37 A in the doublet state and 2.49 A in the

symmetry. quartet state. The computed Fe–S bond length of bothThe roots of the CI-matrix were used to calculate the states are in good agreement with the Fe–S bond length in

˚frequencies of the electronic transitions. The electric-di- the cryogenic P450cam compound I structure is 2.360.2 Apole matrix elements were used to calculate oscillator [4] (I. Schlicting, personal communication).strengths for each transition corresponding to the intensity In both states, there is some variation in the 4-Fe–Nof the transition. Together these quantities, a list of pyrrole distances. This small variation and the additionalfrequencies, oscillator strengths, and contributions to the substituents present on the porphyrin ring leads to aintensity from the x, y, and z components of the transition reduction in symmetry from the D idealized symmetry of4h

moment comprise the calculated spectrum of each species.These results can therefore be presented as ‘d function’spectral lines (stick figures) in a frequency/ intensity plot inwhich the position on the abscissa axis indicates the Table 1

afrequency and the height represents its intensity, as well as DFT optimized geometries of oxyferryl methylmercaptide protopor-phyrin IX CYP450 compound I model in doublet and quartet spin statesin tabular form.

In addition, a simple model spectrum was generated Geometric Protoporphyrin IX (heme)quantityfrom the sums of uniform width Gaussians convoluted Oxyferryl Bond order Oxyferryl Bond order

with the ‘d function’ spectral lines. In this procedure, a (S51/2) (Mayer) (S53/2) (Mayer)Gaussian was convoluted with each spectral line and the

Fe–S 2.37 0.47 2.49 0.32results summed to generate a total spectral contour. The Fe–O1 1.66 1.31 1.67 1.35amplitude at the Gaussian center was determined by the Fe–N1 1.99 0.34 2.03 0.34

Fe–N2 2.02 0.34 2.03 0.33oscillator strength of that underlying spectral line. TheFe–N3 2.07 0.32 2.02 0.33primary data are the spectral line intensities and fre-Fe–N4 2.03 0.34 2.01 0.34quencies but the convoluted band contours are generated inC2–S 1.82 0.48 1.82 0.51

order to make the most consistent comparisons with the N2–Fe–O 93 90experimental spectra. ,S–Fe–O1 165 172

,C–S1–Fe 111 111Assignment of each of these spectral lines in terms of,C–S–Fe–N4 163 167the type of excitations contributing to them was also made.

aThere is no case where a single excitation is responsible Distances in angstroms and angles in degrees.


the porphine model. This reduction in symmetry is more very similar despite the difference in spin in the two statespronounced for the protoporphyrin IX doublet state com- and indicate comparable electrostatic environments aboutpared to the quartet. the compound I forms in both spin states.

Table 2 presents the calculated difference in energy The finding of an antiferromagnetic doublet ground statebetween the optimized lowest doublet and quartet elec- is consistent with the direct experimental evidence for atronic states of the oxyferryl protoporphyrin IX P450 doublet state from the ESR of CPO-I [18]. In this study,compound I obtained from the DFT calculations at this total spin represented by the observed ESR signal waslevel of theory. As seen in this table, a doublet is the found to correspond to a maximum of one unpaired spin.ground state with the quartet state 2.5 kcal /mol higher in The authors conclude that the broad band with a g value ofenergy. Although a small difference, it is larger than that 1.73 could only result from large coupling from an electroncalculated for comparable HRP-I heme models where the in an a orbital.2u

two states were found to be essentially degenerate both in The finding in this study of some unpaired spin densityour own work and in the prior work of Kuromichi and in a porphyrin p orbital of CPO compound I is alsoco-workers [28,39]. The ordering of these virtually degen- consistent with experimental deductions from EPR anderate spin states is highly dependent on the functional Raman studies of both peroxidases as well as modelemployed in DFT calculations with the quartet being the systems. Raman studies of compound I of chloroperoxid-ground state in calculations employing hybrid functionals ase and horseradish peroxidase have been both beenincorporating additional Hartree-Fock exchange. interpreted in terms of porphyrin p cation radical character

Also reported in Table 2 is the unpaired spin density [40,41]. The assignment of which of the two nearlydistribution. These results clearly shows that these two degenerate porphyrin p orbitals, a (p) or a (p), is2u 1u

states are the antiferromagnetic and ferromagnetic coupled involved in compound I has been a vexing problem [42].partners, respectively, of the Fe=O (S51)–porphyrin p Given the near degeneracy of the a (p) and a (p)2u 1u

(S51/2) cation radical. In both states there are approxi- orbitals, their relative energies may be dictated by manymately two unpaired parallel spins on the Fe=O center and factors including the nature and position of porphyrina single unpaired spin delocalized in the a porphyrin peripheral substituents, the nature of the axial ligands to2u

orbital and the S p orbital. This delocalization is due to the the iron as well as more subtle factors such as environment1 1* *mixing of these orbitals in the highest occupied molecular and temperature [41]. Our own studies of the a and a2u 1u

orbital (HOMO). The major difference in the two partners, compound I forms indicate them to be quite similar asis that in the antiferromagnetic doublet state, this spin is regards the excess spin density on the Fe and O atomsantiparallel to that on the Fe=O center and in the ferromag- [28]. DFT calculations of an octaethyl porphyrin com-

1*netic quartet state it is parallel. pound I species with an axial mercapto ligand gives an a1u

Also given in Table 2 are the calculated net charges on ground state, consistent with the general pattern expectedthe iron, the oxygen and sulfur ligands and on the for b-substituted radical cations [28]. Larger changes inporphyrin ring atoms for these doublet and quartet states. electronic state structural descriptions, however, occur as aThese charges are derived from the calculated the molecu- result of doming rather than by changes in the symmetry oflar electrostatic potential (MEP). These net charges are the porphyrin p radical cation [43,44]. No evidence to date

indicates a functional difference in the two compound Iforms. The calculated values of the quadrupole splitting of21.2 mm/s for the antiferromagnetic doublet ground state

2of the A radical cation found here is in good agreementTable 2 2u

DFT relative energies, unpaired spin distributions of doublet and quartet with the experimental value for CPO-I of 1.02 mm/s.electronic states of oxyferryl methyl mercaptide protoporphyrin IX The finding of an antiferromagnetic doublet ground statemodels of CYP450 compound I using the more realistic protoporphyrin IX methyl mer-

Doublet Quartet capto model of P450-I /CPO-I is consistent with ourRelative energy(kcal /mol): 0.0 2.5 previous studies of a simpler porphine model for thisq(MEP-derived charges) species using the same level of theory [24]. It is alsoFe 2.50 2.38 consistent with several other previous DFT based calcula-O 20.58 20.60 tions for a simple porphine model of P450-I /CPO-IS 20.39 20.34

[23,25]. All DFT studies to date have found approximatelyCH3 0.21 0.10two unpaired parallel spins on the Fe=O moiety but differA (porphyrin atoms) 21.74 21.542u

in the extent to which the remaining radical center is aUnpaired spin density

porphyrin p radical cation and the extent of unpaired spinon atomic centersdensity on the S. While it has been conjectured that greaterFe 0.88 1.03

O 0.89 1.01 radical character is significantly basis set dependent, theS 20.53 0.62 use of B3LYP or B3PW91 hybrid functionals with theA (porphyrin atoms) 20.24 0.342u basis set used in this study also recovers a ground state


with more extensive S radical character (D. Harris, un-published results).

3.2. INDO calculations of protoporphyrin IX CYP450compound I at DFT optimized geometries

INDO/ROHF/CI calculations at the DFT geometrywere made for the oxyferryl protoporphyrin IX-compoundI species that is a heme model for both CYP450cam-I andCPO-I. These calculations were made to serve as a basisfor the calculation of the spectrum of the antiferromagneticdoublet ground state in order to compare it with ex-perimental spectra attributed to CPO-I and CYP450cam-I.

Shown in Table 3, are the relative energies and unpairedspin distributions of the lowest energy doublet and quartetstates. Consistent with the DFT results, the ground stateobtained from these INDO/ROHF/CI procedures is an Fig. 2. The different configurations significantly contributing to the

doublet ground state description of compound in the DFT optimizedantiferromagnetic doublet with a low lying ferromagneticgeometry. Only configurations with normalized coefficients greater thanquartet partner only 1.8 kcal /mol higher in energy, com-0.02 are shown. The p–d and p–d orbitals correspond to two sets ofi 'parable to the 2.5 kcal /mol obtained using the DFTorthogonal p orbitals. These are formed by overlap of S–p ,Fe–d , Opx xz xmethods. The consistency of these results provides addi- orbitals (to form the p–d p) and S–p ,Fe–d , Op atomic orbitals (toi y yz y

tional support for the previously proven ability of the form the p–d molecular orbitals.'

INDO Hamiltonian to accurately predict relative energiesof heme electronic states of different spin states [45].

The INDO/ROHF/CI calculations also reveal that the tional doublet ground state found was used as the referenceantiferromagnetically coupled doublet is a multiconfigura- configuration and the calculations were performed usingtional state. As shown in Fig. 2 a number of configurations the same INDO/ROHF/CI method that led to this mul-make a significant contribution to the doublet ground state. ticonfigurational description of the doublet ground state.As seen in this figure, the dominant configurations (64%) The calculated spectra in the vis–UV region of this groundinvolves single electron occupation of the two Fe=O state of the protoporphyrin IX model of CYP450 com-orbitals and the a porphyrin p orbital. However, there is pound I at the DFT optimized geometry are shown in Figs.2u

some contribution (26%) from configurations in which the 3–5.third single occupied orbital is the a porphyrin p orbital. Fig. 3 shows the two components of the split Soret band1u

The net result is a state with predominant a radical cation as well as the Q band and all the underlying transitions2u

character with a small admixture of a radical cation. contributing to each. The solid line was obtained by a1u

uniform width Gaussian convolution of each of the calcu-3.3. INDO-ROHF-CI spectra of oxyferryl methyl lated transition components.mercaptide protoporphyrin IX compound I Fig. 4 shows the computed spectra of the Soret region

2for the A radical cation protoporphyrin IX model of2u

For these calculations, the lowest energy multiconfigura- CYP450 compound I and for comparison, the corre-

Table 3Calculated relative energies and unpaired spin distributions of anti-ferromagnetic doublet and ferromagnetic quartet states using INDO/ROHF/CI

Antiferromagnetic Ferromagneticdoublet quartet

Relative energies (kcal /mol) 0 1.8

Unpaired spin distributionsAtom center

aFe 0.97 1.47 (1.47)aO 0.32 0.49 (0.47)aS 20.19 0.57 (0.59)aPorphyrin atoms 20.29 0.46 (0.49)

a Included in parentheses are the ROHF unpaired spin distribution for Fig. 3. Calculated electronic excitations in the Soret and Q band regionsthe quartet state for comparison with the results from ROHF/CI calcula- of the ground state doublet of CYP450 compound I together with thetions. convoluted bands obtained from them.


Fig. 5. (A) Calculated convoluted spectra in the visible Q(a,b) bandregion of the ground state doublet of CYP450 compound I and the lowspin resting state. The resting state Q band components have been labeledas QR and compound I Q bands as QI. (B) The correspondingexperimental spectra from Ref. [19].

spectrum (B) and the spectrum of the transient species (A)in the Soret region reported by Egawa and co-workersduring the reaction of m-chloroperbenzoic acid with cyto-chrome CYP450cam. While the calculated positions of thepeaks in both spectra differ from the absolute experimentalvalues by about 50–60 nm, the relative shifts and overallband contour are comparable. Specifically, the main peakcomputed for the Soret band in compound I is 60 nm blueshifted with respect to the peak in the computed resting

Fig. 4. (A) Calculated convoluted Soret region spectra of the ground state state spectra. This calculated shift in the Soret band regiondoublet of CYP450 compound I and the low spin resting form. (B) The between compound I and the resting state spectra comparescorresponding experimental spectra from Ref. [19].

well with the experimentally observed differences in thesespectra.

sponding computed spectrum of the low spin ferric resting Fig. 5A shows the calculated Q band contributions inform of the protoporphyrin IX model of P450. The two the heme compound I model spectrum and for comparisoncalculated peaks in the compound I species have been the calculated Q band contributions in the low spin restingidentified as components of a split Soret by virtue of the state.dominant porphyrin p→p* character in the underlying These results shows the calculated compound I Q bandstransitions. are red-shifted with respect to those observed for the

These calculated spectra in Fig. 4A can be compared resting state. Specifically, the low ferric resting state Qwith the observed spectra shown in Fig. 4B. This ex- bands at 600 nm are red shifted to 690 nm in the modelperimental spectra (B) shows the low spin resting state P450 compound I. The bands labeled as the Q band


components in the computed spectrum have been identified pound I of chloroperoxidase (CPO-I). The B(Soret) andby assigning the transitions comprising the band. In Q(a,b) band absorption maxima of the transientparticular, the computed compound I (at 690 nm) and CYP450cam species, at 367 and 694 nm, were very similarresting state bands (at 600 nm) so labeled were mixtures of to those reported for chloroperoxidase at 367 and 688 nm.3a (p)→4e (p*) and a (p)→4e (p*) excitations. The Thus, the calculated spectra identify both transient species2u g 1u g

computed bands appearing in the region near 540 nm have as the oxyferrryl form. The greater stability of the CPO-Ias their origin Fe d–d* transitions in the resting state and species and the common heme unit shared with thein the compound I spectra. CYP450s, provide further support that this common origin

These calculated spectra can be compared with the identified by the comparison with the calculated spectra isexperimental Q band spectra shown in Fig. 5B derived correct.from transient spectra of the species reported by Egawa Finally, as shown in Fig. 6, the computed spectrum ofand co-workers during the reaction of m-chloroperbenzoic HRP-I has been reported [28] and is reproduced here. Asacid with CYP450cam. As shown by comparisons of the shown in this figure, there is a distinct 29-nm blue shift incalculated Q region spectrum in Fig. 5A and the ex- the Soret maximum of P450-I compared to that of HRP-I.perimental spectrum in Fig. 5B, the calculated red shift of This result is in good agreement with the 33-nm spectral80 nm is in close agreement with the experimental shift in shift between these two species observed experimentallythe Q band spectra of 90 nm from 580 nm in the resting [11]. The computed results taken together support thestate (B) to 690 nm in the spectra of a transient species (A) previously published assignment by Egawa and co-workersassumed to be compound I. of their transient spectrum as being due to P450cam

In summary, as shown in Figs. 4 and 5, the magnitude compound I.and direction of the calculated shifts in both the Soret andQ band region between compound I and the resting statespectra compare well with the experimentally observed 4. Conclusionsspectra by Egawa et al. [19]. This consistency providesadditional support for the identification of the transient The optimized geometry of an oxyferryl protoporphyrinspecies of unknown structure with that of CYP450 com- IX methyl mercaptide complex, a heme model of bothpound I [19]. CYP450 compound I and chloroperoxidase compound I

In addition to the good agreement between the calcu- was computed using nonlocal density functional theory.2lated and observed spectra, additional evidence for The DFT ground state was determined to be a A ground2u

CYP450 compound I as the origin of the transient spectra state doublet with antiferromagnetically (antiparallel) cou-observed in CYP450cam is provided by the its close pled electron spin distributed on the two Fe=O (S51)resemblance to the transient spectra attributed to com- orbitals, and on the a porphyrin p2Sp (S51/2) orbital.2u

Fig. 6. The computed spectra of P450-I and HRP-I in the computed Soret region. The figure indicates a shift in the excitations principally determining theSoret maximum.


The analogous quartet partner of this three-unpaired spin calculated using the INDO/ROHF/CI method and com-system was computed to be 2.5 kcal /mol higher in energy pared with experiment. The main component of the splitat its optimized geometry. Soret in the computed compound I spectra is blue shifted

INDO/ROHF/CI calculations were then performed at by 60 nm with respect to the computed resting state Soretthe doublet and quartet DFT optimized geometries. Consis- transition. The Q(ab) band spectra calculated for por-tency between these two methods is important because the phyrin CYP450 compound I models were found to beINDO method was used to calculate the spectrum of the about 80 nm red shifted with respect to the computedprotoporphyrin IX CYP450 compound I species. resting state Q bands. The computed spectral shifts are in

Similar to the DFT results, an antiferromagnetic doublet very good agreement with those observed in the reportedground state was found with its ferromagnetic quartet state spectra of transient species in the reaction of m-chloro-partner about 1.8 kcal /mol higher in energy comparable to perbenzoic acid with cytochrome P450cam and in thethe difference of 2.5 kcal /mol from the DFT results. These reaction of peracetic acid with chloroperoxidase both ofresults provide additional support for the previous proven which that lead to the direct formation of a compound Iability of the INDO Hamiltonian to accurately predict species with the identical heme site. Thus, the calculatedrelative energies of heme electronic states of different spin spectra identify both transient species as the oxyferrylstates. form. The greater stability of the CPO-I species and the

Additional insight into the nature of the two states was common heme unit shared with the CYP450s provideobtained from the explicit CI calculations made using the further support for the identification of CYP450cam-I asINDO method. From these results, the lowest energy the origin of the observed transient spectrum. Finally,quartet state was found to be a predominantly a single while the electronic state descriptions and functionalconfiguration state. By contrast, the lowest energy anti- measures [48] of P450cam-I and HRP-I [11] are notferromagnetic doublet state, as shown in Fig. 2, has radically different, the INDO/ROHF/CI study indicates asignificant contributions from a number of configurations. proximal ligand effect on excitation spectra in agreementThe most important configurations (64%) have a single with experiment [28].electron occupancy in the two Fe=O orbitals and in the a2u

porphyrin-S p orbital. There is, however, some contribu-tion (26%) from configurations in which the singly oc- 5. Abbreviationscupied porphyrin orbital is the a porphyrin p orbital.1u

1 1* *Thus, the doublet ground state is neither pure a or a in BPW91 Becke’s 1988 nonlocal exchange12u 1u

nature. This complexity might confound deductions about Perdew-Wang (1991) gradient-cor-the nature of compound I in CYP450 based on Raman rected correlation functionalmarker bands. What may in fact be measured is the relative B3LYP Becke’s three-parameter hybrid ex-

1 1* *importance of the a versus a ‘character’ in the change-correlation functional with2u 1u

ground state of these systems. This relative weight is likely Lee, Yang, Parr. nonlocal correlationto be a function of porphyrin geometry and substituents. CPO chloroperoxidase

The unpaired spin on the S and a atoms of the CPO-I chloroperoxidase compound I2u

porphyrin ring is the result of the mixing of their atomic DFT density functional theoryorbitals in the same HOMO. In previous calculations, we DZVP valence double-zeta basis set plus onehave demonstrated that this same mixing in the ferrous set of polarization functions on allcarbonyl CYP450 heme complex is the origin of the split atomsSoret and the red shift of one component to P450cam that ENDOR electron nuclear double resonanceis the signature of this family of enzymes [46]. In addition, spectroscopythis characteristic is consistent with the observed ESR ESR electron spin resonance spectroscopy

¨spectra and Mossbauer spectra of CPO-I [17,18]. EXAFS extended X-ray absorption fine struc-In both states, the ferryl oxygen has significant radical ture spectroscopy

character, indicating oxidation of substrates occurs via HRP horseradish peroxidaseradical-mechanisms. As shown in early studies in our HRP-I horseradish peroxidase compound Ilaboratory [47] the radical nature of the ferryl oxygen atom INDO/ROHF/CI Intermediate neglect of differentialfound here is consistent with an H-abstraction mechanism overlap (INDO), restricted open shellfor hydroxylation and with a two-step epoxidation mecha- Hartree-Fock (ROHF), configurationnism involving asymmetric addition of the oxygen atom to interaction (CI)one carbon of a substrate double bond to form a tetrahedral P450cam P450 isozyme from Pseudomonasintermediate. putida

The spectra of the low spin ferric resting form of P450(CYP450) a family of heme proteins havingCYP450 and of the antiferromagnetic multiconfigurational monooxygenase catalytic functiondoublet ground state of the compound I species was P450-I P450 compound I


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Documents

Calculation of the electronic structure and spectra of model cytochrome P450 compound I