Eur. J. Biochem. 96, 119-123 (1979)
Mossbauer Spectroscopic Investigations of Photodissociated Myoglobin-CO at Low Temperatures Hans-Ernst MARCOLIN, Reiner RESCHKE, and Alfred TRAUTWEIN Angewandte Physik, Universitiit des Saarlandes, Saarbrucken (Received April 27, 1978/January 30, 1979)
Myoglobin-CO (MbCO) has been photodissociated at low temperatures. The photoproduct Mb* has been identified by Mossbauer spectroscopy as a ferrous high-spin complex with Mossbauer parameters which are different from those of deoxy-myoglobin. From the time-dependent change of the linewidth of the Mb* Mossbauer spectrum at 5 K over a time interval of 8 h, we conclude that there exist several slightly different Mb* conformations with different recombination characteristics. In order to obtain a convenient time resolution of the recombination behavior, we have investigated the time-dependence of the Mb* absorption within a time interval of lo2- 5 . lo4 s after the photodissociation, with a Mossbauer drive of constant velocity. The resulting recombination data have then been analyzed with various approximation steps. It is shown that the interpretation of experimental data on the basis of two independent exponentials leads to pre-exponential frequency factors which are of the order of A1 = 10 s-' and A2 = lo5 k 5 . lo's-'. The attempt to interpret the experimental data on the basis of a distribution of energies seems more plausible and is in agreement with the existence of several slightly different Mb* conformations. The corresponding frequency factor A is about 4 s-' and is therefore in disagreement with the value of A (of about lo7 s-') which was derived from optical recombination data by Austin et al. within a time interval of lop6- lo3 s after the photodissociation. Typical activation energies for the recombination process Mb* + CO + MbCO are 8 kJ/mol. At low temperatures ( T < 46 K) the recombination behavior is explained by quantum mechanical tunnelling.
The low-temperature recombination behavior of photodissociated myoglobin-CO (MbCO), as exemplified by absorbance measurements [ 1 - 41, especially in the tunnelling region, has been the subject of controversy during the last few years. The low-temperature recombination of the relatively large CO molecule with the heme iron has been described by the quantummechanical tunnelling process. We therefore thought it might be worth investigating this low-temperature recombination using the Mossbauer technique.
EXPERIMENTAL PROCEDURE Protohemin, enriched to 90 % with 57Fe, was synthesized from protoporphyrin IX dimethyl ester and 57Fe203(90.42 % 57Fe) by the ferrous sulfate/acetic Abbreviations. Mb, myoglobin ; MbCO, carbon monoxide myoglobin; Mb*, product of photodissociation of MbCO at low temperature.
acid method [5] and was purified chromatographically according to procedures described elsewhere [6]. Metmyoglobin was purified from sperm whale skeletal muscle by the method of Kendrew and Parrish [7] and recrystallized with ammonium sulfate. Apomyoglobin was prepared by removing protohemin from a 1 solution of metmyoglobin with methylethyl ketone according to the method of Teale [8], modified by Breslow [9]. The reconstitution was carried out by mixing 0.44% apomyoglobin in solution and an equivalent amount of 57Fe-enriched protohemin in borate buffer, pH 9.47, for 1 h at l O T , and the mixture was dialyzed against distilled water, the insoluble matter being removed by filtration. To check the 57Fe-enriched metmyoglobin crystallographically single crystals were grown, which were found to be of the type A crystal reported by Kendrew and Parrish [ 5 ] . No intensity difference was observed between the native and reconstituted metmyoglobin crystals. The crystals were then dissolved in water, desalted by a Sephadex G-25 column and lyophilized. Myoglobin was then prepared using standard methods.
120
Mossbauer Study of Photodissociated Myoglobin-CO below 80 K
Table 1. E.t-perimentul M5sshuuer parameters of deoxjmyoglohin ( M h ) , myoglohin-CO ( MhCO), und .f'the pliotodissociated adduct i M h * ) Values of the isomer shift 6 were measured relative to metallic iron at 300 K. Values in brackets have been obtained using Voigt curves. all other values have been obtained using Lorentzian curves
7K
6
Solvent
Remarks
dist. HzO, pH 7 glycerol/water, pH 7 dist. HzO, pH 7
measured 20 h measured 16 h measured 1 h, irradiated 4 min measured 7 h, irradiated 4 min measured 9 h after the measurement has been performed at 5 K Spartalian et al. [12]
mm s C 1 ~
4.2 5 5 5
46 4.2
8
r
AEQ
Material
C~
~
-
~~~
2.174 f 0.004 0.370 k 0.005 2.27 k 0.03 0.35 k 0.01 2.14 k 0.03 0.355 k 0.004 2.25 k 0.02 0.368 k 0.005 2.191 (2.330)
Mb Mb Mb* MbCO Mb* M bCO Mb* MbCO Mb*
_
C
_
0.429 _+ 0.006 0.282 0.008 0.35 k 0.03 0.35 k 0.02 0.390 k 0.008 0.390 f 0.008 0.310 k 0.009 0.310 f 0.009 0.31 (0.22)
~
~~
~
~
C~ ~
0.98
T
\ P
0.96
0.944
, -3
I
t
-2
-1 0 velocity, Y
I (mrnis)
2
~~~~
0.921 k 0.009 0.278 k 0.008 0.89 k 0.03 0.27 k 0.02 0.93 k 0.04 0.28 _+ 0.02 0.89 k 0.04 0.28 f 0.02 0.888 (0.888)
3
Fig. 1 . Tlpicul Mijsshauer spectrum .J'u "Fe-cnriched myogluhin-CO sumple of3-mm thickness at 5 K qfier irrudiuring the prohe with white light 1450 W)/?)r 4 min. (During the ircadiation with white light the temperature increased by 0.25 K). The quadrupole doublet indicated by the arrow corresponds to the photoflash product Mb*, the other quadrupole doublet to MbCO
RESULTS AND DISCUSSION 57Fe-enriched myoglobin-CO was photodissociated at 5 K with a photoflash of white light (Xe lamp, 450 W, Osram XBO 450) and investigated by Mossbauer transmission spectroscopy [lo] before and after the irradiation with light. Mossbauer spectra were obtained with two different drive systems, one in constant-acceleration mode and one in constant-velocity mode. Details oYthe experimental procedure have been published elsewhere [I I]. A typical spectrum obtained in the constant-acceleration mode of the photodissociated material at 5 K, as shown in Fig. 1, has two quadrupole doublets, one with the parameters of MbCO and one with the parameters of the phgtodissociated adduct (Mb*). In Table 1 we summarize the Mossbauer parameters 'quadrupole splitting' ( A EQ), 'isomer shift' (a), and 'line width' (r )of deoxygenated myoglobin (Mb), of MbCO and of Mb*, depending on solvent and temperature. The results obtained for Mb* (glycerol/water, pH = 7) are close to the values
~
glycerol/water, pH 7 glycerol/water, pH 7 phosphate buffer, pH 7
obtained for Mb* (phosphate buffer, pH = 7) by Spartalian et al. [I21 and Maeda et al. [13,14] and to the values of the deoxygenated M b (distilled HzO, pH = 7). From isomer shift and quadrupole splitting of Mb* we conclude that the heme iron in Mb* is in the ferrous high-spin state as in Mb. The differences in the subspectra obtained, i.e. in 6 and AEQ between Mb* (distilled Hz0, pH = 7) and M b (distilled Hz0, pH = 7), directly reflect the difference in electronic structure of the heme iron within the two compounds; this is an advantage of the Mossbauer method compared to optical spectroscopy. This difference in electronic structure might be caused by the CO molecule: in Mb* the CO molecule remains in the heme pocket after photodissociation and has measurable influence over the electronic structure of the heme iron, whereas in the case of M b the CO is removed from the heme pocket. A study of the recombination of CO with Mb* at 5 K (measuring a spectrum each hour in the constantacceleration mode) reveals a reproduceable change of 6, r and AEQ of the Mb* spectrum (Fig.2). (Note that the minimum obtainable r is 0.19 mm s-' due to the Heisenberg relation between linewidth and lifetime of the Mossbauer state in 57Fe; this minimum value might be broadened due to instrumentation and absorber thickness up to about 0.25 mm s-'.) Our interpretation of this result is that several slightly different subspectra contribute to the spectrum measured during the first hour after the photodissociation has taken place, and that the number of subspectra decreases with time. A possible explanation of this is (a) that the flashed-off CO molecule has been trapped at different sites within the heme pocket of different myoglobin molecules (forming different conformational states of Mb*), and (b) that the recombination of C O with the heme iron is different for these different conformational states. This aspect is in qualitative
H.-E. Marcolin. R. Reschke. and A. Trdutwein 0.45
3
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2
2 0.35
i+
A
+++Ht ,"*++
+
+
+
+t++
++++t +t+
++
++t
T = 5K
l i
E E
L
0.30
'1 0i
0.25
1
2 0.90
i
1 '-
2
E E
v
a 0.85
2.35
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+*;t
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,
++ +*u
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+
+t
++
Tz59K
Oi_
I
G a
7~46K
1
D
C ,
r
,
,
,
,
t
,
0 1 2 - 3 4 5 6 7 8
0
5
10 Time,[
Time, f (h)
Fig. 2. E.\-per.imental Mosshauer parameters oj'Mh* at 5 K depending on time after the probe has heen irradiated with light. Each spectrum was collected over a time period of 1 h. (A) Line width; (B) isomer shift; (C) quadrupole splitting
agreement with the finding of Austin et al. [l] that the recombination of CO with Mb* is governed by a distribution rather than by a sum of two independent exponentials as proposed by Iizuka et al. [4]. In the constant-velocity mode, with the velocity indicated by the arrow in Fig. 1, we were also able to investigate the recombination behavior of CO with Mb* at elevated temperatures. Fig. 3 shows recombination curves at 5 K, 4 6 K , 5 9 K , and 65 K. The analysis of these curves has been performed (a) using two independent exponentials (as in the analysis by Iizuka et al. [4] of optical recombination data), and (b) using a distribution of independent exponentials (as in the analysis by Austin et al. [ l ]of optical recombination data). The main feature of analysis (a) is that we obtain recombination rates k l and k z , as shown in Fig.4, with pre-exponential factors A1 = 10 8 s - ' , A z = 10st5.102s-1,andenergiesEl =5.44* 1.26kJ mol-', Ez = 12.14 & 5.26 kJ mol-', respectively, of the Arrhenius law k(T) = Aexp(- E/RT). While the values for El and E2 are close to the value (Epeak % 10 kJ m o l - ' ) found by Austin et al. [l], the value for A l deviates considerably from that reported by the Illinois group [I] ( A = 10' s-'). We continued our analysis (b) of recombination curves N ( r ) with a distribution of independent exponentials according to [l]:
*
15
20
(h)
Fig. 3. Time-dependence o f t h e intcnsit~~ ofthe Mh* line i I-A,/A, indicated in Fig. I by an arrow, at various temperatures
'1 N ( t ) = R T S - - g ( k ) exp(-kt) dk o k
),
=
(1)
From Eqn (1) we derive a distribution of recombination rates k : k g(k) = ----L-' { N ( t ) }, (2) RT where L -
'
is the inverse Laplace transformation.
l - ( A u / A m )of Fig.3 is proportional to N ( t ) . The pro-
cedure was applied to the recombination curves at. 46 K, 59 K and 65 K (Fig. 3) leading to fit-parameters n and to [Eqn (l)], which are related to the frequency factor A and to the peak energy Epeakof the energy distribution by [I]
(3) Plotting In (n/fo) versus l / T yields Epeak = 4.94 kJ mol-' and A = 3.7 s - ' . It is important to note that Eqn (3) includes the approximation that A is constant over the entire energy spectrum g ( E ) (which is given in Fig.5 and is compared with that obtained by Austin et al. [I]). The analysis of N ( 2 ) on the basis of a finite number of independent exponentials, on the other hand, allows the determination of independent fre-
122
Mossbauer Study of Photodissociated Myoglobin-CO below 80 K
+---t-
3.5 4.8 6.1 7.4 E z R J In k i A ( k J i r n o l )
5
0
0.025
0.200
0.050
I ir
( ~ - 1 )
Fig. 4. Rcc~ombinutionrates k , (T) and k l ( T ) which ,fbllon>,from analyzing e.uperimenta1 data of A,(t,T)/A, on the basis of two independent exponentials
quency factors Ai. Comparing A1 = 10 i 8 sC1, A z = lo5 f 5 . lo2 s-' from analysis (a) and A % 4s-' from analysis (b) with A % lo7 s-' from Austin et al. [l], we conclude that the results derived depend on the analytical model applied and probably also on the time interval, At, during which the recombination was investigated ( A t is 102-5 . lo4 s in the present study and was lo3 s in the study of Austin et al. [11h In the temperature range T < 46 K the recombination rates are too high compared to values which one would expect from an Arrhenius law. Furthermore, the recombination rates are nearly temperature-independent within T < 46 K (horizontal line in Fig.4); this in fact can be attributed to the tunnelling of CO molecules [ l ] through a potential barrier with height AE. The recombination rate for the temperatureindependent tunnelling process ( T + 0), assuming a three-dimensional spherical potential barrier with height A E and thickness d, can be approximated by [15]:
[:
k , = A, exp --+TE
- Ekin)
d
]
(4)
where A , is the frequency factor which describes the attempts of the CO molecule to penetrate the potential wall, E k i n is the kinetic energy of the molecule and m its mass. In order to obtain an estimate of d and A , we treat the CO molecule as a free particle within the heme pocket; then A , may be approximated by [15]:
where uo is an average velocity which may be derived from kinetic gas theory, and YO is the radius of the spherical potential barrier with height AE. This estimate leads to an upper limit of A,, since the CO molecule is more likely to be trapped in the heme than able to move free. Neglecting E k i n in Eqn (4) (at 5 K E k i n
0 7.1
8.4
9.7
11.0 12.3 E (kJ/mOI)
13.6
14.9
Fig. 5. ( A ) Energy ~listributioncurve g(E) iihich re.sult.s,fi-oni analj;ing Miisshuuer recombination data at T = 46 K , 59 K and 65 K , using the same power la~.,for N(t) as Austin er al. [ I ] have used.fbr the analysis uf their optical data. ( B ) Energ-v distribution curve g ( E ) which has been derived hy Austin et al. ,from optical recornhination curves
is about 1/10 of A E ) and using ro = 0.5 nm (0.25 nm) we derive the thickness d to bed = 0.072 nm (0.070 nm). An alternative estimate might be interesting to note. Assuming that the thickness of the potential wall is correlated with the distance A x of iron from the heme plane in M b (it was reported to be 0.055 nm [16]), we derive from Eqn (4) A , values of about 10 s-', lo5 s-' and 10l2 s-', respectively, for A x = 0.02, 0.04 and 0.08 nm. We realize that the realistic value for A x of 0.055 nm leads to an A t value of the order of lo7 s-', consistent with the value derived by Austin et al. [l] but inconsistent with our value derived from analysis (b). This discrepancy is probably due to the crude approximation for the tunnelling process, described by Eqns (4) and (9,due to the different time intervals during which the recombination was investigated by Austin et al. [ l ] and by us, and due to the limitation of the Mossbauer method when being applied to the present problem. An overall conclusion of our study is that Mossbauer spectroscopy is very limited in resolving relatively fast recombination processes due to the poor statistics obtained within narrow time intervals, compared to optical techniques; however, it has the advantage with respect to optical spectroscopy of yielding individual spectra of the photoproduct Mb* out of a mixture of MbCO and Mb* at very low temperatures, where the lifetime of Mb* is relatively long. Fruitful discussion with Prof. H. Frauenfelder, with Dr A. Alfsen and with Dr Y. Maeda are gratefully acknowledged. The entire preparational 57Fe-enrichmentprocedure was performed by
H.-E. Marcolin, R. Reschke, and A. Trautwein Prof. Y. Morita, who supplied us with lyophilized samples, and to whom we are very much indebted. The preparation of myoglobinCO from lyophilized material was kindly performed by D r W. Nastainczyk. This work was supported by Deutsche Forschungsgemeinschufi.
REFERENCES I. Austin, R. H., Beeson, K. W., Eisenstein, L., Frauenfelder, H., Gunsalus, 1. C. & Marshall, V. P. (1974) Phys. Rev. Lett. 32, 403 - 405. 2. Austin, R. H., Beeson, K. W., Eisenstein, L., Frauenfelder, H. & Gunsalus, I. C. (1975) Biochemistrv, 14, 5355 - 5373. 3. Alberding. N., Austin, R. H., Beeson, K . W., Chan, S . S., Eisenstein, L., Frauenfelder, H. & Nordlund, T. M. (1976) Science ( Wa.~h.D.C.) IY2, 1002- 1003. 4. Iizuka, T., Yamamoto, H., Kotani, M. & Yonetani, T. (1974) Biochim. Biophqs. A m , 351, 182- 195. 5 . Falk, J. E. (1964) Porphjrin and Metalloporphyrin, p. 133, Elsevier, Amsterdam.
123 6 . Maeda, Y., Morita, Y. & Yoshida, C . (1971) J . Bktcl7rm. (TO~JW 70,) 509-514. 7. Kendrew, J. C. & Parrish, R. G. (1956) Proc. R. Soc. (Lond.) A238, 305 - 324. 8. Teale, F. W. J. (1959) Biophys. Biochim. Acta, 35, 543. 9. Breslow, E. (1964) J . Biol. Chem. 239, 486-496. 10. Giitlich, P., Link, R. & Trautwein, A. (1978) Miissbauer Spectroscopy and Transition Metal Chemistry, Springer-Verlag, Heidel berg. 11. Marcolin, H. E., Reschke, R. & Trautwein, A. (1977) 2. Natur,fbrsch. 32c, 683 - 695. 12. Spartalian, K., Lang, G. & Yonetani, T. (1976) Biochim. Biophj.7. Acts, 428, 28 1 - 290. 13. Madea, Y., Harami, T., Sakai, H. & Morita, Y. (1977) Proceedings of the International Conjerence on Miisshauer Sprctroscopy (Barb, D. & Tarinh, D., eds) pp. 299-300, Revue Romaine de Physique, Bucharest. 14. Maeda, Y., Harami, T., Sakai, H. & Morita, Y. (1978) Proc. 6th Int. Biophys. (bngr. p. 408, Science Council of Japan, Kyoto. 15. Blochinzew, B. I. (1953) Grundlagen der Quantenmec~hnnik, Deutscher Verlag der Wissenschaften, Berlin. 16. Phillips, S . E. V. (1978) Nature (Lond.) 273, 247-248.
H.-E. Marcolin, R. Reschke. and A. Trautwein, Angewandte Physik, Fachbereich 12.1, Universitit des Saarlandes, D-6600 Saarbriicken 1 1. Federal Republic of Germany
Mossbauer Spectroscopic Investigations of Photodissociated Myoglobin-CO at Low Temperatures Hans-Ernst MARCOLIN, Reiner RESCHKE, and Alfred TRAUTWEIN Angewandte Physik, Universitiit des Saarlandes, Saarbrucken (Received April 27, 1978/January 30, 1979)
Myoglobin-CO (MbCO) has been photodissociated at low temperatures. The photoproduct Mb* has been identified by Mossbauer spectroscopy as a ferrous high-spin complex with Mossbauer parameters which are different from those of deoxy-myoglobin. From the time-dependent change of the linewidth of the Mb* Mossbauer spectrum at 5 K over a time interval of 8 h, we conclude that there exist several slightly different Mb* conformations with different recombination characteristics. In order to obtain a convenient time resolution of the recombination behavior, we have investigated the time-dependence of the Mb* absorption within a time interval of lo2- 5 . lo4 s after the photodissociation, with a Mossbauer drive of constant velocity. The resulting recombination data have then been analyzed with various approximation steps. It is shown that the interpretation of experimental data on the basis of two independent exponentials leads to pre-exponential frequency factors which are of the order of A1 = 10 s-' and A2 = lo5 k 5 . lo's-'. The attempt to interpret the experimental data on the basis of a distribution of energies seems more plausible and is in agreement with the existence of several slightly different Mb* conformations. The corresponding frequency factor A is about 4 s-' and is therefore in disagreement with the value of A (of about lo7 s-') which was derived from optical recombination data by Austin et al. within a time interval of lop6- lo3 s after the photodissociation. Typical activation energies for the recombination process Mb* + CO + MbCO are 8 kJ/mol. At low temperatures ( T < 46 K) the recombination behavior is explained by quantum mechanical tunnelling.
The low-temperature recombination behavior of photodissociated myoglobin-CO (MbCO), as exemplified by absorbance measurements [ 1 - 41, especially in the tunnelling region, has been the subject of controversy during the last few years. The low-temperature recombination of the relatively large CO molecule with the heme iron has been described by the quantummechanical tunnelling process. We therefore thought it might be worth investigating this low-temperature recombination using the Mossbauer technique.
EXPERIMENTAL PROCEDURE Protohemin, enriched to 90 % with 57Fe, was synthesized from protoporphyrin IX dimethyl ester and 57Fe203(90.42 % 57Fe) by the ferrous sulfate/acetic Abbreviations. Mb, myoglobin ; MbCO, carbon monoxide myoglobin; Mb*, product of photodissociation of MbCO at low temperature.
acid method [5] and was purified chromatographically according to procedures described elsewhere [6]. Metmyoglobin was purified from sperm whale skeletal muscle by the method of Kendrew and Parrish [7] and recrystallized with ammonium sulfate. Apomyoglobin was prepared by removing protohemin from a 1 solution of metmyoglobin with methylethyl ketone according to the method of Teale [8], modified by Breslow [9]. The reconstitution was carried out by mixing 0.44% apomyoglobin in solution and an equivalent amount of 57Fe-enriched protohemin in borate buffer, pH 9.47, for 1 h at l O T , and the mixture was dialyzed against distilled water, the insoluble matter being removed by filtration. To check the 57Fe-enriched metmyoglobin crystallographically single crystals were grown, which were found to be of the type A crystal reported by Kendrew and Parrish [ 5 ] . No intensity difference was observed between the native and reconstituted metmyoglobin crystals. The crystals were then dissolved in water, desalted by a Sephadex G-25 column and lyophilized. Myoglobin was then prepared using standard methods.
120
Mossbauer Study of Photodissociated Myoglobin-CO below 80 K
Table 1. E.t-perimentul M5sshuuer parameters of deoxjmyoglohin ( M h ) , myoglohin-CO ( MhCO), und .f'the pliotodissociated adduct i M h * ) Values of the isomer shift 6 were measured relative to metallic iron at 300 K. Values in brackets have been obtained using Voigt curves. all other values have been obtained using Lorentzian curves
7K
6
Solvent
Remarks
dist. HzO, pH 7 glycerol/water, pH 7 dist. HzO, pH 7
measured 20 h measured 16 h measured 1 h, irradiated 4 min measured 7 h, irradiated 4 min measured 9 h after the measurement has been performed at 5 K Spartalian et al. [12]
mm s C 1 ~
4.2 5 5 5
46 4.2
8
r
AEQ
Material
C~
~
-
~~~
2.174 f 0.004 0.370 k 0.005 2.27 k 0.03 0.35 k 0.01 2.14 k 0.03 0.355 k 0.004 2.25 k 0.02 0.368 k 0.005 2.191 (2.330)
Mb Mb Mb* MbCO Mb* M bCO Mb* MbCO Mb*
_
C
_
0.429 _+ 0.006 0.282 0.008 0.35 k 0.03 0.35 k 0.02 0.390 k 0.008 0.390 f 0.008 0.310 k 0.009 0.310 f 0.009 0.31 (0.22)
~
~~
~
~
C~ ~
0.98
T
\ P
0.96
0.944
, -3
I
t
-2
-1 0 velocity, Y
I (mrnis)
2
~~~~
0.921 k 0.009 0.278 k 0.008 0.89 k 0.03 0.27 k 0.02 0.93 k 0.04 0.28 _+ 0.02 0.89 k 0.04 0.28 f 0.02 0.888 (0.888)
3
Fig. 1 . Tlpicul Mijsshauer spectrum .J'u "Fe-cnriched myogluhin-CO sumple of3-mm thickness at 5 K qfier irrudiuring the prohe with white light 1450 W)/?)r 4 min. (During the ircadiation with white light the temperature increased by 0.25 K). The quadrupole doublet indicated by the arrow corresponds to the photoflash product Mb*, the other quadrupole doublet to MbCO
RESULTS AND DISCUSSION 57Fe-enriched myoglobin-CO was photodissociated at 5 K with a photoflash of white light (Xe lamp, 450 W, Osram XBO 450) and investigated by Mossbauer transmission spectroscopy [lo] before and after the irradiation with light. Mossbauer spectra were obtained with two different drive systems, one in constant-acceleration mode and one in constant-velocity mode. Details oYthe experimental procedure have been published elsewhere [I I]. A typical spectrum obtained in the constant-acceleration mode of the photodissociated material at 5 K, as shown in Fig. 1, has two quadrupole doublets, one with the parameters of MbCO and one with the parameters of the phgtodissociated adduct (Mb*). In Table 1 we summarize the Mossbauer parameters 'quadrupole splitting' ( A EQ), 'isomer shift' (a), and 'line width' (r )of deoxygenated myoglobin (Mb), of MbCO and of Mb*, depending on solvent and temperature. The results obtained for Mb* (glycerol/water, pH = 7) are close to the values
~
glycerol/water, pH 7 glycerol/water, pH 7 phosphate buffer, pH 7
obtained for Mb* (phosphate buffer, pH = 7) by Spartalian et al. [I21 and Maeda et al. [13,14] and to the values of the deoxygenated M b (distilled HzO, pH = 7). From isomer shift and quadrupole splitting of Mb* we conclude that the heme iron in Mb* is in the ferrous high-spin state as in Mb. The differences in the subspectra obtained, i.e. in 6 and AEQ between Mb* (distilled Hz0, pH = 7) and M b (distilled Hz0, pH = 7), directly reflect the difference in electronic structure of the heme iron within the two compounds; this is an advantage of the Mossbauer method compared to optical spectroscopy. This difference in electronic structure might be caused by the CO molecule: in Mb* the CO molecule remains in the heme pocket after photodissociation and has measurable influence over the electronic structure of the heme iron, whereas in the case of M b the CO is removed from the heme pocket. A study of the recombination of CO with Mb* at 5 K (measuring a spectrum each hour in the constantacceleration mode) reveals a reproduceable change of 6, r and AEQ of the Mb* spectrum (Fig.2). (Note that the minimum obtainable r is 0.19 mm s-' due to the Heisenberg relation between linewidth and lifetime of the Mossbauer state in 57Fe; this minimum value might be broadened due to instrumentation and absorber thickness up to about 0.25 mm s-'.) Our interpretation of this result is that several slightly different subspectra contribute to the spectrum measured during the first hour after the photodissociation has taken place, and that the number of subspectra decreases with time. A possible explanation of this is (a) that the flashed-off CO molecule has been trapped at different sites within the heme pocket of different myoglobin molecules (forming different conformational states of Mb*), and (b) that the recombination of C O with the heme iron is different for these different conformational states. This aspect is in qualitative
H.-E. Marcolin. R. Reschke. and A. Trdutwein 0.45
3
0.40
2
2 0.35
i+
A
+++Ht ,"*++
+
+
+
+t++
++++t +t+
++
++t
T = 5K
l i
E E
L
0.30
'1 0i
0.25
1
2 0.90
i
1 '-
2
E E
v
a 0.85
2.35
C
4
+*;t
+t,+.'
. 0)
E
2.30
2 2.25
,
++ +*u
t*+*
+
+t
++
Tz59K
Oi_
I
G a
7~46K
1
D
C ,
r
,
,
,
,
t
,
0 1 2 - 3 4 5 6 7 8
0
5
10 Time,[
Time, f (h)
Fig. 2. E.\-per.imental Mosshauer parameters oj'Mh* at 5 K depending on time after the probe has heen irradiated with light. Each spectrum was collected over a time period of 1 h. (A) Line width; (B) isomer shift; (C) quadrupole splitting
agreement with the finding of Austin et al. [l] that the recombination of CO with Mb* is governed by a distribution rather than by a sum of two independent exponentials as proposed by Iizuka et al. [4]. In the constant-velocity mode, with the velocity indicated by the arrow in Fig. 1, we were also able to investigate the recombination behavior of CO with Mb* at elevated temperatures. Fig. 3 shows recombination curves at 5 K, 4 6 K , 5 9 K , and 65 K. The analysis of these curves has been performed (a) using two independent exponentials (as in the analysis by Iizuka et al. [4] of optical recombination data), and (b) using a distribution of independent exponentials (as in the analysis by Austin et al. [ l ]of optical recombination data). The main feature of analysis (a) is that we obtain recombination rates k l and k z , as shown in Fig.4, with pre-exponential factors A1 = 10 8 s - ' , A z = 10st5.102s-1,andenergiesEl =5.44* 1.26kJ mol-', Ez = 12.14 & 5.26 kJ mol-', respectively, of the Arrhenius law k(T) = Aexp(- E/RT). While the values for El and E2 are close to the value (Epeak % 10 kJ m o l - ' ) found by Austin et al. [l], the value for A l deviates considerably from that reported by the Illinois group [I] ( A = 10' s-'). We continued our analysis (b) of recombination curves N ( r ) with a distribution of independent exponentials according to [l]:
*
15
20
(h)
Fig. 3. Time-dependence o f t h e intcnsit~~ ofthe Mh* line i I-A,/A, indicated in Fig. I by an arrow, at various temperatures
'1 N ( t ) = R T S - - g ( k ) exp(-kt) dk o k
),
=
(1)
From Eqn (1) we derive a distribution of recombination rates k : k g(k) = ----L-' { N ( t ) }, (2) RT where L -
'
is the inverse Laplace transformation.
l - ( A u / A m )of Fig.3 is proportional to N ( t ) . The pro-
cedure was applied to the recombination curves at. 46 K, 59 K and 65 K (Fig. 3) leading to fit-parameters n and to [Eqn (l)], which are related to the frequency factor A and to the peak energy Epeakof the energy distribution by [I]
(3) Plotting In (n/fo) versus l / T yields Epeak = 4.94 kJ mol-' and A = 3.7 s - ' . It is important to note that Eqn (3) includes the approximation that A is constant over the entire energy spectrum g ( E ) (which is given in Fig.5 and is compared with that obtained by Austin et al. [I]). The analysis of N ( 2 ) on the basis of a finite number of independent exponentials, on the other hand, allows the determination of independent fre-
122
Mossbauer Study of Photodissociated Myoglobin-CO below 80 K
+---t-
3.5 4.8 6.1 7.4 E z R J In k i A ( k J i r n o l )
5
0
0.025
0.200
0.050
I ir
( ~ - 1 )
Fig. 4. Rcc~ombinutionrates k , (T) and k l ( T ) which ,fbllon>,from analyzing e.uperimenta1 data of A,(t,T)/A, on the basis of two independent exponentials
quency factors Ai. Comparing A1 = 10 i 8 sC1, A z = lo5 f 5 . lo2 s-' from analysis (a) and A % 4s-' from analysis (b) with A % lo7 s-' from Austin et al. [l], we conclude that the results derived depend on the analytical model applied and probably also on the time interval, At, during which the recombination was investigated ( A t is 102-5 . lo4 s in the present study and was lo3 s in the study of Austin et al. [11h In the temperature range T < 46 K the recombination rates are too high compared to values which one would expect from an Arrhenius law. Furthermore, the recombination rates are nearly temperature-independent within T < 46 K (horizontal line in Fig.4); this in fact can be attributed to the tunnelling of CO molecules [ l ] through a potential barrier with height AE. The recombination rate for the temperatureindependent tunnelling process ( T + 0), assuming a three-dimensional spherical potential barrier with height A E and thickness d, can be approximated by [15]:
[:
k , = A, exp --+TE
- Ekin)
d
]
(4)
where A , is the frequency factor which describes the attempts of the CO molecule to penetrate the potential wall, E k i n is the kinetic energy of the molecule and m its mass. In order to obtain an estimate of d and A , we treat the CO molecule as a free particle within the heme pocket; then A , may be approximated by [15]:
where uo is an average velocity which may be derived from kinetic gas theory, and YO is the radius of the spherical potential barrier with height AE. This estimate leads to an upper limit of A,, since the CO molecule is more likely to be trapped in the heme than able to move free. Neglecting E k i n in Eqn (4) (at 5 K E k i n
0 7.1
8.4
9.7
11.0 12.3 E (kJ/mOI)
13.6
14.9
Fig. 5. ( A ) Energy ~listributioncurve g(E) iihich re.sult.s,fi-oni analj;ing Miisshuuer recombination data at T = 46 K , 59 K and 65 K , using the same power la~.,for N(t) as Austin er al. [ I ] have used.fbr the analysis uf their optical data. ( B ) Energ-v distribution curve g ( E ) which has been derived hy Austin et al. ,from optical recornhination curves
is about 1/10 of A E ) and using ro = 0.5 nm (0.25 nm) we derive the thickness d to bed = 0.072 nm (0.070 nm). An alternative estimate might be interesting to note. Assuming that the thickness of the potential wall is correlated with the distance A x of iron from the heme plane in M b (it was reported to be 0.055 nm [16]), we derive from Eqn (4) A , values of about 10 s-', lo5 s-' and 10l2 s-', respectively, for A x = 0.02, 0.04 and 0.08 nm. We realize that the realistic value for A x of 0.055 nm leads to an A t value of the order of lo7 s-', consistent with the value derived by Austin et al. [l] but inconsistent with our value derived from analysis (b). This discrepancy is probably due to the crude approximation for the tunnelling process, described by Eqns (4) and (9,due to the different time intervals during which the recombination was investigated by Austin et al. [ l ] and by us, and due to the limitation of the Mossbauer method when being applied to the present problem. An overall conclusion of our study is that Mossbauer spectroscopy is very limited in resolving relatively fast recombination processes due to the poor statistics obtained within narrow time intervals, compared to optical techniques; however, it has the advantage with respect to optical spectroscopy of yielding individual spectra of the photoproduct Mb* out of a mixture of MbCO and Mb* at very low temperatures, where the lifetime of Mb* is relatively long. Fruitful discussion with Prof. H. Frauenfelder, with Dr A. Alfsen and with Dr Y. Maeda are gratefully acknowledged. The entire preparational 57Fe-enrichmentprocedure was performed by
H.-E. Marcolin, R. Reschke, and A. Trautwein Prof. Y. Morita, who supplied us with lyophilized samples, and to whom we are very much indebted. The preparation of myoglobinCO from lyophilized material was kindly performed by D r W. Nastainczyk. This work was supported by Deutsche Forschungsgemeinschufi.
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123 6 . Maeda, Y., Morita, Y. & Yoshida, C . (1971) J . Bktcl7rm. (TO~JW 70,) 509-514. 7. Kendrew, J. C. & Parrish, R. G. (1956) Proc. R. Soc. (Lond.) A238, 305 - 324. 8. Teale, F. W. J. (1959) Biophys. Biochim. Acta, 35, 543. 9. Breslow, E. (1964) J . Biol. Chem. 239, 486-496. 10. Giitlich, P., Link, R. & Trautwein, A. (1978) Miissbauer Spectroscopy and Transition Metal Chemistry, Springer-Verlag, Heidel berg. 11. Marcolin, H. E., Reschke, R. & Trautwein, A. (1977) 2. Natur,fbrsch. 32c, 683 - 695. 12. Spartalian, K., Lang, G. & Yonetani, T. (1976) Biochim. Biophj.7. Acts, 428, 28 1 - 290. 13. Madea, Y., Harami, T., Sakai, H. & Morita, Y. (1977) Proceedings of the International Conjerence on Miisshauer Sprctroscopy (Barb, D. & Tarinh, D., eds) pp. 299-300, Revue Romaine de Physique, Bucharest. 14. Maeda, Y., Harami, T., Sakai, H. & Morita, Y. (1978) Proc. 6th Int. Biophys. (bngr. p. 408, Science Council of Japan, Kyoto. 15. Blochinzew, B. I. (1953) Grundlagen der Quantenmec~hnnik, Deutscher Verlag der Wissenschaften, Berlin. 16. Phillips, S . E. V. (1978) Nature (Lond.) 273, 247-248.
H.-E. Marcolin, R. Reschke. and A. Trautwein, Angewandte Physik, Fachbereich 12.1, Universitit des Saarlandes, D-6600 Saarbriicken 1 1. Federal Republic of Germany
