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On the electron transfer between cytochrome c molecules as observed by nuclear magnetic resonance
JOURNAL
OF MAGNETIC
RESONANCE
7,66-73 (1972)
On the Electron Transfer Between Cytochrome c Molecules as Observed by Nuclear Magnetic Resonance R. K. GUPTA, S. H. KOENIG, AND A. G . REDFIELD* IBM Thomas J. Watson Research Center, Yorktown Heights, New York 10598
Presentedat the Fourth International Symposiumon Magnetic Resonance, Israel, August, 1971 PulsedFourier transform NMR studieson mixed solutions of the two oxidation statesof cytochromec revealthat the rate of exchangeof oxidation statesby electron transfer is sensitive to the conformation of the protein molecules.The electron transfer process in vitro is found to arise from binary collisions. At low ionic strengths,the rate is affectedby the electrostaticchargeon the protein molecules. INTRODUCTION
Cytochrome c plays an important role in vivo as a link in the m itochondrial electrontransport chain. The protein has also proven to be particularly amenableto study via the recently developed Fourier transform NMR techniques (Z-5). The goal of our current work, of which this is a preliminary report, is to elucidate the electron-transfer mechanism in the light of the protein structure. The usual approach in studying the structure-function relationship is to alter the chemical structure slightly and then observe the effect on the function. For cytochrome c this approach requires the incorporation of m o d ified cytochrome c into cytochrome c depleted m itochondria with a subsequent study of the restoration of respiration. However, altered function of cytochrome c would only be reflectedin the total respiratory processif electron transfer through cytochrome c is the rate-determining step for electron transfer in m itochondria. Furthermore, it would be necessaryto separatethe effect of altered electron transferfunction from altered binding properties of m o d ified cytochrome c which may also affect the total respiratory processin vivo. In vitro methods are available that approximate the in viva function of cytochrome c in varying degrees.O f particular interest to us is the existenceof an exchangeof oxidation states between ferri- and ferrocytochrome
c involving an electron transfer in vitro which
can be directly observedand quantitated by double nuclear magnetic resonancestudies of mixed solutions of both oxidation states (1). Since the cytochrome c molecules in the
m itochondrion also alternate between their two oxidation states as electron transport occurs, it may be expectedintuitively that the dynamic mechanismof the in viva process is similar to that of the in vitro transfer. This similarity has not beendemonstratedas yet. Another attractive feature of cytochrome c is its great stability with respect to pH and temperature changes.Ferrocytochrome c retains its native conformation over the * On leave, during 1971-72,at the Biochemistry Department, University of California, Berkeley, California 94720. 0
1972 by Academic
Press, Inc.
66
ELECTRON TRANSFER IN CYTOCHROME
C MOLECULES
67
pH range 4-12 and ferri over the range pH 3-9. The conformational changes at these extremes are reversible. The protein changesirreversibly only at pH’s below 1 and above 13. In what follows, C3+(N), C2+(N) refer to forms of ferri- and ferrocytochrome c most abundant at neutral pH. C3+(L), C2+(L) refer to the reversible acidic pH conformation of ferricytochrome c with transition point pH z 3 and that of ferrocytochrome c with transition point pH E 4. C3+(H), C2+(H) refer to the reversible basic pH conformation of ferricytochrome c with transition point pH E 9 and that of ferrocytochrome c with transition point pH z 12. THEORY
The NMR absorption spectrum of ferrocytochrome c differs from that of ferricytochrome c. The latter is paramagnetic and shows hyperfine-shifted absorptions which are absent in the diamagnetic ferro form, and the NMR spectrum of a mixture of ferri- and ferrocytochrome c consists of the superimposed resonancesof the two forms. In order to detect electron transfer between ferri- and ferrocytochrome c, we selectively saturate the resonance of a group of spins in protein molecules of one oxidation state and study the transfer of this saturation to the resonance of the same spins in molecules of the other oxidation state in an equilibrium mixture of the two states in solution. Such a transfer is observable since the two oxidation statesinterconvert, via electron exchange, at a rate comparable to the spin-lattice relaxation rate of protons (referred to as moderately rapid) (6). We can, in general, monitor the kinetics of the interconversion of any two, or more, conformations occurring at moderately rapid rates by experimenting on a steady-state mixture. If two rapidly interconverting forms are simultaneously present at equilibrium, a time-averaged NMR spectrum of the two is observed. Very slow interconversion, on the other hand, gives rise to a simple superposition of the two spectra with no double resonance effects. In cases of either slow (S) or rapid (F) processes, one can only put upper or lower limits on the rate, respectively. We would briefly review the basis for our measurements of moderately rapid (M) rates below. Among the many interesting features of the native cytochrome c NMR spectra is the presence of a well-resolved upfield-shifted methyl group resonance of the methionine coordinated to the face of the heme in both oxidation states (7,8). The shift arises from induced diamagnetism of the heme ring in the reduced state, and predominantly from hyperfine interaction (presumably of contact type occurring via a spin polarization mechanism) in the oxidized state (4). Further, the methionine methyl group spins are relaxed via hyperfine interaction with the unpaired electron in the oxidized state and have a short spin-lattice relaxation time (-2 mseconds) in contrast to a significantly longer time in the reduced state (-200 mseconds). The exchange time in a typical solution is, in general, so much longer than 2 mseconds that we may regard the methyl group spins as being instantaneously relaxed in the oxidized state. For the reduced-state magnetization we then have
where M,R, A4: are the magnetizations in the reduced and oxidized state, respectively, with equilibrium values Mt, M,O. Tf is . t h e spin-lattice relaxation time in the isolated
68
GUPTA, KOENIG, AND REDFIELD
reduced state. rR and TV are the exchange times in the reduced and oxidized states, respectively. Since Tf’ (spin-lattice relaxation time in the oxidized state) ~0, then Mf z M,O, and since at thermal equilibrium, (MO”M,R) = (TJ~,& Eq. [I] can be rewritten as
This analysis shows that in a mixture of oxidation states, when the oxidized state has a very short spin-lattice relaxation time, the return of the reduced-state magnetization to equilibrium following a perturbation will be exponential with apparent spin-lattice relaxation time TTu given by (TfJ-’
= (Tf)-’ + (T&I.
EXPERIMENTAL
131
RESULTS
Experiments reported here were done at room temperature (25’) unless otherwise specified. Measurements were made at 100 MHz employing a home-built pulsed Fourier transform NMR spectrometer (9). Electron-transfer rates were obtained from the experimental values of the exchange time TV which in turn was calculated from the measured values of Tf and Tfa for the methionine methyl resonance via Eq. [3]. T, measurements were made employing a 180”-90” pulse sequence (20) separated by a variable time t. Numerical values for Tl were obtained from the values of t that gave zero signal after the 90” pulse (the “zero-crossing” method). The absolute values of T, may be systematically in error by 115 %, but the relative values of Tl have uncertainties -5 %. Interconversion of Various Monomeric Forms of Cytochrome c
We experimented on a mixture of the two oxidation states of cytochrome c in solution near its isoelectric point (pH z IO), where C3+(N), C3+(H) and C2+(N) exist simultaneously. The observed NMR spectrum is then a weighted superposition of three distinct spectra. Upon saturating the hyperhne shifted heme ring methyl resonancesof either C3+(N) or C3+(H) we searched for a double NMR effect in the region of the spectrum where the ring methyl resonancesof the other forms were expected.The results were negative for the equilibrium between C3+(N) and C3+(H) and between C2+(N) and C3+(H) showing that such forms interconvert only very slowly on our time scale. A positive effect was, on the other hand, seen for the equilibrium between C3+(N) and C2+(N) (see Fig. 1) showing moderately rapid electron-transfer linked interconversion of the neutral pH forms even at the isoelectric point. Further studies of the NMR spectra at low pH, their relaxation and double resonance properties lead us to the following interconversion diagram:
c3+ (L) I
c2+ (L)
c3+ (N) -h3+(H)
69
ELECTRONTRANSFERINCYTOCHROMECMOLECULES HDO
FIG. 1. A difference spectrum showing the presence of a double NMR effect between the resonances of the (I?+(N) and G+(N) forms of cytochrome c (pH x 10, temp. = 25°C). The trace was obtained averaging and Fourier-transforming the free induction decay signal following a 90” pulse. A saturating double irradiation pulse alternated between the two methyl resonances near -35 and -32 ppm belonging to C3+(N) form. The difference spectrum was obtained by adding and subtracting the alternate sweeps during the averaging process. Saturation was transferred to the methyl resonances of C’+(N) at -2.1 and -3.8 ppm as shown. (Reference for field shifts is the standard DSS methyl resonance.)
Electron-Transfer
Process
Experimental results on the rate of the electron-transfer process are summarized below. (i) We detect the presenceof a moderately rapid electron transfer even in a sample well electrodialyzed to its isoelectric point (pH x 10) to remove any small ions from solution. (ii) At constant ionic strength near neutral pH the rate of exchangeprocess shows first-order dependence on the cytochrome c concentration and one can calculate a bimolecular rate constant for the process given by k = (T~[ox])-~ where [ox] is the concentration of oxidized cytochrome c. (iii) There is no observable isotope effect in the kinetics in going from HZ0 to D,O solvent.
I FIG. 2. pH dependence of the electron-transfer rate at low ionic strength (5.05M 3*
HEPES).
70
GUPTA, KOENIG, AND REDFIELD
(iu) In the absence of any appreciable concentration of small ions, the rate is dependent on pH in the range investigated (pH 6-11) (pH changed by adding just sufficient amounts of HEPES or NaOD as needed). This dependence is shown in Fig. 2. The rate increases with increasing pH peaking near the isoelectric point (pH z 10) and then it falls. (0) The rate of the exchange process near neutral pH is strongly affected by the presence of small ions, in contrast to the rate at the isoelectric point where there is no effect of small ions, within the experimental error. The dependence of the rate at pH 7 on salt concentration is shown in Fig. 3 plotted US(ionic strength)‘j2.
I 20 (IONIC STRENGTH ~10~) ‘I2
FIG. 3. Graph showing the ionic strength dependence of the rate of electron transfer near neutral pH.
(vi) The rate at high ionic strength (-1M) varies with pH in a manner shown in Fig. 4. Here one obtains a fairly constant rate up to about pH 8.5 which gradually falls off to zero with increasing pH. KCI, KBr, KC103 all effect the rate near neutral pH in a similar fashion.
FIG. 4. pH dependence of the electron-transfer rate at high ionic strength (-1M KCl).
ELECTRON TRANSFER IN CYTOCHROME
C MOLECULES
71
(u/i) The rate is dependent on temperature. An Arrhenius plot yields an activation energy of -10 kcal for the exchange process at neutral pH and low ionic strength. DISCUSSION
It follows from the interconversion diagram that only the neutral pH form of cytochrome c (and neither the basic nor acidic forms) is capable of transferring electrons at a moderately rapid rate. We have shown earlier that perturbing the oxidized cytochrome c in the neutral conformation with cyanide also has an inhibitory effect on exchange (I). Thus, electron transfer between cytochrome c molecules is sensitive to the conformation of the protein and the possibility exists of using it as a novel approach to study structurefunction relationship. The basic interest in any electron transfer problem is, however, in the elucidation of the physical mechanism of the process. For cytochrome c this would mean an understanding of the paths for flow of electrons into and out of the protein molecule and the nature of the activated complex existing during electron transfer. Although NMR does not answer all our questions in this regard, it does yield the following information. The exchange process does not require the mediation of small ions. The exchange of oxidation states occurs via a direct process resulting from binary collisions as evidenced by the first-order dependenceof rates on cytochrome c concentration. The absenceof an isotope effect indicates that no proton motion is likely to be involved or that, at least, the proton motion is not the rate limiting step for the process. It is difficult to visualize direct transfer of an electron from the heme iron of one macromolecule to another, since the iron is buried deep in the protein. However, the X-ray structure of cytochrome c has revealed that one edge of the heme is exposed to the solvent (II), which suggeststhe possibility of direct transfer through the edge of the heme ring. The extensive delocalization of the unpaired electron to the heme periphery in cytochrome c lends support to this view (7). One expects the rate at basic pH to be dependent on pH becausethe exchange-active form of the oxidized protein C3+(N) changes to an inactive form C)+(H) with pK = 9. So one expects to seea fairly constant rate below pH M 9, and a progressive decreasein the exchange rate at higher pH as the concentration of the exchange-active species decreases.However, this does not happen at least in the absenceof small ions. The rate at low ionic strength reachesa peak value near the isoelectric point (pH z 10) where the concentration of the exchange-active species has dropped to about 10 % of its neutral pH value. Obviously some other effect is coming into play. The rate constants obtained after correction for the concentration of the exchange-active speciesare P”(pH
x 10) = 2 x 104,k”““(pH = 7) w lo3 liter mole-’ second-‘.
There is, thus, a difference of an order of magnitude in the rate constants at pH 7 and pH 10. The only difference in the exchange-active form of the protein at pH 10 and pH 7 lies in its charge-state. Figure 5 shows an approximate charge us pH curve for cytochrome c. Any net charge on the protein molecule was assumed to be uniformly smeared over the protein surface for purposes of calculating its effect on the dissociation of constituent amino acids. At pH x 10, each protein molecule is electrically neutral, but at pH 7 it bears -+lO electron units of electrostatic charge. On the basis of this observation, we believe that for electron transfer to take place, the oxidized and reduced
72
GUPTA, KOENIG, AND REDFIELD
protein molecules have to approach quite closely to each other for the donor orbitals of reduced cytochrome c to overlap effectively with the acceptor orbitals of oxidized cytochrome c. When the protein molecules are charged, their distance of closest approach is limited due to electrostatic repulsion. This limitation results in a decrease in the electron transfer rate. Our qualitative model is supported by our observation that the rate at pH 7, but not at pH 10, is affected by the presenceof small ions. At pH w 10, the macromolecules are electrically neutral, and therefore the presenceof small ions does not affect the rate. At other pH values, the presenceof small ions can shield the charge on the protein. Further support for our model comes from the observation that the rate of exchange appears to
+201~1
-20’;
I 4
I 6
I 8
I IO
I 12
14
PH
FIG. 5. Approximate curve showing the dependence of the electrostatic charge on the cytochrome c molecules with pH.
behave in the expected manner with pH (i.e., the rate constant appears to be roughly independent of pH, when corrected for varying concentration of the exchange-active specieswith pH) at high salt (-l&f), when all charge effects will be well screened. It is tempting to attempt a crude calculation of the diEusion limited rate for the exchange process. At pH 10, where the protein molecules bear no net charge, the rate constant assuming electron transfer during every binary collision (k,,,,) is given by (12). kdiff = (87rdDN/103)w 5 x lo9 liter mole-’ second-‘,
141
where d is the distance of closest approach (essentially the diameter of the protein molecules (w 34 A)), D is the diffusion constant for cytochrome c, (w lO-‘j cm*/second) and N is Avogadro’s number (6 x 1023/male).The fact that observed rate constant k,,,(2 x 104) is several orders of magnitude less than this suggests that only those collisions for which the colliding molecules have a certain specific orientation result in electron transfer. Taking the orientation factor into account [kobs= kdirf (area of exchange site/total surface area)z] yields for the area of the exchange site a value of ~0.2 % of the total surface area or 7 A* corresponding to a circle of diameter 3 A. This estimate ignores any local electrostatic interactions which may exist at the isoelectric point. Furthermore, the transfer will be aided by any slight association between oxidized and reduced forms but impeded by the probable need (23) for the two forms to be in similar conformation during transfer, rather than in different conformation indicated by X-ray diffraction (14).
ELECTRON TRANSFER IN CYTOCHROME C MOLECULES
73
CONCLUSION
We have shown that electron transfer in vitro between cytochrome c molecules of different oxidation statesoccurs with moderate rapidity only when the protein molecules are in their native conformation. The transfer rate is maximum at the isoelectric point (pH M 10) when the net charge on the protein is zero, and decreasesas the net charge increases,the more so the less the charge is shielded by small ions. The electron exchange observed by NMR may well involve the same pathways that are imporant in electron transport in vivo. If so, then charge effects may be similarly important in vivo, and in vitro studies of electron exchange between cytochrome c molecules of different oxidation state, or between cytochrome c and other protein molecules, can be a unique technique for studying an important biological function. ACKNOWLEDGMENT We thank Dr. D. W. Schaefer for the use of his computer program to calculate charge vs pH curve. REFERENCES 1. R. K. GUPTA AND A. G. REDFIELD, Science 169,1204 (1910). 2. R. K. GUPTA AND A. G. REDFIELD,Biochem. Biophys. Res. Commun. 41,213 (1910).
3. R. K. GUPTA AND A. G. REDFIELD,“Proc. 1910 Wenner Gren Symp. Struct. Funct. Oxidation Reduction Enzymes,” Pergamon Press, Oxford, in press. 4. A. G. REDFIELDAND R. K. GUPTA, “Proc. Cold Spring Harbor Symp. Quant. Biol.,” 36,44X(1911). 5. R. K. GUPTA AND S. H. KOENIG, Biochem. Biophys. Res. Commun., 45,1134 (1911). 6. S. FORSENAND R. A. HOFFMANN,J. Chem. Phys. 39,2892 (1963). 7. K. WUTHRICH,Proc. Nat. Acad. Sci. U.S. 63,lOll (1969). 8. C. C. MCDONALD, W. D. PHILLIPS, AND S. N. VINOGRADOV, Biochem. Biophys. Res. Commun. 36, 442 (1969). 9. A. G. REDRELD AND R. K. GUPTA, Aduan. Magn. Resonance, 1911, in press; A. G. REDFIELD AND R. K. GUPTA,J. Chem. Phys. 54,1418 (1911). 10. R. L. VOLD, J. S. WAUGH, M. P. KLEIN, AND D. E. PHELPS,J. Chem. Phys. 48,3831(1968). 11. R. E. DICKERSON, T. TAKANO, D. EISENBERG,0. B. KALLAI, L. SAMPSON,A. COOPER, AND E. MARGOLIASH,J. Biol. Chem. 246,151l (1911). 12. S. W. BENSON,“The Foundations of Chemical Kinetics,” p. 493ff, McGraw-Hill, New York, 1960. 13. F. A. COLON AND G. WILKINSON, “Advanced Inorganic Chemistry,” Second Edition, p. 118, Interscience, New York, 1966. 14. T. TAKANO, R. SWANSON,0. B. KALLAI, AND R. E. DICKERSON,“Proc. Cold Spring Harbor Symp. Quant. Biol.,” 36,397 (1911).
OF MAGNETIC
RESONANCE
7,66-73 (1972)
On the Electron Transfer Between Cytochrome c Molecules as Observed by Nuclear Magnetic Resonance R. K. GUPTA, S. H. KOENIG, AND A. G . REDFIELD* IBM Thomas J. Watson Research Center, Yorktown Heights, New York 10598
Presentedat the Fourth International Symposiumon Magnetic Resonance, Israel, August, 1971 PulsedFourier transform NMR studieson mixed solutions of the two oxidation statesof cytochromec revealthat the rate of exchangeof oxidation statesby electron transfer is sensitive to the conformation of the protein molecules.The electron transfer process in vitro is found to arise from binary collisions. At low ionic strengths,the rate is affectedby the electrostaticchargeon the protein molecules. INTRODUCTION
Cytochrome c plays an important role in vivo as a link in the m itochondrial electrontransport chain. The protein has also proven to be particularly amenableto study via the recently developed Fourier transform NMR techniques (Z-5). The goal of our current work, of which this is a preliminary report, is to elucidate the electron-transfer mechanism in the light of the protein structure. The usual approach in studying the structure-function relationship is to alter the chemical structure slightly and then observe the effect on the function. For cytochrome c this approach requires the incorporation of m o d ified cytochrome c into cytochrome c depleted m itochondria with a subsequent study of the restoration of respiration. However, altered function of cytochrome c would only be reflectedin the total respiratory processif electron transfer through cytochrome c is the rate-determining step for electron transfer in m itochondria. Furthermore, it would be necessaryto separatethe effect of altered electron transferfunction from altered binding properties of m o d ified cytochrome c which may also affect the total respiratory processin vivo. In vitro methods are available that approximate the in viva function of cytochrome c in varying degrees.O f particular interest to us is the existenceof an exchangeof oxidation states between ferri- and ferrocytochrome
c involving an electron transfer in vitro which
can be directly observedand quantitated by double nuclear magnetic resonancestudies of mixed solutions of both oxidation states (1). Since the cytochrome c molecules in the
m itochondrion also alternate between their two oxidation states as electron transport occurs, it may be expectedintuitively that the dynamic mechanismof the in viva process is similar to that of the in vitro transfer. This similarity has not beendemonstratedas yet. Another attractive feature of cytochrome c is its great stability with respect to pH and temperature changes.Ferrocytochrome c retains its native conformation over the * On leave, during 1971-72,at the Biochemistry Department, University of California, Berkeley, California 94720. 0
1972 by Academic
Press, Inc.
66
ELECTRON TRANSFER IN CYTOCHROME
C MOLECULES
67
pH range 4-12 and ferri over the range pH 3-9. The conformational changes at these extremes are reversible. The protein changesirreversibly only at pH’s below 1 and above 13. In what follows, C3+(N), C2+(N) refer to forms of ferri- and ferrocytochrome c most abundant at neutral pH. C3+(L), C2+(L) refer to the reversible acidic pH conformation of ferricytochrome c with transition point pH z 3 and that of ferrocytochrome c with transition point pH E 4. C3+(H), C2+(H) refer to the reversible basic pH conformation of ferricytochrome c with transition point pH E 9 and that of ferrocytochrome c with transition point pH z 12. THEORY
The NMR absorption spectrum of ferrocytochrome c differs from that of ferricytochrome c. The latter is paramagnetic and shows hyperfine-shifted absorptions which are absent in the diamagnetic ferro form, and the NMR spectrum of a mixture of ferri- and ferrocytochrome c consists of the superimposed resonancesof the two forms. In order to detect electron transfer between ferri- and ferrocytochrome c, we selectively saturate the resonance of a group of spins in protein molecules of one oxidation state and study the transfer of this saturation to the resonance of the same spins in molecules of the other oxidation state in an equilibrium mixture of the two states in solution. Such a transfer is observable since the two oxidation statesinterconvert, via electron exchange, at a rate comparable to the spin-lattice relaxation rate of protons (referred to as moderately rapid) (6). We can, in general, monitor the kinetics of the interconversion of any two, or more, conformations occurring at moderately rapid rates by experimenting on a steady-state mixture. If two rapidly interconverting forms are simultaneously present at equilibrium, a time-averaged NMR spectrum of the two is observed. Very slow interconversion, on the other hand, gives rise to a simple superposition of the two spectra with no double resonance effects. In cases of either slow (S) or rapid (F) processes, one can only put upper or lower limits on the rate, respectively. We would briefly review the basis for our measurements of moderately rapid (M) rates below. Among the many interesting features of the native cytochrome c NMR spectra is the presence of a well-resolved upfield-shifted methyl group resonance of the methionine coordinated to the face of the heme in both oxidation states (7,8). The shift arises from induced diamagnetism of the heme ring in the reduced state, and predominantly from hyperfine interaction (presumably of contact type occurring via a spin polarization mechanism) in the oxidized state (4). Further, the methionine methyl group spins are relaxed via hyperfine interaction with the unpaired electron in the oxidized state and have a short spin-lattice relaxation time (-2 mseconds) in contrast to a significantly longer time in the reduced state (-200 mseconds). The exchange time in a typical solution is, in general, so much longer than 2 mseconds that we may regard the methyl group spins as being instantaneously relaxed in the oxidized state. For the reduced-state magnetization we then have
where M,R, A4: are the magnetizations in the reduced and oxidized state, respectively, with equilibrium values Mt, M,O. Tf is . t h e spin-lattice relaxation time in the isolated
68
GUPTA, KOENIG, AND REDFIELD
reduced state. rR and TV are the exchange times in the reduced and oxidized states, respectively. Since Tf’ (spin-lattice relaxation time in the oxidized state) ~0, then Mf z M,O, and since at thermal equilibrium, (MO”M,R) = (TJ~,& Eq. [I] can be rewritten as
This analysis shows that in a mixture of oxidation states, when the oxidized state has a very short spin-lattice relaxation time, the return of the reduced-state magnetization to equilibrium following a perturbation will be exponential with apparent spin-lattice relaxation time TTu given by (TfJ-’
= (Tf)-’ + (T&I.
EXPERIMENTAL
131
RESULTS
Experiments reported here were done at room temperature (25’) unless otherwise specified. Measurements were made at 100 MHz employing a home-built pulsed Fourier transform NMR spectrometer (9). Electron-transfer rates were obtained from the experimental values of the exchange time TV which in turn was calculated from the measured values of Tf and Tfa for the methionine methyl resonance via Eq. [3]. T, measurements were made employing a 180”-90” pulse sequence (20) separated by a variable time t. Numerical values for Tl were obtained from the values of t that gave zero signal after the 90” pulse (the “zero-crossing” method). The absolute values of T, may be systematically in error by 115 %, but the relative values of Tl have uncertainties -5 %. Interconversion of Various Monomeric Forms of Cytochrome c
We experimented on a mixture of the two oxidation states of cytochrome c in solution near its isoelectric point (pH z IO), where C3+(N), C3+(H) and C2+(N) exist simultaneously. The observed NMR spectrum is then a weighted superposition of three distinct spectra. Upon saturating the hyperhne shifted heme ring methyl resonancesof either C3+(N) or C3+(H) we searched for a double NMR effect in the region of the spectrum where the ring methyl resonancesof the other forms were expected.The results were negative for the equilibrium between C3+(N) and C3+(H) and between C2+(N) and C3+(H) showing that such forms interconvert only very slowly on our time scale. A positive effect was, on the other hand, seen for the equilibrium between C3+(N) and C2+(N) (see Fig. 1) showing moderately rapid electron-transfer linked interconversion of the neutral pH forms even at the isoelectric point. Further studies of the NMR spectra at low pH, their relaxation and double resonance properties lead us to the following interconversion diagram:
c3+ (L) I
c2+ (L)
c3+ (N) -h3+(H)
69
ELECTRONTRANSFERINCYTOCHROMECMOLECULES HDO
FIG. 1. A difference spectrum showing the presence of a double NMR effect between the resonances of the (I?+(N) and G+(N) forms of cytochrome c (pH x 10, temp. = 25°C). The trace was obtained averaging and Fourier-transforming the free induction decay signal following a 90” pulse. A saturating double irradiation pulse alternated between the two methyl resonances near -35 and -32 ppm belonging to C3+(N) form. The difference spectrum was obtained by adding and subtracting the alternate sweeps during the averaging process. Saturation was transferred to the methyl resonances of C’+(N) at -2.1 and -3.8 ppm as shown. (Reference for field shifts is the standard DSS methyl resonance.)
Electron-Transfer
Process
Experimental results on the rate of the electron-transfer process are summarized below. (i) We detect the presenceof a moderately rapid electron transfer even in a sample well electrodialyzed to its isoelectric point (pH x 10) to remove any small ions from solution. (ii) At constant ionic strength near neutral pH the rate of exchangeprocess shows first-order dependence on the cytochrome c concentration and one can calculate a bimolecular rate constant for the process given by k = (T~[ox])-~ where [ox] is the concentration of oxidized cytochrome c. (iii) There is no observable isotope effect in the kinetics in going from HZ0 to D,O solvent.
I FIG. 2. pH dependence of the electron-transfer rate at low ionic strength (5.05M 3*
HEPES).
70
GUPTA, KOENIG, AND REDFIELD
(iu) In the absence of any appreciable concentration of small ions, the rate is dependent on pH in the range investigated (pH 6-11) (pH changed by adding just sufficient amounts of HEPES or NaOD as needed). This dependence is shown in Fig. 2. The rate increases with increasing pH peaking near the isoelectric point (pH z 10) and then it falls. (0) The rate of the exchange process near neutral pH is strongly affected by the presence of small ions, in contrast to the rate at the isoelectric point where there is no effect of small ions, within the experimental error. The dependence of the rate at pH 7 on salt concentration is shown in Fig. 3 plotted US(ionic strength)‘j2.
I 20 (IONIC STRENGTH ~10~) ‘I2
FIG. 3. Graph showing the ionic strength dependence of the rate of electron transfer near neutral pH.
(vi) The rate at high ionic strength (-1M) varies with pH in a manner shown in Fig. 4. Here one obtains a fairly constant rate up to about pH 8.5 which gradually falls off to zero with increasing pH. KCI, KBr, KC103 all effect the rate near neutral pH in a similar fashion.
FIG. 4. pH dependence of the electron-transfer rate at high ionic strength (-1M KCl).
ELECTRON TRANSFER IN CYTOCHROME
C MOLECULES
71
(u/i) The rate is dependent on temperature. An Arrhenius plot yields an activation energy of -10 kcal for the exchange process at neutral pH and low ionic strength. DISCUSSION
It follows from the interconversion diagram that only the neutral pH form of cytochrome c (and neither the basic nor acidic forms) is capable of transferring electrons at a moderately rapid rate. We have shown earlier that perturbing the oxidized cytochrome c in the neutral conformation with cyanide also has an inhibitory effect on exchange (I). Thus, electron transfer between cytochrome c molecules is sensitive to the conformation of the protein and the possibility exists of using it as a novel approach to study structurefunction relationship. The basic interest in any electron transfer problem is, however, in the elucidation of the physical mechanism of the process. For cytochrome c this would mean an understanding of the paths for flow of electrons into and out of the protein molecule and the nature of the activated complex existing during electron transfer. Although NMR does not answer all our questions in this regard, it does yield the following information. The exchange process does not require the mediation of small ions. The exchange of oxidation states occurs via a direct process resulting from binary collisions as evidenced by the first-order dependenceof rates on cytochrome c concentration. The absenceof an isotope effect indicates that no proton motion is likely to be involved or that, at least, the proton motion is not the rate limiting step for the process. It is difficult to visualize direct transfer of an electron from the heme iron of one macromolecule to another, since the iron is buried deep in the protein. However, the X-ray structure of cytochrome c has revealed that one edge of the heme is exposed to the solvent (II), which suggeststhe possibility of direct transfer through the edge of the heme ring. The extensive delocalization of the unpaired electron to the heme periphery in cytochrome c lends support to this view (7). One expects the rate at basic pH to be dependent on pH becausethe exchange-active form of the oxidized protein C3+(N) changes to an inactive form C)+(H) with pK = 9. So one expects to seea fairly constant rate below pH M 9, and a progressive decreasein the exchange rate at higher pH as the concentration of the exchange-active species decreases.However, this does not happen at least in the absenceof small ions. The rate at low ionic strength reachesa peak value near the isoelectric point (pH z 10) where the concentration of the exchange-active species has dropped to about 10 % of its neutral pH value. Obviously some other effect is coming into play. The rate constants obtained after correction for the concentration of the exchange-active speciesare P”(pH
x 10) = 2 x 104,k”““(pH = 7) w lo3 liter mole-’ second-‘.
There is, thus, a difference of an order of magnitude in the rate constants at pH 7 and pH 10. The only difference in the exchange-active form of the protein at pH 10 and pH 7 lies in its charge-state. Figure 5 shows an approximate charge us pH curve for cytochrome c. Any net charge on the protein molecule was assumed to be uniformly smeared over the protein surface for purposes of calculating its effect on the dissociation of constituent amino acids. At pH x 10, each protein molecule is electrically neutral, but at pH 7 it bears -+lO electron units of electrostatic charge. On the basis of this observation, we believe that for electron transfer to take place, the oxidized and reduced
72
GUPTA, KOENIG, AND REDFIELD
protein molecules have to approach quite closely to each other for the donor orbitals of reduced cytochrome c to overlap effectively with the acceptor orbitals of oxidized cytochrome c. When the protein molecules are charged, their distance of closest approach is limited due to electrostatic repulsion. This limitation results in a decrease in the electron transfer rate. Our qualitative model is supported by our observation that the rate at pH 7, but not at pH 10, is affected by the presenceof small ions. At pH w 10, the macromolecules are electrically neutral, and therefore the presenceof small ions does not affect the rate. At other pH values, the presenceof small ions can shield the charge on the protein. Further support for our model comes from the observation that the rate of exchange appears to
+201~1
-20’;
I 4
I 6
I 8
I IO
I 12
14
PH
FIG. 5. Approximate curve showing the dependence of the electrostatic charge on the cytochrome c molecules with pH.
behave in the expected manner with pH (i.e., the rate constant appears to be roughly independent of pH, when corrected for varying concentration of the exchange-active specieswith pH) at high salt (-l&f), when all charge effects will be well screened. It is tempting to attempt a crude calculation of the diEusion limited rate for the exchange process. At pH 10, where the protein molecules bear no net charge, the rate constant assuming electron transfer during every binary collision (k,,,,) is given by (12). kdiff = (87rdDN/103)w 5 x lo9 liter mole-’ second-‘,
141
where d is the distance of closest approach (essentially the diameter of the protein molecules (w 34 A)), D is the diffusion constant for cytochrome c, (w lO-‘j cm*/second) and N is Avogadro’s number (6 x 1023/male).The fact that observed rate constant k,,,(2 x 104) is several orders of magnitude less than this suggests that only those collisions for which the colliding molecules have a certain specific orientation result in electron transfer. Taking the orientation factor into account [kobs= kdirf (area of exchange site/total surface area)z] yields for the area of the exchange site a value of ~0.2 % of the total surface area or 7 A* corresponding to a circle of diameter 3 A. This estimate ignores any local electrostatic interactions which may exist at the isoelectric point. Furthermore, the transfer will be aided by any slight association between oxidized and reduced forms but impeded by the probable need (23) for the two forms to be in similar conformation during transfer, rather than in different conformation indicated by X-ray diffraction (14).
ELECTRON TRANSFER IN CYTOCHROME C MOLECULES
73
CONCLUSION
We have shown that electron transfer in vitro between cytochrome c molecules of different oxidation statesoccurs with moderate rapidity only when the protein molecules are in their native conformation. The transfer rate is maximum at the isoelectric point (pH M 10) when the net charge on the protein is zero, and decreasesas the net charge increases,the more so the less the charge is shielded by small ions. The electron exchange observed by NMR may well involve the same pathways that are imporant in electron transport in vivo. If so, then charge effects may be similarly important in vivo, and in vitro studies of electron exchange between cytochrome c molecules of different oxidation state, or between cytochrome c and other protein molecules, can be a unique technique for studying an important biological function. ACKNOWLEDGMENT We thank Dr. D. W. Schaefer for the use of his computer program to calculate charge vs pH curve. REFERENCES 1. R. K. GUPTA AND A. G. REDFIELD, Science 169,1204 (1910). 2. R. K. GUPTA AND A. G. REDFIELD,Biochem. Biophys. Res. Commun. 41,213 (1910).
3. R. K. GUPTA AND A. G. REDFIELD,“Proc. 1910 Wenner Gren Symp. Struct. Funct. Oxidation Reduction Enzymes,” Pergamon Press, Oxford, in press. 4. A. G. REDFIELDAND R. K. GUPTA, “Proc. Cold Spring Harbor Symp. Quant. Biol.,” 36,44X(1911). 5. R. K. GUPTA AND S. H. KOENIG, Biochem. Biophys. Res. Commun., 45,1134 (1911). 6. S. FORSENAND R. A. HOFFMANN,J. Chem. Phys. 39,2892 (1963). 7. K. WUTHRICH,Proc. Nat. Acad. Sci. U.S. 63,lOll (1969). 8. C. C. MCDONALD, W. D. PHILLIPS, AND S. N. VINOGRADOV, Biochem. Biophys. Res. Commun. 36, 442 (1969). 9. A. G. REDRELD AND R. K. GUPTA, Aduan. Magn. Resonance, 1911, in press; A. G. REDFIELD AND R. K. GUPTA,J. Chem. Phys. 54,1418 (1911). 10. R. L. VOLD, J. S. WAUGH, M. P. KLEIN, AND D. E. PHELPS,J. Chem. Phys. 48,3831(1968). 11. R. E. DICKERSON, T. TAKANO, D. EISENBERG,0. B. KALLAI, L. SAMPSON,A. COOPER, AND E. MARGOLIASH,J. Biol. Chem. 246,151l (1911). 12. S. W. BENSON,“The Foundations of Chemical Kinetics,” p. 493ff, McGraw-Hill, New York, 1960. 13. F. A. COLON AND G. WILKINSON, “Advanced Inorganic Chemistry,” Second Edition, p. 118, Interscience, New York, 1966. 14. T. TAKANO, R. SWANSON,0. B. KALLAI, AND R. E. DICKERSON,“Proc. Cold Spring Harbor Symp. Quant. Biol.,” 36,397 (1911).