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Factors affecting photoinduced electron transfer in a donor—acceptor pair (DA) incorporated into bovine serum albumin
J. Photo&em.
Photobiol. A: Chem., 83 (1994) 229-236
229
Factors affecting photoinduced electron transfer in a donor-acceptor pair (D-A) incorporated into bovine serum albumin V.R. Vogel”, E.T. Rubtsovaa, G.I. Likhtenshteinb and K. Hideg” “Xnstiluteof Chemical Physics, Russian Academy of Science, 142432 Chemogolovka (Russian
Federation)
bBen-Gurion Universiry of the Negev. P.0. Box 653, 84105 Beer-Sheva (Israel) ‘Central Laborafoty of Chemistry, University of Pees, P.O. Box 99, H-7644 Pees {Hungary) (Received November 23, 1993; accepted February 22, 1994)
Abstract A donor-acceptor hybrid molecule (D-A) consisting of a 1-dimethyl-aminonaphthalene-S-sulphonate group in an excited singlet state (donor) and a nitroxide radical (acceptor) was incorporated into a hydrophobic cavity of bovine serum albumin (BSA). The kinetEcs of reversible and irreversible intramolecular electron transfer from the donor to the acceptor were monitored by luminescence and ESR techniques in the temperature range from 77 K to 300 K. The temperature dependence of the micropolarity and the intramolecular dynamics in the vicinity of the donor and the acceptor groups were monitored by fluorescence and ESR techniques, respectively. The Arrhenius dependence of the reversible ET constant (IQ was non-linear and the apparent activation energy (I&,) changes from 0 eV (at T=77-100 K) to 0.25 eV (at T=298 K). The temperature region of the E,, increase (100-240 K) was close to the temperature of the increase of relaxation shift of the donor luminescent spectra (T=lOO-240 K). On the basis of the obtained kinetic data and of the data of the micropolarity in the vicinity of the donor and the acceptor groups, values of standard Gibbs energy (AC”) change, the reorganization energy (&), the resonance integral (V,,) and the Frank-Condon factor for reversible electron transfer were estimated. The role of molecular dynamics in the vicinity of the donor and the acceptor groups is also discussed.
1. Introduction Electron transfer reactions in proteins have been investigated extensively during the 20 years [l-17]. There are several reasons why specialists in different fields pay attention to this problem. First, the elementary steps in life-giving processes cataiysed by metallo-enzymes involve electron transfer (ET) reactions. These include biological oxidation, oxidative- and photo-phosphorylation, fixation of atmospheric nitrogen, photo-oxidation of water etc. Second, ET includes important photobiological and radiobiological processes which lead to damage of proteins and other biological structures. Finally, ET is attractive and the simplest model for development of a theory of elementary steps of reactions in proteins, including enzymatic ones. In spite of the progress that has been achieved, there is not a great deal known about factors that affect electron transfer in proteins. The specific organization of protein globules leads to a number of factors, such as a mosaic of local dynamics, polarity and the capacity to delocalize electrons. On reviewing the literature it may be stated that the principal shortcoming in recent approaches to
the problem appears to be underestimation of the specific properties of proteins. At present, the wide methodological and theoretical techniques of modern chemical physics have been used for the study of ET in proteins. Nevertheless, limitations exist at al1 stages and these are the subject of active research. Some beginnings have been made in this direction by studying systems with known structure, such as donor-acceptor pairs in protein globules, hybrid molecules consisting of a photoactive luminescence chromophore in the excited state acting as a donor, and a quinone or nitroxide radical (NR) acting as an acceptor [13,18-211. The latter system gives, in particular, a unique possibility for monitoring the kinetics of ET, local dynamics and polarity [13,19-211. The NR can serve as a probe of the rigidity and local mobility of certain portions of proteins and may be used for study of the conformational dynamics of macromolecular breathing. The NR are extremely useful to characterize the microscopic polarity, e.g. an apparent dielectric constant, or to measure the donor-acceptor capacity of an
1010.6030/94/$07.00 @ 1994 Elsevier Science S.A. All rights reserved SSDI 1010-6030(94)03822-C
environment in a determined region of a biological object. On the other hand, the analysis of fluorescent and phosphorescent spectra of chromophores provides information about microviscosity and local dynamics in the vicinity of the centre studied. The main objective of the research proposed was to estimate the main parameters governing electron transfer in protein modified by a donor-acceptor pair on the basis of data on kinetics, micropolarity and local dynamics. With this aim an experimental investigation of the temperature dependence of the kinetics of photoinduced ET in donor-acceptor pairs (D-A) of the following type incorporated in a hydrophobic cavity of protein of bovine serum albumin (BSA) was carried out. D
A
CI%
In the hybrid molecule (D-A) the excited fragment of chromophore (D) serves as a donor and the nitroxide fragment (A) as an acceptor. The reversible ET rate constant was measured by the degree of fluorescence quenching of the D fragment. The same fragments of the D-A molecule allow monitoring of the micropolarity and the molecular dynamics of the medium in the vicinity of these fragments by ESR methods (the analysis of the intensity and the line shape of the spectra) and by fluorescence (the measurement of the relaxation shift of the Auorescence spectra). The irreversible ET rate constant was measured by ESR earlier [l&20] from the experiments on the photoreduction kinetics of the nitroxide group of D-A to nitro-oxylamine (D-AH) by following the decrease of intensity of the central component of the ESR spectra after irradiation.
2. Experimental
details
2.1. Materials 1-dimethyl-aminonaphthalene-.5-sulphonate covalently bound with nitroxide radical from Reanal (Hungary) and bovine serum albumin from Sigma were used without further purification. All solutions were prepared using distilled water, and ethylene glycol of analytical grade was distilled prior to use.
2.2. Sample preparation The modification of bovine serum albumin by the D-A molecules was performed in 0.1 M phosphate buffer with pH = 7.1, and the D-A binding was controlled by absorption spectroscopy. The absorption spectra were recorded by spectrophotometer Specord M40 (DDR). The addition of D-A to an aqueous solution of the protein causes a concentration-dependent blue shift in the 495 nm absorption band and a small decrease in the maximum absorbence. This shift in the absorption band is consistent with the D-A molecule experiencing a less polar solvent environment than the aqueous phase, as is expected when the D-A molecule is included in the protein hydrophobic cavity. The association constant (=3x 10’ MP’) of the D-A: protein complex was calculated using Scatchard’s method. The final protein concentration in the samples was 2 X 1K4 M, and the D-A concentration was 4 X lo-’ M. All samples were protected from light during the preparation and experiments were carried out with freshly prepared protein-DA samples dissolved in a glassy ethylene glycol-water mixture (1:l) and degassed by the freeze-pump-thaw technique to a final vacuum of less than lop5 mbar.
2.3. ESR measurements The ESR spectra were recorded on a Radiopan SE/X-2544 spectrometer (X-band). Quartz capillaries of 2 mm diameter containing 20 ~1 solution of D-A-protein complex were placed in the cavity of an ESR spectrometer equipped with a window for irradiation and a temperature unit. ESR spectra were recorded with the following instrument settings: a modulation amplitude of 0.3 mT, a microwave power of 15 dB and a sweep width of 10 mT.
2.4. Fhorescence measurements Luminescence spectra were recorded using a modified Aminco-Bowman (USA) spectrofluorimeter equipped with a thermostatted cell holder based on nitrogen gas flow temperature regulation. Quartz capillaries of 3 mm diameter containing 40 ~1 solution of D-A-protein complex were placed in a cell holder of an spectrofluorimeter. The registration of fluorescence proceeded at right angles to the excitation light. The 0.5 mm slit was used for excitation of fluorescence of the donor group of D-A when the steady state fluorescence spectra were obtained.
KR. Vogel et al. I Photoinduced ET in u D-A pair in bovine serum albumin
3. Results 3.1: Kinetics of reversible ET
The experimental dependencies of the fluorescence intensity of the sample containing the D-AH-protein complex (ID_.& and of the sample containing the D-A-protein complex (IDA) vs. temperature are presented in Fig. 1. The reversible ET in the D-A molecules was accompanied by a quenching of the fluorescence of the donor singlet state according to the following scheme: P-AI
Tf
$
I
[D*-A] k-l
k.,
(D”A3’
-%
{D+-A-],,,,
es
&
[Xl
k-2
P--AH1
1T
hv
UT,
p-AH]*
where hu is the quantum of exciting light, q is the donor fluorescent lifetime, k, and k_, are the rate constants of direct and reverse intramolecular electron transfer, k2 is the solvation rate constant and k-, is the reverse charge transfer in molecule after solvation. The DC--A- species participates in the following stages of protonation and is then reduced by an unidentified amino acid residue X and/or solution impurities. 50
1 1, rel.un.
231
According to this scheme the rate constant of reversible electron transfer may be calculated from the fluorescence intensity of D-A molecules (ID_*) and completely reduced form D-AH molecules (I-& by the equation: K, = &AH
- &4)~MH0/~f~&4H&4
(1)
where the &-’ and TVvalues are taken at room temperature. The temperature dependence of K, calculated from these data by eqn. (1) is presented in Fig. 2 and in its Arrhenius form in Fig. 2(b). 3.2. Measurements relaxation shifr
of donor fluorescence
The medium dynamic states were investigated using the method of measuring relaxation shifts of fluorescence spectra maximum VS. temperatures [22,23]. Figure 3 shows a typical plot of the fluorescence maximum (A,,‘) of the excited donor as a function of temperature. The shift in (A,,? to shorter wavelengths as the temperature decreased can be attributed to a decrease in solvent (protein media) stabilization of the emitting excited state when the rigidity of the medium increases. At room temperature in fluid D-A-protein samples, the solvent reorientation time (7,) is rapid compared with the excited state lifetime, allowing full stabilization of the more polar excited state, and this emission is from the solvent-stabilized S, excited state. However, as the rigidity of the medium increases with decreasing of temperature, solvent (protein microsurrounding media) reorientation becomes much slower than the lifetime of the S1 state, leading to a shorter wavelength emission which originates from a less stabilized excited state. 3.3. ESR measurements of molecular dynamics Figure 4 presents the temperature dependence of the rotational correlation times (7J of a spin fragment of D-A molecule incorporated in BSA. The correlation times r, in a fast motion region of the spin group were calculated from the ESR spectra using the formula 1241: 2x10s %=
A&[(h,/h_,)“Z-
l]
(2)
where AH0 is the width of the central component in Gauss, and ho and h- 1 are the intensities of the spectral components. T. K Fig. 1. The temperature dependencies of the intensity of steady state fluorescence spectra measured for samples containing a solution of the D-A protein complex, t,, {curve 1) and for the same sample after irradiation (completely photoreduced), fDmnH (curve 2).
3.4. Estimation of apparent micropolatity The value of apparent dielectric constant (E) in the microenvironment of a D-A molecule in the protein was determined experimentally from the relaxation shift of fluorescence spectra of the
232
KR. VogeI et aI. I Photoinduced ET in a D-A pair in bovine serum albumin
Fig. 3. The temperature dependence of the relaxation shift of the maximum of steady state fluorescence spectrum near excited chromophore fragments of D-A molecules in BSA.
b 1/T =
10QE -1
2.5 q
2.0 : q
1.5 :
,.,i’”
1.0 -
0.0
0.2
0.4
0.6
0.0
I.0
1.2
1.4 0.5 -
Fig. 2. (a) The temperature dependence of the intramolecular ET rate constant K, for D-A molecules incorporated into bovine serum albumin (1:l ethylene glycol-water mixture). @) The Arrhenius dependence of the intramolecular ET rate constant K. for D-A molecules incorporated into bovine serum albumin.
fluorescence label octadecilaminoeosine (ODE) [25] attached to BSA in the following manner. It is known [23] that the maximal value of the shift Ah,, = (hT_,, K- A,_,,K), where A is the wavelength of the maximum of the fluorescence spectra, is correlated with the value of e for the liquid. From the comparison of the values of AAm= for
Fig. 4. The temperature dependence of the Brownian diffusion correlation time rc calculated from the ESR spectra of nitroxide fragments of D-A molecules in BSA.
octadecilaminoeosine in a number of liquids [25] with the value of Ah,,= 28 nm for the complex of octadecilaminoeosine molecules with BSA, the conclusion can be drawn that the c1 = 11 to 13.
VR
Vogel et al. / Photoinduced ET in a D-A pair in bovine serum albumin
Another value of E = 63 has been determined from the value of the HFS constant of the ESR spectra of the nitroxide fragment of a D-A molecule attached to BSA by the method described in [24]. For comparison, the value of e for a 50% water-ethylene glycol mixture is equal to 65. Thus these data indicate that the D-A molecule is partly embedded in a hydrophobic region of the protein globule and that the process of intramolecular electron transfer proceeds on a ‘Lmicrointerface” with variable polarity.
4. Discussion
tribution form the orientational component in solvation remains open, assuming l = 2 at 77 K would lead to AG,,,‘= - 1.7 eV at 77 K. Consequently, the overall change A(AG,,,“) due to orientational solvation with the decrease of the temperature from 300 K to 77 K would equal A(AG,,,‘) = - 1.7 eV. Using these data the diagram of Gibbs energy levels for these reactions can be constructed (Fig. 5). The present estimate of Gibbs energy indicates that the value AGO for reversible direct ET equals 0 to 0.5 eV at 77 K. Now we will discuss the kinetics of reversible ET presented in Fig. 2. At low temperatures, when kT
The data mentioned above allow us to estimate the main factors affecting ET in a donor-acceptor pair D-A incorporated in cavity BSA and to discuss the role of molecular dynamics in the vicinity of the donor and the acceptor groups. 4. I. Estimation of Gibbs energy AGo At room temperature, when solvation after the process of producing photo-initiated charge pairing is rapid, the value of the Gibbs energy can be calculated by the following equation: AC&P
= ED,,+‘” - (En+,a’” + E,,*) -
c(rD + rAk)
(3)
[271. The value of e2/e1(rD+rA) was estimated to be equal to 0.025 V for r, = r, = 4 8, and E = 13 (see Section 3). Taking into account these adduced data the value of AG297K”= - 1.75 eV was determined from eqn. (3). Now we can estimate the energy of solvation of [D+-A-] using the Born equation: = e2/2[r, - ‘( 1- l/eJ + r, - ‘( 1 - UE~)]
4.2. Estimation of the resonance integral V,, For a hybrid donor-acceptor (D-X-A) molecule with a bridge group (X) the value of the resonance integral can be estimated from the formula: rIx
(6)
/
For calculation of AG,,, K the following experimental data were used: ED* = 2.4 eV, determined from the fluorescence spectra at T=77 K, E D/D+ ‘12= 0 . 7.5 eV for 1-dimethyl- aminonaphtalene in acetonitrile [26] and E,+,A1n=0.3 eV for a nitroxide radical in a 96% water-ethanol mixture
AG_P
(51
where FC denotes the Frank-Condon factor and V,,, is the resonance integral. As is seen from the experimental dependence in Fig. 2, the rate constant K. is independent of temperature up to T=lOO K.
v=v,
e2
(4)
where E, = 13 and c2= 63 (see Section 3). The estimation gives the value AG,rv”= -3.4 eV. ASsuming that this value of AG,,,* corresponds to the change of AGo due to the solvation and taking into account that at 77 K the question of a con-
233
where V, is the resonance integral at van der Waals contact of the donor (D) and the acceptor (A) fragments and ‘yi are the attenuation parameters for a given bond or atom which is a part of the bridge group. As has been shown [28], the value of the attenuation parameter yi can be derived from data on spin exchange between two paramagnetic groups or paramagnetic ions in biradicals and complexes with radical ligands. According to [28J, the values of 15 are equal to ‘yc =7, y,., =3. Applying eqn. (6) to the donor-acceptor hybrid molecule with V,=3 eV [29] and taking into account that the spin density of odd electrons in the N-O group is distributed between the N and the 0 atoms with a distribution coefficient (Ye,N = 0.5 leads to r/ D-A= (3 x0.5)/(3
X 73) = 1.5 X 10m3 eV
(7)
4.3. Estimation of Frank-Condon factor FC Using the experimental value KI = 7.2 X lo8 s and calculating the value of V,, by the empirical method, the value of FC may be estimated using
KR.
234
Vogel et al. I Photoinduced
ET in cz D-A
AG*C evl
pair
in bovine ~erw-n albumin
I X+C D--AID
4
I
_elrx
1
3
2
I
0
x
!
CD-A9
+
-=A
IX
+ca
+A->l&
elax
-1
-2
-3 Fig. 5. Diagram of the Gibbs energy levels for ET in D-A molecules amino acid residue and/or solution impurities.
eqn. (5). Then eV_l.
the value
of FC equals
l-10-’
4.4. me intermediate temperature region urul Ihe correlation with molecular medium dynamics The temperature range from T=lOO K to T=240 K is emphasized in Fig. 2 and the temperature dependence of K, is non-linear in Arrhenius’s coordinates {Fig. 2(b)). Apparently, in this intermediate region the ET is complicated by the low frequency of conformational fluctuations of protein and of solvent polarization. In accordance with ref. 11, we can consider the ET transition in this intermediate temperature region in the twodimensional free-energy surface representation of the transition state. In this model, one coordinate is an internal reactive coordinate of the solute and the second coordinate is a solvent polarization coordinate. The transition state lies at the intersection of the potential surfaces U&, q} and U,{Q, q}, and a two-dimensional free- energy surface is presented. The reaction can be described by the diffusion reaction equation, in which the solvation (or conformational) coordinate Q diffuses via Brownian motion under the influence of a potential U(Q) while the reaction of ET occurs at each Q with rate constant K(Q) {determined by the traditional theory of radiationless transition) during the diffusion. As a result the ET between the “instant energy levels” and the “effective value of AGO” of this reaction becomes a function of
in ESA at various
temperatures,
where X is an unidentified
salvation and conformational coordinates. The effects of solvation and/or conformational relaxation dynamics on the apparent ET rate constant are persistent in this intermediate temperature region. At temperatures 10&150 K, when K1 and K_, > l/T, we have the multiexponential transition. At temperatures 150-250 K we may consider the time-scale of reactions to be about the time-scale of salvation, and then iY1= l/r,. Finally, at higher temperatures equilibrium for all degrees of freedom occurs and we can use the traditional expression for the rate constant. As we can see from Section 3, the changes in the relaxation shift of the fluorescence spectrum of the naphthalene fragment of the D-A molecule (Fig. 3) takes place at T= 150 K, and on the other hand the changes of correlation time of the rotational movements 7C of the nitroxide fragment of the D-A molecule (Fig. 4) take place at T > 240 K only. As is known, the temperature of the glass transition of a 50% water-ethylene glycol mixture is also about 240 K. These changes confirm the presence of the orientational and diffusional movcmcnts of medium molecules with correlation frequencies about 10P7-lo-“’ s-’ in this temperature region. This conclusion is confirmed also by literature data on the slower molecular dynamics registered in this temperature range by ESR and phosphorescence methods using various labels and probes
KR. Vogel et al. / Photoinduced ET in a D-A pair in bovine semm albumin
incorporated [28,3O-311.
into the albumin
hydrophobic
cavity
4.5. Reversible ET in the high-temperature region: T>250 K If the reaction proceeds at temperatures close to room temperature, and kT B ho, then equilibrium probably occurs for all of degrees of freedom. Let us use the Marcus equation, assuming for more simplicity that the reaction is non-adiabatic:
v2 Kt, = (kT,Q-I)‘n
(AGO+&)’ exp
-
4E,kT
(3)
4.6. Estimation of reorganization energy E, First, we can evaluate the activation energy from consideration of the linear part of Arrhenius’s dependence (Fig. 2(b)) as E, = 0.25 eV. Assuming AGO= -1.75 eV, the value of the reorganization energy E, was determined to be about 0.8 eV. The calculation shows that the reaction proceeds in the “inverted” manner at room temperature. The value of E, for the reaction can be estimated independently by the Marcus equation, taking into account the volume of reagents [32]: E, = (l/n2 - 1/es)e2/2( l/rD + l/r* - 2/rDA
235
tron transfer at low and high tcmperaturcs, but at intermediate temperatures the mechanism of the process seems to be more complicated, owing to slow conformational and solvation processes. As a consequence, this leads to the fact that the kinetics of ET and the “effective values” of such thermodynamic parameters as AGo and E, become some complex function of temperature and depend on the character of the relaxational dynamics of medium. Further, the molecular dynamics of protein determining the conditions of solvation of primarily separated charges of [D+-A-] is the essential factor for the irreversible process of photoinduced electron transfer reaction causing the irreversible reduction of nitroxide radical in this system.
Acknowledgments
This investigation was supported in part by a grant from Russian state research programme 08.05: “Newest methods of bioengineering, subprogram engineering in Enzymology”.
References
+ [rD3+ rA3]/2rDA4)
(9) where r, and r, are the radii of the donor D and the acceptor A and rDA is the distance between D and A. The use of this equation for the system studied is very difficult, because the reaction proceeds on the interface of media characterized by various “effective” dielectric constants. So, for a rough estimate let us use the mean value: E, = {El, +E,)/2, where E,, and Ezr are calculated by eqn. (9) in media with Ed= 11 and ~~=65 respectively, The estimate gives the value E,= 0.9 eV. 4.7. Estimation of V,, from the rate constant at room temperature Using the experimental value of K, at 300 K, K, = 9.7 x 10’ s-l and the above calculated values of AGO and E,, we can estimate the resonance integral from eqn. (8) (V,,=S X lo-’ eV); nevertheless, the value of V,, at room temperature proves to be more than that at low temperatures by more than an order of magnitude. 5. Conclusions The
perature
theory of ET is able to describe the temdependence of the intramolecular elec-
1 J.J. Hopfield, Proc. Nati.Acad. Sci. USA, 71 (1974) 3640-3644. 2 3. Jortner, /. Chem. Phys., 64 (1976) 4860-4867. 3 R.R. Dogonadze, A.M. Kuznetsov and J. Ulstrup, J. Theor. BioL, 69 (1977) 239-263. D. De Vault, Q. Rev. Biophys., 13 (1980) 387-564, J.M. Nocek, N. Liang, S.A. Wallin, A.G. Mauk and B.M. Hofman, J. Am. Chum Sm., I12 (1990) 1623-1627. F. Parak, E.N. Frolov, A.A. Kononenko, R.L. Mossbauer, V.I. Goldansky and A.B. Rubin, FEBS Let& 117 (1980) 368-374. 7 SE. Peterson-Kennedy, J.L. McGourty and B.M. Hoffman, i Am. Chem. Sm., 1% (1984) 5010-5012. 8 E.G. Petrov, Physics of Electron Transfer, Naukova dumka, Kiev, 1984 (in Russian). 9 V.R. Vogel, G-1. Likhtenstein and A.I. Kotelnikov, BLofizika, 30 (1985) 394399. 10 R.A. Marcus and N. Sutin, Biochem. Biophys. Actu, 811(1985) 265322. 11 J-I. Sumi and R.A. Marcus, J. Chem. Phys., 84 (1986) 4895-4907 12 J.A. Cowan, R.K. Upmacis, D.N. Beratan, J.N. Onuchic and H.B. Gray, Ann. NYAcod. Sci., 550 (1988) 68-84. 13 G.I. Likhtenstein, Chemical Physics ofMetalloenzyme Ca~aalysis, Springer, Heidelberg, 1988. 14 G.L. McLendon, Act. Chem. Res., 21 (1988) 16&167. 15 L.H. Zang and AH. Maki, J. Am. Chem Sac., 212 (1990) 434w350. 16 M.D. Newton, Ctiem. Rev., 91 (1991) 767-792. 17 B. Cartling, .7. Chem. Phys., 95 (1991) 317-323. 18 I.M. Bystryak, G.I. Likhtenstcin, A.I. Kotclnikov, H.O. Hankovski and K. Hidcg, Zh. FL&Khim., II (1986) 27962802. 19 G.I. Likhtenstein, SM. Bystryak, A.I. Kotelnikov, xhim. Fti, 5 (1990) 697-705.
236
V.R. Vogel et al. I Photoinduced ET in a D-A pair in bovine serum albumin
20 E.T. Rubtsova, V.R. Vogel, D.B. Kbudyakov, A.I. Kotelnikov and G.I. Likhtenstein, B~jizi&z~, 38 (1993) 222-227. 21 O.V. Belonogova, G.1. Likhtenstein and V.I. Krinichnyi, Eur. 3. Biophys., in press. 22 N.G. Bakhshiev and Yu.T. Masurenko, Opfika and Spectroskopia, 28 (1970) 905-913. principles of Fluorescence Spectroscopy, New 23 J.R. Iakowicq York, Plenum, 1983. 24 L. Berliner and J. Reuben (eds.), Spin Labeling. Theory and Application, Plenum, New York, 1989. 25 S.M. Bysttyak, A.I. Kotelnikov, G.I. Likhtenstein and V.I. Nikulin, Z/z. priwnd. Spektrosk., 52 (1990) 225-230. 26 C.K. Mann and K. Barnes, Electrochemical Reactions in Nonaqueous Systems, Marcel Dekker, New York, 1970.
27 I-B. Volodarskii, LA. Grigoriev, S.A. Dikanov, V.A. Resnikov and G.I. Shukin, lmidasolin nitmxy! radicals, Nauka, Novosibirsk, 1988. 28 G.I. Likbtenstein, Biophysiml Lube&g Methods in Molecular Biology, Cambridge University Press, Cambridge, 1993. 29 V.M. Berdnikov and GA. Bogdanchikov, Z/I. I&&n. Fit., 53 (1979) 273-283. V.R. Vogel, G.I. Likhtenstein, A.A. 30 A.I. Kotelnikov, Kononenko, P.P. Noks, N.P. Grishanova and A.B. Rubin, Molekuly~muya biologiyu, 17 (1983) 846-854. 31 G-1. Likhtenstein, V.R. Bogatyrenko and A.V. Kulikov, Bioj&a, 37 (1983) 585-589. 32 Yu.1. Kbarkatz, ElektrockJzimiya 12 (1976) 592-595.
Photobiol. A: Chem., 83 (1994) 229-236
229
Factors affecting photoinduced electron transfer in a donor-acceptor pair (D-A) incorporated into bovine serum albumin V.R. Vogel”, E.T. Rubtsovaa, G.I. Likhtenshteinb and K. Hideg” “Xnstiluteof Chemical Physics, Russian Academy of Science, 142432 Chemogolovka (Russian
Federation)
bBen-Gurion Universiry of the Negev. P.0. Box 653, 84105 Beer-Sheva (Israel) ‘Central Laborafoty of Chemistry, University of Pees, P.O. Box 99, H-7644 Pees {Hungary) (Received November 23, 1993; accepted February 22, 1994)
Abstract A donor-acceptor hybrid molecule (D-A) consisting of a 1-dimethyl-aminonaphthalene-S-sulphonate group in an excited singlet state (donor) and a nitroxide radical (acceptor) was incorporated into a hydrophobic cavity of bovine serum albumin (BSA). The kinetEcs of reversible and irreversible intramolecular electron transfer from the donor to the acceptor were monitored by luminescence and ESR techniques in the temperature range from 77 K to 300 K. The temperature dependence of the micropolarity and the intramolecular dynamics in the vicinity of the donor and the acceptor groups were monitored by fluorescence and ESR techniques, respectively. The Arrhenius dependence of the reversible ET constant (IQ was non-linear and the apparent activation energy (I&,) changes from 0 eV (at T=77-100 K) to 0.25 eV (at T=298 K). The temperature region of the E,, increase (100-240 K) was close to the temperature of the increase of relaxation shift of the donor luminescent spectra (T=lOO-240 K). On the basis of the obtained kinetic data and of the data of the micropolarity in the vicinity of the donor and the acceptor groups, values of standard Gibbs energy (AC”) change, the reorganization energy (&), the resonance integral (V,,) and the Frank-Condon factor for reversible electron transfer were estimated. The role of molecular dynamics in the vicinity of the donor and the acceptor groups is also discussed.
1. Introduction Electron transfer reactions in proteins have been investigated extensively during the 20 years [l-17]. There are several reasons why specialists in different fields pay attention to this problem. First, the elementary steps in life-giving processes cataiysed by metallo-enzymes involve electron transfer (ET) reactions. These include biological oxidation, oxidative- and photo-phosphorylation, fixation of atmospheric nitrogen, photo-oxidation of water etc. Second, ET includes important photobiological and radiobiological processes which lead to damage of proteins and other biological structures. Finally, ET is attractive and the simplest model for development of a theory of elementary steps of reactions in proteins, including enzymatic ones. In spite of the progress that has been achieved, there is not a great deal known about factors that affect electron transfer in proteins. The specific organization of protein globules leads to a number of factors, such as a mosaic of local dynamics, polarity and the capacity to delocalize electrons. On reviewing the literature it may be stated that the principal shortcoming in recent approaches to
the problem appears to be underestimation of the specific properties of proteins. At present, the wide methodological and theoretical techniques of modern chemical physics have been used for the study of ET in proteins. Nevertheless, limitations exist at al1 stages and these are the subject of active research. Some beginnings have been made in this direction by studying systems with known structure, such as donor-acceptor pairs in protein globules, hybrid molecules consisting of a photoactive luminescence chromophore in the excited state acting as a donor, and a quinone or nitroxide radical (NR) acting as an acceptor [13,18-211. The latter system gives, in particular, a unique possibility for monitoring the kinetics of ET, local dynamics and polarity [13,19-211. The NR can serve as a probe of the rigidity and local mobility of certain portions of proteins and may be used for study of the conformational dynamics of macromolecular breathing. The NR are extremely useful to characterize the microscopic polarity, e.g. an apparent dielectric constant, or to measure the donor-acceptor capacity of an
1010.6030/94/$07.00 @ 1994 Elsevier Science S.A. All rights reserved SSDI 1010-6030(94)03822-C
environment in a determined region of a biological object. On the other hand, the analysis of fluorescent and phosphorescent spectra of chromophores provides information about microviscosity and local dynamics in the vicinity of the centre studied. The main objective of the research proposed was to estimate the main parameters governing electron transfer in protein modified by a donor-acceptor pair on the basis of data on kinetics, micropolarity and local dynamics. With this aim an experimental investigation of the temperature dependence of the kinetics of photoinduced ET in donor-acceptor pairs (D-A) of the following type incorporated in a hydrophobic cavity of protein of bovine serum albumin (BSA) was carried out. D
A
CI%
In the hybrid molecule (D-A) the excited fragment of chromophore (D) serves as a donor and the nitroxide fragment (A) as an acceptor. The reversible ET rate constant was measured by the degree of fluorescence quenching of the D fragment. The same fragments of the D-A molecule allow monitoring of the micropolarity and the molecular dynamics of the medium in the vicinity of these fragments by ESR methods (the analysis of the intensity and the line shape of the spectra) and by fluorescence (the measurement of the relaxation shift of the Auorescence spectra). The irreversible ET rate constant was measured by ESR earlier [l&20] from the experiments on the photoreduction kinetics of the nitroxide group of D-A to nitro-oxylamine (D-AH) by following the decrease of intensity of the central component of the ESR spectra after irradiation.
2. Experimental
details
2.1. Materials 1-dimethyl-aminonaphthalene-.5-sulphonate covalently bound with nitroxide radical from Reanal (Hungary) and bovine serum albumin from Sigma were used without further purification. All solutions were prepared using distilled water, and ethylene glycol of analytical grade was distilled prior to use.
2.2. Sample preparation The modification of bovine serum albumin by the D-A molecules was performed in 0.1 M phosphate buffer with pH = 7.1, and the D-A binding was controlled by absorption spectroscopy. The absorption spectra were recorded by spectrophotometer Specord M40 (DDR). The addition of D-A to an aqueous solution of the protein causes a concentration-dependent blue shift in the 495 nm absorption band and a small decrease in the maximum absorbence. This shift in the absorption band is consistent with the D-A molecule experiencing a less polar solvent environment than the aqueous phase, as is expected when the D-A molecule is included in the protein hydrophobic cavity. The association constant (=3x 10’ MP’) of the D-A: protein complex was calculated using Scatchard’s method. The final protein concentration in the samples was 2 X 1K4 M, and the D-A concentration was 4 X lo-’ M. All samples were protected from light during the preparation and experiments were carried out with freshly prepared protein-DA samples dissolved in a glassy ethylene glycol-water mixture (1:l) and degassed by the freeze-pump-thaw technique to a final vacuum of less than lop5 mbar.
2.3. ESR measurements The ESR spectra were recorded on a Radiopan SE/X-2544 spectrometer (X-band). Quartz capillaries of 2 mm diameter containing 20 ~1 solution of D-A-protein complex were placed in the cavity of an ESR spectrometer equipped with a window for irradiation and a temperature unit. ESR spectra were recorded with the following instrument settings: a modulation amplitude of 0.3 mT, a microwave power of 15 dB and a sweep width of 10 mT.
2.4. Fhorescence measurements Luminescence spectra were recorded using a modified Aminco-Bowman (USA) spectrofluorimeter equipped with a thermostatted cell holder based on nitrogen gas flow temperature regulation. Quartz capillaries of 3 mm diameter containing 40 ~1 solution of D-A-protein complex were placed in a cell holder of an spectrofluorimeter. The registration of fluorescence proceeded at right angles to the excitation light. The 0.5 mm slit was used for excitation of fluorescence of the donor group of D-A when the steady state fluorescence spectra were obtained.
KR. Vogel et al. I Photoinduced ET in u D-A pair in bovine serum albumin
3. Results 3.1: Kinetics of reversible ET
The experimental dependencies of the fluorescence intensity of the sample containing the D-AH-protein complex (ID_.& and of the sample containing the D-A-protein complex (IDA) vs. temperature are presented in Fig. 1. The reversible ET in the D-A molecules was accompanied by a quenching of the fluorescence of the donor singlet state according to the following scheme: P-AI
Tf
$
I
[D*-A] k-l
k.,
(D”A3’
-%
{D+-A-],,,,
es
&
[Xl
k-2
P--AH1
1T
hv
UT,
p-AH]*
where hu is the quantum of exciting light, q is the donor fluorescent lifetime, k, and k_, are the rate constants of direct and reverse intramolecular electron transfer, k2 is the solvation rate constant and k-, is the reverse charge transfer in molecule after solvation. The DC--A- species participates in the following stages of protonation and is then reduced by an unidentified amino acid residue X and/or solution impurities. 50
1 1, rel.un.
231
According to this scheme the rate constant of reversible electron transfer may be calculated from the fluorescence intensity of D-A molecules (ID_*) and completely reduced form D-AH molecules (I-& by the equation: K, = &AH
- &4)~MH0/~f~&4H&4
(1)
where the &-’ and TVvalues are taken at room temperature. The temperature dependence of K, calculated from these data by eqn. (1) is presented in Fig. 2 and in its Arrhenius form in Fig. 2(b). 3.2. Measurements relaxation shifr
of donor fluorescence
The medium dynamic states were investigated using the method of measuring relaxation shifts of fluorescence spectra maximum VS. temperatures [22,23]. Figure 3 shows a typical plot of the fluorescence maximum (A,,‘) of the excited donor as a function of temperature. The shift in (A,,? to shorter wavelengths as the temperature decreased can be attributed to a decrease in solvent (protein media) stabilization of the emitting excited state when the rigidity of the medium increases. At room temperature in fluid D-A-protein samples, the solvent reorientation time (7,) is rapid compared with the excited state lifetime, allowing full stabilization of the more polar excited state, and this emission is from the solvent-stabilized S, excited state. However, as the rigidity of the medium increases with decreasing of temperature, solvent (protein microsurrounding media) reorientation becomes much slower than the lifetime of the S1 state, leading to a shorter wavelength emission which originates from a less stabilized excited state. 3.3. ESR measurements of molecular dynamics Figure 4 presents the temperature dependence of the rotational correlation times (7J of a spin fragment of D-A molecule incorporated in BSA. The correlation times r, in a fast motion region of the spin group were calculated from the ESR spectra using the formula 1241: 2x10s %=
A&[(h,/h_,)“Z-
l]
(2)
where AH0 is the width of the central component in Gauss, and ho and h- 1 are the intensities of the spectral components. T. K Fig. 1. The temperature dependencies of the intensity of steady state fluorescence spectra measured for samples containing a solution of the D-A protein complex, t,, {curve 1) and for the same sample after irradiation (completely photoreduced), fDmnH (curve 2).
3.4. Estimation of apparent micropolatity The value of apparent dielectric constant (E) in the microenvironment of a D-A molecule in the protein was determined experimentally from the relaxation shift of fluorescence spectra of the
232
KR. VogeI et aI. I Photoinduced ET in a D-A pair in bovine serum albumin
Fig. 3. The temperature dependence of the relaxation shift of the maximum of steady state fluorescence spectrum near excited chromophore fragments of D-A molecules in BSA.
b 1/T =
10QE -1
2.5 q
2.0 : q
1.5 :
,.,i’”
1.0 -
0.0
0.2
0.4
0.6
0.0
I.0
1.2
1.4 0.5 -
Fig. 2. (a) The temperature dependence of the intramolecular ET rate constant K, for D-A molecules incorporated into bovine serum albumin (1:l ethylene glycol-water mixture). @) The Arrhenius dependence of the intramolecular ET rate constant K. for D-A molecules incorporated into bovine serum albumin.
fluorescence label octadecilaminoeosine (ODE) [25] attached to BSA in the following manner. It is known [23] that the maximal value of the shift Ah,, = (hT_,, K- A,_,,K), where A is the wavelength of the maximum of the fluorescence spectra, is correlated with the value of e for the liquid. From the comparison of the values of AAm= for
Fig. 4. The temperature dependence of the Brownian diffusion correlation time rc calculated from the ESR spectra of nitroxide fragments of D-A molecules in BSA.
octadecilaminoeosine in a number of liquids [25] with the value of Ah,,= 28 nm for the complex of octadecilaminoeosine molecules with BSA, the conclusion can be drawn that the c1 = 11 to 13.
VR
Vogel et al. / Photoinduced ET in a D-A pair in bovine serum albumin
Another value of E = 63 has been determined from the value of the HFS constant of the ESR spectra of the nitroxide fragment of a D-A molecule attached to BSA by the method described in [24]. For comparison, the value of e for a 50% water-ethylene glycol mixture is equal to 65. Thus these data indicate that the D-A molecule is partly embedded in a hydrophobic region of the protein globule and that the process of intramolecular electron transfer proceeds on a ‘Lmicrointerface” with variable polarity.
4. Discussion
tribution form the orientational component in solvation remains open, assuming l = 2 at 77 K would lead to AG,,,‘= - 1.7 eV at 77 K. Consequently, the overall change A(AG,,,“) due to orientational solvation with the decrease of the temperature from 300 K to 77 K would equal A(AG,,,‘) = - 1.7 eV. Using these data the diagram of Gibbs energy levels for these reactions can be constructed (Fig. 5). The present estimate of Gibbs energy indicates that the value AGO for reversible direct ET equals 0 to 0.5 eV at 77 K. Now we will discuss the kinetics of reversible ET presented in Fig. 2. At low temperatures, when kT
The data mentioned above allow us to estimate the main factors affecting ET in a donor-acceptor pair D-A incorporated in cavity BSA and to discuss the role of molecular dynamics in the vicinity of the donor and the acceptor groups. 4. I. Estimation of Gibbs energy AGo At room temperature, when solvation after the process of producing photo-initiated charge pairing is rapid, the value of the Gibbs energy can be calculated by the following equation: AC&P
= ED,,+‘” - (En+,a’” + E,,*) -
c(rD + rAk)
(3)
[271. The value of e2/e1(rD+rA) was estimated to be equal to 0.025 V for r, = r, = 4 8, and E = 13 (see Section 3). Taking into account these adduced data the value of AG297K”= - 1.75 eV was determined from eqn. (3). Now we can estimate the energy of solvation of [D+-A-] using the Born equation: = e2/2[r, - ‘( 1- l/eJ + r, - ‘( 1 - UE~)]
4.2. Estimation of the resonance integral V,, For a hybrid donor-acceptor (D-X-A) molecule with a bridge group (X) the value of the resonance integral can be estimated from the formula: rIx
(6)
/
For calculation of AG,,, K the following experimental data were used: ED* = 2.4 eV, determined from the fluorescence spectra at T=77 K, E D/D+ ‘12= 0 . 7.5 eV for 1-dimethyl- aminonaphtalene in acetonitrile [26] and E,+,A1n=0.3 eV for a nitroxide radical in a 96% water-ethanol mixture
AG_P
(51
where FC denotes the Frank-Condon factor and V,,, is the resonance integral. As is seen from the experimental dependence in Fig. 2, the rate constant K. is independent of temperature up to T=lOO K.
v=v,
e2
(4)
where E, = 13 and c2= 63 (see Section 3). The estimation gives the value AG,rv”= -3.4 eV. ASsuming that this value of AG,,,* corresponds to the change of AGo due to the solvation and taking into account that at 77 K the question of a con-
233
where V, is the resonance integral at van der Waals contact of the donor (D) and the acceptor (A) fragments and ‘yi are the attenuation parameters for a given bond or atom which is a part of the bridge group. As has been shown [28], the value of the attenuation parameter yi can be derived from data on spin exchange between two paramagnetic groups or paramagnetic ions in biradicals and complexes with radical ligands. According to [28J, the values of 15 are equal to ‘yc =7, y,., =3. Applying eqn. (6) to the donor-acceptor hybrid molecule with V,=3 eV [29] and taking into account that the spin density of odd electrons in the N-O group is distributed between the N and the 0 atoms with a distribution coefficient (Ye,N = 0.5 leads to r/ D-A= (3 x0.5)/(3
X 73) = 1.5 X 10m3 eV
(7)
4.3. Estimation of Frank-Condon factor FC Using the experimental value KI = 7.2 X lo8 s and calculating the value of V,, by the empirical method, the value of FC may be estimated using
KR.
234
Vogel et al. I Photoinduced
ET in cz D-A
AG*C evl
pair
in bovine ~erw-n albumin
I X+C D--AID
4
I
_elrx
1
3
2
I
0
x
!
CD-A9
+
-=A
IX
+ca
+A->l&
elax
-1
-2
-3 Fig. 5. Diagram of the Gibbs energy levels for ET in D-A molecules amino acid residue and/or solution impurities.
eqn. (5). Then eV_l.
the value
of FC equals
l-10-’
4.4. me intermediate temperature region urul Ihe correlation with molecular medium dynamics The temperature range from T=lOO K to T=240 K is emphasized in Fig. 2 and the temperature dependence of K, is non-linear in Arrhenius’s coordinates {Fig. 2(b)). Apparently, in this intermediate region the ET is complicated by the low frequency of conformational fluctuations of protein and of solvent polarization. In accordance with ref. 11, we can consider the ET transition in this intermediate temperature region in the twodimensional free-energy surface representation of the transition state. In this model, one coordinate is an internal reactive coordinate of the solute and the second coordinate is a solvent polarization coordinate. The transition state lies at the intersection of the potential surfaces U&, q} and U,{Q, q}, and a two-dimensional free- energy surface is presented. The reaction can be described by the diffusion reaction equation, in which the solvation (or conformational) coordinate Q diffuses via Brownian motion under the influence of a potential U(Q) while the reaction of ET occurs at each Q with rate constant K(Q) {determined by the traditional theory of radiationless transition) during the diffusion. As a result the ET between the “instant energy levels” and the “effective value of AGO” of this reaction becomes a function of
in ESA at various
temperatures,
where X is an unidentified
salvation and conformational coordinates. The effects of solvation and/or conformational relaxation dynamics on the apparent ET rate constant are persistent in this intermediate temperature region. At temperatures 10&150 K, when K1 and K_, > l/T, we have the multiexponential transition. At temperatures 150-250 K we may consider the time-scale of reactions to be about the time-scale of salvation, and then iY1= l/r,. Finally, at higher temperatures equilibrium for all degrees of freedom occurs and we can use the traditional expression for the rate constant. As we can see from Section 3, the changes in the relaxation shift of the fluorescence spectrum of the naphthalene fragment of the D-A molecule (Fig. 3) takes place at T= 150 K, and on the other hand the changes of correlation time of the rotational movements 7C of the nitroxide fragment of the D-A molecule (Fig. 4) take place at T > 240 K only. As is known, the temperature of the glass transition of a 50% water-ethylene glycol mixture is also about 240 K. These changes confirm the presence of the orientational and diffusional movcmcnts of medium molecules with correlation frequencies about 10P7-lo-“’ s-’ in this temperature region. This conclusion is confirmed also by literature data on the slower molecular dynamics registered in this temperature range by ESR and phosphorescence methods using various labels and probes
KR. Vogel et al. / Photoinduced ET in a D-A pair in bovine semm albumin
incorporated [28,3O-311.
into the albumin
hydrophobic
cavity
4.5. Reversible ET in the high-temperature region: T>250 K If the reaction proceeds at temperatures close to room temperature, and kT B ho, then equilibrium probably occurs for all of degrees of freedom. Let us use the Marcus equation, assuming for more simplicity that the reaction is non-adiabatic:
v2 Kt, = (kT,Q-I)‘n
(AGO+&)’ exp
-
4E,kT
(3)
4.6. Estimation of reorganization energy E, First, we can evaluate the activation energy from consideration of the linear part of Arrhenius’s dependence (Fig. 2(b)) as E, = 0.25 eV. Assuming AGO= -1.75 eV, the value of the reorganization energy E, was determined to be about 0.8 eV. The calculation shows that the reaction proceeds in the “inverted” manner at room temperature. The value of E, for the reaction can be estimated independently by the Marcus equation, taking into account the volume of reagents [32]: E, = (l/n2 - 1/es)e2/2( l/rD + l/r* - 2/rDA
235
tron transfer at low and high tcmperaturcs, but at intermediate temperatures the mechanism of the process seems to be more complicated, owing to slow conformational and solvation processes. As a consequence, this leads to the fact that the kinetics of ET and the “effective values” of such thermodynamic parameters as AGo and E, become some complex function of temperature and depend on the character of the relaxational dynamics of medium. Further, the molecular dynamics of protein determining the conditions of solvation of primarily separated charges of [D+-A-] is the essential factor for the irreversible process of photoinduced electron transfer reaction causing the irreversible reduction of nitroxide radical in this system.
Acknowledgments
This investigation was supported in part by a grant from Russian state research programme 08.05: “Newest methods of bioengineering, subprogram engineering in Enzymology”.
References
+ [rD3+ rA3]/2rDA4)
(9) where r, and r, are the radii of the donor D and the acceptor A and rDA is the distance between D and A. The use of this equation for the system studied is very difficult, because the reaction proceeds on the interface of media characterized by various “effective” dielectric constants. So, for a rough estimate let us use the mean value: E, = {El, +E,)/2, where E,, and Ezr are calculated by eqn. (9) in media with Ed= 11 and ~~=65 respectively, The estimate gives the value E,= 0.9 eV. 4.7. Estimation of V,, from the rate constant at room temperature Using the experimental value of K, at 300 K, K, = 9.7 x 10’ s-l and the above calculated values of AGO and E,, we can estimate the resonance integral from eqn. (8) (V,,=S X lo-’ eV); nevertheless, the value of V,, at room temperature proves to be more than that at low temperatures by more than an order of magnitude. 5. Conclusions The
perature
theory of ET is able to describe the temdependence of the intramolecular elec-
1 J.J. Hopfield, Proc. Nati.Acad. Sci. USA, 71 (1974) 3640-3644. 2 3. Jortner, /. Chem. Phys., 64 (1976) 4860-4867. 3 R.R. Dogonadze, A.M. Kuznetsov and J. Ulstrup, J. Theor. BioL, 69 (1977) 239-263. D. De Vault, Q. Rev. Biophys., 13 (1980) 387-564, J.M. Nocek, N. Liang, S.A. Wallin, A.G. Mauk and B.M. Hofman, J. Am. Chum Sm., I12 (1990) 1623-1627. F. Parak, E.N. Frolov, A.A. Kononenko, R.L. Mossbauer, V.I. Goldansky and A.B. Rubin, FEBS Let& 117 (1980) 368-374. 7 SE. Peterson-Kennedy, J.L. McGourty and B.M. Hoffman, i Am. Chem. Sm., 1% (1984) 5010-5012. 8 E.G. Petrov, Physics of Electron Transfer, Naukova dumka, Kiev, 1984 (in Russian). 9 V.R. Vogel, G-1. Likhtenstein and A.I. Kotelnikov, BLofizika, 30 (1985) 394399. 10 R.A. Marcus and N. Sutin, Biochem. Biophys. Actu, 811(1985) 265322. 11 J-I. Sumi and R.A. Marcus, J. Chem. Phys., 84 (1986) 4895-4907 12 J.A. Cowan, R.K. Upmacis, D.N. Beratan, J.N. Onuchic and H.B. Gray, Ann. NYAcod. Sci., 550 (1988) 68-84. 13 G.I. Likhtenstein, Chemical Physics ofMetalloenzyme Ca~aalysis, Springer, Heidelberg, 1988. 14 G.L. McLendon, Act. Chem. Res., 21 (1988) 16&167. 15 L.H. Zang and AH. Maki, J. Am. Chem Sac., 212 (1990) 434w350. 16 M.D. Newton, Ctiem. Rev., 91 (1991) 767-792. 17 B. Cartling, .7. Chem. Phys., 95 (1991) 317-323. 18 I.M. Bystryak, G.I. Likhtenstcin, A.I. Kotclnikov, H.O. Hankovski and K. Hidcg, Zh. FL&Khim., II (1986) 27962802. 19 G.I. Likhtenstein, SM. Bystryak, A.I. Kotelnikov, xhim. Fti, 5 (1990) 697-705.
236
V.R. Vogel et al. I Photoinduced ET in a D-A pair in bovine serum albumin
20 E.T. Rubtsova, V.R. Vogel, D.B. Kbudyakov, A.I. Kotelnikov and G.I. Likhtenstein, B~jizi&z~, 38 (1993) 222-227. 21 O.V. Belonogova, G.1. Likhtenstein and V.I. Krinichnyi, Eur. 3. Biophys., in press. 22 N.G. Bakhshiev and Yu.T. Masurenko, Opfika and Spectroskopia, 28 (1970) 905-913. principles of Fluorescence Spectroscopy, New 23 J.R. Iakowicq York, Plenum, 1983. 24 L. Berliner and J. Reuben (eds.), Spin Labeling. Theory and Application, Plenum, New York, 1989. 25 S.M. Bysttyak, A.I. Kotelnikov, G.I. Likhtenstein and V.I. Nikulin, Z/z. priwnd. Spektrosk., 52 (1990) 225-230. 26 C.K. Mann and K. Barnes, Electrochemical Reactions in Nonaqueous Systems, Marcel Dekker, New York, 1970.
27 I-B. Volodarskii, LA. Grigoriev, S.A. Dikanov, V.A. Resnikov and G.I. Shukin, lmidasolin nitmxy! radicals, Nauka, Novosibirsk, 1988. 28 G.I. Likbtenstein, Biophysiml Lube&g Methods in Molecular Biology, Cambridge University Press, Cambridge, 1993. 29 V.M. Berdnikov and GA. Bogdanchikov, Z/I. I&&n. Fit., 53 (1979) 273-283. V.R. Vogel, G.I. Likhtenstein, A.A. 30 A.I. Kotelnikov, Kononenko, P.P. Noks, N.P. Grishanova and A.B. Rubin, Molekuly~muya biologiyu, 17 (1983) 846-854. 31 G-1. Likhtenstein, V.R. Bogatyrenko and A.V. Kulikov, Bioj&a, 37 (1983) 585-589. 32 Yu.1. Kbarkatz, ElektrockJzimiya 12 (1976) 592-595.