Electron paramagnetic resonance of the excited triplet state of metal-free and metal-substituted cytochrome c

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Electron paramagnetic resonance of the excited triplet state of metal-free and metal-substituted cytochrome c
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  Biophysical Journal Volume 68June1995 2505-2518 Electron Paramagnetic Resonance of theExcited Triplet State of Metal-Free and Metal-Substituted Cytochrome c P. J. Angiolillo and J. M. Vanderkooi Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 USA ABSTRACT The photoactivated metastable triplet states of the porphyrin (free-base, i.e., metal-free) zinc and tin derivatives of horse cytochrome c were investigated using electron paramagnetic resonance. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range 3.8-150 K are discussed in terms of porphyrin-protein interactions. The zero-field splitting parameters D for the free-base, Zn and Sn derivatives are 465 x 104, 342 x 104, and353 x 104 cm-1, respectively, and are temperature invariant over the temperature ranges studied. An El value at 4 K of 73 x 1 04 cm-1 was obtained for Zncytochrome c, larger than any previously found for Zn porphyrin derivatives of hemeproteins, showing that the heme site of cytochrome c imposes an asymmetric field. Though theElvalue for Zn cytochrome c is large, the geometry of the site appears quite constrained, as indicated by a spectral line shape showing a single species. Intersystem crossing occurred predominantly to the T2) zero-field spin sublevel. EPR line shapechanges with respect to temperature of Zn cyt care interpreted in terms of vibroniccoupling, and a maximum Jahn-Teller crystal-fieldsplitting of approximately 180 cm-' is obtained. Sn cytochrome c in comparison with the Zn protein exhibits a photoactivated triplet line shape that is lesswell resolved in the X-Y region. The El value is approximately 60 x 104 cm-1 at 4 K; its value rapidly tends toward zero with increasing temperature, fromwhich a value for the Jahn-Teller crystal-field splitting of -40 cm-1 is estimated. In contrast to those for the metal cyto- chromes, theEl value for thefree-base derivative was essentially zero at all temperatures studied. This finding is discussed asa consequence of an excited-state tautomerization process that occurs even at 4 K. INTRODUCTION Cytochromes c, single polypeptide chain heme-containing proteins, are key  players in biological electron transportprocesses. All organisms utilizing photosynthesis or mito-chondrial oxidative phosphorylation have c-typecyto- chromes (Pettigrew and Moore, 1987). In mammalian sys- tems, it is the penultimate electron transfer protein, shuttling electrons between its reductase (ubiquinol-cyt c reductase) and cytc oxidase. Cytochromes c are unusual in that not only is the prosthetic heme moiety attached by covalent coordi- nation to the protein through axialligation on thecentral metal but the heme side groups also have covalent bonds to the protein.In the case of mammalian systems these bonds arise through condensationof the two vinylattachments with cysteine 14 and 17. This biological modificationallows theiron in cytochromes c to be replaced without removal of the porphyrin, unlike in other hemeproteins such as myoglobin, hemoglobin, and b-type cytochromes, in which there is no covalent attachment to the protein other than through axialligation. Receivedforpublication 16 November 1994 and infinalfonn 6 March 1995. Address reprintrequests to Dr. JaneVanderkooi, Department of Biochem- istry and Biophysics,University ofPennsylvania, Philadelphia, Pennsyl- vania 19104-6089. Tel.: 215-898-8783; FAX: 215-573-2042; E-mail: Vanderkooi@mscf.med.upenn.edu. Abbreviationsused: cyt c, cytochrome c; H cyt c: metal freecyt c; Zn cyt c: Fe replaced by Zn; Sn cyt c: Fe replaced by Sn; EPR, electronpara- magnetic resonance, ZFS, zero-field splitting; ISC, intersystem crossing; ODMR, opticallydetected magnetic resonance;JT, Jahn-Teller; PP, pro- toporphyrin IX dimethyl ester; MP, mesoporphyrin IX dimethyl ester. C 1995 by the Biophysical Society 0006-3495/95/06/2505/14  2.00 Removing the iron or replacing it with other metals, such as zinc and tin, renders the porphyrin of cytcfluo- rescent and phosphorescent, with emission lifetimes spanningapproximately 6 orders of magnitude (10 ns to 20 ms, respectively, for fluorescence andphosphores- cence). This modification has made it amenable to studies using high-resolution optical spectroscopy (Angiolillo et al., 1982; Logovinsky et al., 1993; Leeson et al., 1994; Friedrich et al., 1994) in attempts to uncover chromophore-protein interactions as manifested inthe electronic and vibronic structure. The further employ- ment of these closed-shell metal derivatives of hemepro- teins as photoactivatable models for energy and electron transfer with its natural and cross-species redox partners has provided a powerfulapproachand continues to grow (Elias et al., 1988; Zang andMaki, 1990; Wallin et al., 1991; Casimiro et al., 1993;Horie et al., 1984; vander Est, 1993; Zhou and Kostic, 1993a,b; Meier et al., 1994). In particular,the excited triplet state of thesederivatives has been implicated in some of the proposed excited-statetransfer mechanisms. Despite the interest in theproperties and reactions of ex- cited states ofporphyrin proteins, information outliningthe photophysics of the excited states, in particularthe meta- stable triplet state, has been lacking. Furthermore,advantagehas not been taken of the information that mightbegleanedabout porphyrin-protein interactions from looking specifi- cally at the triplet state. Zn cyt c and Sn cyt c, like thefree-base porphyrin de- rivative of cyt c (H cyt c), are diamagnetic in the ground state (S = 0). With visible wavelength excitation into the first excitedsinglet manifoldandsubsequent intersystem crossing 2505  Biophysical Journal (ISC), a metastable triplet state is formed. Because the triplet state has a net spin of unity (S = 1), it is amenable to study by electron paramagnetic resonance (EPR) and optically de- tected magnetic resonance (ODMR). In this paper we look at the photoactivated triplet state of the free-base zinc(II) and tin(IV) derivatives of cyt c (horse), from the standpoint of assessing local geometry and the spin dynamics, as manifested in the line-shape and zero field split- ting (ZFS) parameters. Also, through the temperature de- pendencies of the ZFSs, the validity of the vibronic coupling scheme, as it applies to proteins, is investigated. PORPHYRIN TRIPLET PHOTOPHYSICS EPR line shape The magnetic resonanceof the lowest triplet excited state in porphyrins is that of a correlated (S = 1) spin system (Welt- ner, 1983). The spin Hamiltonian is governed mainly by the Zeeman interaction and the dipolar spin-spin interaction of the two electrons in the triplet molecular orbital. The nuclear hyperfine couplings are rarely, if ever, seen in randomly ori- ented triplets in external field because of the large anisotropy. Within the molecular axis system, the total spin Hamiltonian describing the Zeeman interaction and the interactionbe- tween the two spins is HT = 3eH - g   S   S * D   S,  1) where H is the applied magnetic field, S is the total spin, g is the g-value tensor, and D is the ZFS tensor. For most organic triplets, theg-tensor can be replaced with the value for the free electron, ge 2.0023, as the orbital angular momentum is essentially quenched (Schlicter, 1991). In a molecular axis systemchosen such that thezero-fieldtensor is diagonal, the Hamiltonian becomes HT = ge,(3eH   S - XS2 - yS2   ZSt (2) where X, Y, and Z are the eigenvalues of the spin-spin in- teraction matrix and Si  i = x, y, z) are the total spin angular momenta projections along the principal axes in zero mag- netic field. Inasmuch as the ZFS tensor is traceless, i.e., X + Y + Z = 0, the Hamiltonian can be recast by using two independent parameters, D and E, giving thefamiliar phe- nomenological spin Hamiltonian HT   ge3eH*S+D(S -j3S2) + E(S2-S)  3) The ZFS parameter D is a  measure of the electron spatial distribution of the triplet molecular orbital and E is a  meas- ure of distortion from tetragonal symmetry. The line shape for randomly oriented triplets was previ- ously described (Kottis and Lefebvre,1963, 1964; Wasser- man et al., 1964). Foreach case, where the applied magnetic field is aligned with a principal molecular axis, three tran-sitions are possible among the triplet sublevels (Fig. 1). Inthe strong-field limit,the triplet spin eigenfunctions are quan- tized along theaxis of the applied field and are adequately B BlIz 1+1> 10>  - 11> 1000 2000 3000 B (G) 4000 5000 FIGURE 1 Energy diagram forthe triplet-state spin components of the lowest triplet state of a metalloporphyrin as a function of applied magnetic field for one of thethree canonical orientations (H || Z). X-band frequency (-9 GHz) assumed. The exploded view to the left shows the hypothetical splitting of the triplet manifold in zero applied magnetic field in a rhombic environment. The assumptions are D > 0, E > 0. given by 1+1)= 1I1a2), 0) = 1/ 2)1/2 l aI3,2) + Pla2))9 1-1)= P3112), where M = 1, 0, -1 are the Z components of the spin angular momentum along theaxis of quantization and a) and 13) are the one electron spin eigenstates. In zero applied field, the eigenfunctions have their spinquantizedalong the principal axes, which usually correspond to axes of symmetry.For porphyrins, as in other planar aro- matic T-ij* systems, thezaxis is taken asthe out-of-plane axis with the xand y axes lying in plane normally along the axes of symmetry. The zero-field spin states, which diago- nalizethe ZFS tensor, are related to thestrong-field eigen- functions through the followingtransformation (Hameka and Oosterhoff,1958): Tx) =-(2- 1/2 + 1) - I-1) Ty) =i(2-1/2)   + 1) + I -1)), ITZ)= l0). Thus, the eigenfunctions T), TY), and Tz) have energiesX, Y, and Z, respectively, with the following conventional ordering X > Y > Z (van der Waals et al., 1979). Historically,the mostprominent feature seen in the triplet EPR spectrum is analmost isotropic transition at g 4, which corresponds to the pseudo AM = 2 transition + 1) <-> -1)  Fig. 1). Transitions in the AM = 1 region, 2506 Volume 68June1995  EPR of TripletState Porphyrins 0)   + 1) and 0) <-- -1), are symmetrically separated around ge = 2.0023. The anisotropy of the ZFSs, in general, leads to six observable lines or turningpoints in the first derivative spectrum in the ge = 2 region, Z1, Y, Xl, Xh, Yh, and Zh, going fromlow field to high field (Fig. 1). Assuming that D is positive, which is the usual assumption for flat planar aromatics, the 0)   +1) transition has components at field positions displaced from the field position for a free electron  hv/ge,f3) by -D, +(D - 3E)/2, and +(D + 3E)/2. Likewise, the 0) 1-> -1) transition has lines at field positionsdisplaced from ge by + D, -(D - 3E)/2, and -(D + 3E)/2, respectively. Thus, from the resulting  powder spectrum, the ZFS parameters are readily extract- able and are AHz = 21 D 1, AHy = D + E 1, and AHx = D - E I, where AH1  i = x,y, z) are the separations of the pair of transitions(in Gauss). Dynamic Jahn-Teller effect in a  crystal-field For square planar central metal porphyrins, the lowest triplet should be spatially degenerate and in the D4h point group would have a representation of 3E.. The special character- istics of porphyrin-excited triplet states are reviewed by van der Waals et al. (1979). Because a porphyrin with C4 or S4 symmetry is JT unstable, it is subject to symmetry-relieving interactionsresulting in two energy equivalent triplet states. JT instability alone is not enough to relieve the energy de- generacy of the triplet state, but interactions with axial li- gands or porphyrin substituents or interaction with environ- mental asymmetries may stabilize one state with respect to the other by anenergyof 6JT. The resulting  powder pattern EPR spectrum, when 8w» > kBT, will show astatic distortion with a nonzero E I, with a splitting between the Y and X transitions (AHxy) equal to 3 E   However, at temperatures that are comparable with or exceed the JT splitting energy, the X and Y transitionswill merge, with complete coales- cence occurring when both states are equally populated and when the exchange frequency, vjr, is greater than thesepa-ration of the X and Ytransitions, i.e., when vp» > gegfeAHxy/h (Carrington and McLachlan, 1967). This temperature depen-dence of the X and Y transitions has beentermed the dynamic Jahn-Teller effect. One theory to account forthe dynamic JT effect involves coupling to one of the in-plane porphyrin vibrational modes, giving riseto two vibronic triplets separated in energy by SJT (de Groot et al., 1969; van der Waals et al., 1979). The popu- lations of the vibronic triplet states are governed by theBoltz- mann distribution lawand thein-planeanisotropy,as meas- ured by the ZFS parameter E will be temperature dependent. For square planar porphyrinsofD4h symmetry, coupling to either b1g or b2g vibrational modes has the effect of merelychanging the sign of E, with E unchanged. The distortions are equivalent to an interchange of the x and y molecular axes. The ramification is that, as kBT >> 6j, the E value shouldapproach zero and a coalescenceof the Y and X transitions should occur. Thus, thein-planeanisotropy,as measuredby the E ZFS parameter in Eq. 4, reduces to E = Etanh(2kjT)  4) where Eo is the value of E when kBT >> 6JT MATERIALS AND METHODS Horse-heart cyt c (Type III) was purchased from Sigma Chemical Co.  St. Louis, MO). The free-base and metallo derivatives of cyt c were prepared bymethods previously described (Vanderkooi et al., 1976; Vanderkooiand Erecinska, 1975). Protoporphyrin IX dimethyl ester (PP) and mesoporphyrinIX dimethyl ester (MP) were purchased from Porphyrin Products (Logan, UT) or Mid-Century (Posen,IL) andused without further purification. All solvents were spectral grade and degassed before use. Instrumentation EPR wasperformed on a Bruker ESP300E spectrometer. Intracavity illu- mination wasperformed directly through the front louvers with fiber opticsusing a 150-W Kuda quartz-halogen illuminator. Temperatures that ranged from 3.8 K upward were obtained by an Oxford ESR 900 continuous-flow cryostat controlled with an Oxford ITC4 temperature controller. Frequency wasmeasured with a Hewlett-Packard 5350B microwave frequency counter. All experiments were conducted at microwave powers that ensured that there was no saturation of resonances. The spectra are presented as the resultants of light minus dark spectra; this procedure removed the contri- bution of a paramagnetic contamination presentin the cavity. In most cases theirradiation procedure caused no permanent changes in the sample as monitored by absorption andemission spectra before and after intracavity irradiation. For Zn cyt c, inparticular, prolonged irradiation at the highest temperatures led to the appearance of increased free-radicalintensity, which allowed forinternal field calibration. Phosphorescence lifetimes were measured with the instrumentdescribed by Vanderkooi et al. (1987). Lifetimes were measured at 77 K in the same glassymatrices used for EPR with excitation at theSoret maximum andemission measured at the maximum of the (0-0) of the phosphorescence spectrum. Lifetimes were determined fromdecay profiles by analysis with the ASYSTANT program (Macmillan Software Co., New York, NY). RESULTS EPR spectra of Zn, Sn, and H cyt c The EPR spectra of the photoactivated triplet state ofZn, Sn, and H cyt c at 4 K are given in Fig. 2. The AM = 2 region for all threederivatives (Fig 2, left) shows absorptive,nearly isotropicsignals of line width 15 G (AHPP) andg values of 4.038, 4.038, and 4.048 for Zn, Sn, and H cyt c, respectively. The spectrum at 4 K in the AM = 1 region for Zn cyt c (Fig. 2 A, right)exhibits a typical six-line pattern char- acteristic of a randomly oriented nondegenerate triplet state with rhombicsymmetry and possessing well-defined ZFS parameters, D and E , of 342 X 10-4 and 73 X 10-4 cm'1, respectively. The spectral line shapes are narrow, showing that the geometry of the site is quite constrained and that there is effectively onlyone species. The polarization pattern fromlow fieldto high field is aaa-eee. TheSn cyt c spectrum at the same temperature (Fig. 2 B, right) also shows spin polarizationforthe Z transitions, in- dicating that ISC occurs with some degreeof spin selectivity; however, because of the less well-resolved and partially Angiolillo and Vanderkooi 2507  Biophysical Journal  0 16451680 1715 28003150350038504200 B(G) FIGURE 2X-band EPR of the photoactivated triplet state of Zn, Sn, and free-base cyt c at 4 K. Experimental conditions for AM = 1 region: (A) 500-mM Zn cyt c in 50 glycerol aqueous glass, 50-,M KCl pH 7, mi-crowave power 2 uW, modulation amplitude 20 G (100 kHz); (B) 1-mM Sn cyt c in 50 glycerol aqueous glass, 50-mM KCI pH 7, microwavepower 2 ,uW, modulation amplitude 20 G (100 kHz); (C) 1-mM free-base cyt c in 50 glycerol aqueous glass, 50-mM KCl pH 7, microwave power 2 ,uW,modulation amplitude 20 G (100kHz); AM = 21 region: (A) microwave power50 ,uW, (B) microwavepower50 ,uW, (C) microwave power 100 ,uW. For A-C the modulation amplitude is 10 G (100 kHz). a indicates an absorptive transition and e an emissive transition. thermatized high-field XY regionof the Sn cyt c triplet spectrum at 4 K, the exactnature of ISC cannot be de- termined. A D valueof 353 X 10-4 cm'1 is obtained at this temperature. In contrast to those for the tripletin Zn cyt c, the canonical X and Y transitions of the spectrum are broadened even at 4 K, indicating environmental dis- order at the porphyrin site or motional broadening of the vibronic triplet states. The E valuecan be estimated to be -60 X 10-' cm-'. The spectrum at 4 K for H cytc (Fig. 3 C, right) dem- onstrates a four-line  powder pattern with a ZFS parameter E of approximately zero. Such a case arises when there is in-plane symmetry. The polarizationpattern of the free-basederivative from low fieldto high field is aa-ee. A D valueof 465 X 10-4 cm'1 is obtained for H cyt c. For Zn cyt c the triplet EPR spectral features were inde- pendent of concentration in the range 30 ,iM to 1 mM. For reasons of sensitivity, only concentrations >100 ,uM could beused forthe Sn and free-base derivatives, but their spectratoo showed an independence of concentration. It can be con- cluded that the porphyrin, by virtue of being buried in the heme crevice of cyt c, createsa system whereby the chro- mophore can be considered to be in an infinitelydilute matrix without the complicationof chromophore-chromophore en- ergy transfer. Temperature dependence Fig. 3 shows the AM = 1 region of the triplet state of Zn cyt c, under steady-state illumination, asa function of 3000320034003600 3800 B(G) FIGURE 3 X-band EPR of the photoactivated tripletstate of Zn cyt c. (A) 4 K,  B 20K, (C) 40 K, (D) 60K, (E) 105K, (F) 130K, (G) 150 K. Experimental conditions:concentration -1 mM in 50 glycerol aqueous glass, 50-mM KCl glass. Modulationamplitude20G, modulationfrequency 100 kHz, and microwave power as follows: (A) 2 ,LW, (B) 10,uW, (C) 100 AW, (D) 10 mW, (E) 50 mW, (F) 100 mW, (G) 200 mW. AM 1=2 IT Volume 68 June 19952508 IAMI=l 12D ID+M. .l A ID-3EI Izi h Z, aa ee ae B 1.,L,A.kL r--r j wr aae aa C; c a e e Z. x Xyh Zh  EPR of Triplet State Porphyrins temperature. The ZFS parameter D is independent of tem- perature from 4 to 150 K. The Z transition linewidth forthe low-field and high-field transitions is 30 G and is also in- dependent of temperature. A temperature dependence is seen in that the spin polarization, evident at 4 K, gives way to thermal equilibrium by 60 K, as indicated by the high-field emissions becoming absorptive in nature when the -1) and + 1)spinsublevels become appreciably populated with in- creasing temperature (see Fig. 1). Consistent with this in- terpretation, the AM = 2 transition signal demonstrates an increasing intensity relative to the AM = 1 region with increasing temperature (data not shown). The line width for the Y and X transitions at 4 K are 26 and 34 G, respectively. Unlike the Z components of the field positions, which were invariant with temperature,the X and Y canonical transitions exhibit a temperature dependence, and the changes inline shape giveevidence of motional broadening from 4 to 150 K. In this temperature range, the X and Ycanonical transitions show a tendency to coalesce, and there is the emergence of transitions (shownby arrows in Fig. 3 D), indicating that there are tripletstates possessing an effective axial symme- try, i.e., E = 0. Fig. 4 (inset) shows the field positions of the canonical transitions X, Y, and Z determined from thedata presented in Fig. 3. From these data, the ZFS parameters were determined. Fig. 4 shows aplot of the nonzero E as a function of T-1, with thesolid curve representing the functional dependence of E with temperature given by Eq. 5 with a JT splitting, 6JT Of 180 cm-'. 75 70F   4 65 I C 60 60 r-455 45- 40- 0.050.1 0.15 0.2 0.25 The AM = 1 region of the photoactivated triplet state of Sn cyt c asa function of temperature is presented inFig. 5. The D ZFS parameter, as in Zn cyt c, is independent of temperatureover the range 4-60 K. At 4 K thehigh-field Y transition is absorptive,signifying that at 4 K there is not the degreeof non-Boltzmann entryinto the triplet manifold thatexists for the Zn derivative. By 60 K there is almost complete coalescence of the X and Y transitions, yielding an axially symmetric line shape (E = 0). An estimated valueof E of approximately 60 X 10-4,cm-1 was obtained from the 10- and 20-K spectra, where the X and Y transitions are visible (shown by the asterisksin Fig. 5). Because axial symmetry is achieved by 60 K, the JT splitting can be 3000 3200 34003600 3800 B(G) 1/T (1/K) FIGURE 4 E zero-field splitting parameter asa function of reciprocal absolute temperature (K-1) for Zn cyt c. 0 Experimental conditionsgiven in Fig. 2. Solid curve, a fit to 73 tanh (180/2kBT). Inset: stationary field position asa function of temperature. FIGURE 5 X-band EPR spectra of the photoactivated triplet state of Sn cyt c. (A) 4 K, (B) 10K,(C) 20 K, (D) 40 K, (E) 60 K. Experimental conditions: concentration -1 mM in 50 glycerol aqueous glass, 50-mM KCI, pH 7, modulationamplitude 20 G,modulationfrequency100 kHz, microwave power (A)2 ,W, (B) 10 ,uW, (C)500 ,uW, (D) 5 mW, (E) 20 mW. 38003600 3400 -D 3200 3000 --*-*-* -- 020 40 60 80 100 120 140 160 T (K) M (). Angiolillo and Vanderkooi 2509  OMON 0 N . , I I I
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