Low-frequency-dependent effects of oscillating magnetic elds on radical pair recombination in enzyme kinetics

C. Eichwald and J. Walleczeka)

Department of Radiation Oncology, Biolelectromagnetics Laboratory, School of MedicineAO38, Stanford University, Stanford, California 94305-5124

Received 16 December 1996; accepted 27 June 1997 A model of an enzyme reaction cycle that includes the generation of a transient spin-correlated radical pair state is discussed. The recombination yield of the radical pair state is altered by external magnetic elds radical pair mechanism . In this theoretical study, the response behavior of the enzyme to pulsed magnetic elds as well as combinations of static and sinusoidally oscillating magnetic elds is investigated by using an approach that combines enzyme kinetics with magnetic eld-sensitive spin kinetics. Calculations show that the enzyme behaves like a frequency sensor that is responsive at lower eld frequencies but less responsive at frequencies that are faster than the time scales inherent to the kinetic properties of the reaction cycle. There is a characteristic transition region in the frequency domain that reects the enzymes relaxation behavior to time-dependent external perturbations. The transition region is characterized by using methods based on the theory of externally driven systems, including Floquet theory and the calculation of correlation functions. Model simulations suggest that time-dependent magnetic elds could be used as a tool to study the response behavior of magnetic eld-sensitive enzymes. 1997 American Institute of Physics. S0021-9606 97 02437-9

I. INTRODUCTION

Transient kinetic methods have long been utilized for studying enzymes for an overview see, for example, Refs. 13 . Chemical relaxation methods make it possible to determine important information regarding enzyme mechanisms and the underlying kinetics. In relaxation techniques external perturbations are applied to the system, including variations of temperature, pressure, and electric elds.1 These external parameters may be varied either periodically sinusoidally or in the form of rectangular pulses , or in a single step impulse. In this contribution a method is discussed that utilizes time-dependent magnetic elds for investigating the kinetics of magnetic eld-sensitive enzymes. The background for this study is based on experimental ndings that revealed static magnetic eld effects on the in vitro activity of B 12-dependent enzymes.48 Specically, in the case of ethanolamine ammonia lyase it was shown that the magnetic eld-dependent step is recombination of a transient spincorrelated radical pair that is formed in the reaction cycle of the enzyme 5 -deoxyadenosyl, cob II alamin - radical pair produced by enzyme-induced homolysis of the CCo bond .6,8 More recently, magnetic eld effects were reported on the redox cycle of horseradish peroxidase.9 An interpretation of these experimental ndings is based on the radical pair mechanism.7 In this mechanism the magnetic eld modulates the recombination yield of spin-correlated radical pairs that are formed as intermediates in chemical or biochemical reaction pathways.1013 The radical pair mechanism is a well-established tool for investigating the kinetics of reactions that proceed via free radical-dependent steps.7,1015
a

Author to whom correspondence should be addressed.

Based on the experimental ndings a prototypical model of a magnetic eld-sensitive enzyme system has been developed.16 The model is based on an approach that combines enzyme kinetics with magnetic eld-dependent spin kinetics. It qualitatively reproduces the reported biphasic magnetic eld effect on the in vitro activity of B 12-dependent ethanolamine ammonia lyase.16 Calculations show that the combination of physical and biochemical mechanisms magnetic eld modulation of radical pair recombination probability and kinetic processes within the enzyme reaction cycle, respectively is a necessary prerequisite for a qualitative as well as a quantitative interpretation of magnetic eld effects on enzyme activity.16 In this contribution the original model is extended to enable studying time-dependent magnetic eld perturbations, including pulsed elds as well as combinations of static and sinusoidally oscillating elds. Motivation for these investigations is based on an earlier hyphothesis that suggested the possibility of low-frequency-dependent magnetic eld effects in biological systems via radical pair recombination processes in enzyme kinetics.14,17 In this case the radical pair mechanism serves as the primary coupling mechanism of the magnetic eld to the enzyme, whereas dependencies on the eld frequency are related to the kinetic properties of the enzyme reaction cycle. These interactions are nonresonant in nature, meaning that there is no resonant coupling of the oscillating magnetic eld to the radical pair system. The response behavior of the enzyme to time-dependent magnetic eld perturbations is studied. Results show that the enzyme behaves like a frequency sensor that is responsive at lower eld frequencies but less responsive at frequencies that are faster than the time scales inherent to the kinetic properties of the reaction cycle. There is a characteristic transition
1997 American Institute of Physics 4943

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C. Eichwald and J. Walleczek: Oscillating magnetic eld effects

region that reects the enzymes relaxation behavior to timedependent external perturbations. Results suggest that timedependent magnetic elds could be used as a tool for studying the relaxation behavior of magnetic eld-sensitive enzyme systems.
II. OUTLINE OF THE MODEL AND CALCULATIONS

The following enzyme reaction scheme is investigated:16

k1 k2

E S
k 1

ES
k 2

k3 S

E S k isc
k3 k4

ES * E ,

P 1

E S

where the forward backward rate constants are denoted as k i (k i ), and the other symbols represent the following: E, enzyme, (ES), enzymesubstrate complex prior to radical pair generation, S/T (E S), enzymesubstrate complex where a spin-correlated radical pair exists, (ES) * , enzyme substrate complex prior to product release, S, substrate, and P, product. The superscript denotes spin multiplicity S, singlet, T, triplet , and k isc is a magnetic eld-dependent rate constant characterizing singlettriplet mixing. A shorthand notation, T (E S), representing all three triplet states T T 1 ,T 0 ,T 1 is used the index refers to the magnetic quantum number . The enzyme reaction scheme, Eq. 1 , represents a prototypical approach for describing magnetic eld effects on enzyme activity.7,16 Radical pair generation is via k 2 , and recombination via k 2 . The rate constant k 3 characterizes spin-independent forward reaction coordinate motion. It is used here to represent the lifetime of the spin-correlated radical pair state. It is assumed that the magnetic eld sensitivity arises solely from the modulation of the population of the singlet and triplet states. All subsequent processes preceding product release via k 4 are not considered to exhibit a requirement for spin correlation.16 The enzyme reaction scheme exhibits transitions between different states that operate on very different time scales. This is a result of combining processes related to chemical kinetics and to magnetic elddependent spin kinetics. The rate constants k 2 radical pair recombination , k 3 forward reaction coordinate motion , and k isc singlettriplet mixing represent fast time scales in the 1100 ns time domain.16 For example, for the B 12-dependent systems a rate constant for radical pair recombination in the order of k rec 109 s 1 was estimated.4,8 The remaining rate constants are related to the much slower processes within the enzyme reaction cycle typical catalytic rate constants are in the range 10 105 s 1 .13 The introduction of an effective rate constant, k isc , characterizing singlettriplet mixing is based on the assumption of steady-state kinetics for describing the radical pair system. Under these conditions singlettriplet interconversion may be represented by an effective rate constant.18 This phenomenological approach has been utilized before in theoretical

studies for interpreting magnetic eld effects. Examples include effects on photosynthetic reactions in the D1D2 reaction center complex, and magnetic eld inuences on the uorescence decay induced by ash photolysis in the reaction of pyrene and dicyanobenzene.18,19 To simplify calculations in the following, we assume that substrate concentration is buffered at a constant level, S S 0 . Alternatively one may assume that substrate concentration is much greater than enzyme concentration. The concentrations of the radical pair states, S/T (E S), are small and rapidly converge to the steady-state value, because the rate constants k 2 , k 3 , and k isc are much greater than the other rate constants. Therefore it is possible to eliminate these variables adiabatically. Taking into account these assumptions and dening x (ES) / E total , y (ES) * T / E total , z ( S (E S) (E S) )/ E total , t(k 1 S 0 )t, one obtains the following set of equations from the enzyme reaction scheme, Eq. 1 : dx dt dy dt 1 1 c1 c 2 c 3 f k isc x y, 2

c 2 f k isc x c 5 y,

3 4

z c 3 f k isc x, where f k isc 1 2k isc /k 3 , 1 c 4 2 c 4 /k isc /k 3

k 1/ and the following parameters are used: c 1 (k 1 S 0 ), c 2 k 2 /(k 1 S 0 ), c 3 k 2 /k 3 , c 4 k 2 /k 3 , c 5 k 4 /(k 1 S 0 ). The relation c 2 f (k isc ) in Eqs. 2 3 represents an efk2 f(kisc), that is related to the fective rate constant, k 2,eff forward ux in the enzyme reaction cycle. The magnetic eld changes the ratio of net forward ux to form a product to the net rate of nonproductive substrate dissociation.7,16 In the case of time-dependent magnetic elds the effective rate constant k 2,eff becomes time dependent via the time dependency of k isc . In the following it is assumed that the temporal variations of the magnetic eld are much slower than the radical pair lifetime, which typically is in the range of 1100 ns.1013,15 Consequently, within this time frame the radical pair senses a quasistatic magnetic eld.15,20 Therefore, spin evolution of the radical pair may be regarded as a quasi-steady-state process on the time scale of the magnetic eld variations. In this special case it is not necessary to explicitly solve the quantum-statistical equations describing spin evolution of the radical pair by including timedependent magnetic elds. Rather these equations may be solved under static magnetic eld conditions, and the actual value of the magnetic ux density may be substituted to calculate the effect of a slowly varying magnetic eld. Under the conditions outlined above, Eqs. 2 5 remain appropriate as a model for describing inuences of time-dependent magnetic elds on enzyme kinetics. To ob-

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tain an explicit expression for the function f (k isc ), Eq. 5 , the effective rate constant k 2,eff may be related to the radical pair recombination probability:16 k 2,eff k 2 1 PR . 6

x i p i t exp

F it

10

In this relation (1 P R ) represents the probability that the radical pair does not recombine. Consequently, the enzyme reaction cycle advances into the forward direction. By comparison one nds that f (k isc ) (1 P R ). The approach, Eq. 6 , enables the calculation of P R by using well-established models known from the theory of radical pair recombination. One possibility is to use the exponential model, which assumes that the radicals separate after a certain period of time and subsequent re-encounters are neglected in this case 1/k 3 is the mean frequency of inter-radical distance changes .11,12 In the simplest case of a singletradical pair precursor with only one spin 1/2 magnetic nucleus present and restriction to the high eld limit B A h , where B is magnetic ux density and A h is the hyperne interaction constant this model yields11 PR B k k
2 2

where p i are functions with the same period as f (k isc ), and F i are the characteristic exponents of the system, Eq. 9 Floquet exponents, i 1,2 . The sum of the Floquet exponents is given by the following theorem:21
F 1 F 2

1 T

Trace P t dt
0

1 c1 c5

c 2 c 3 f k isc

T,

11

where T is the period of P(t). The expression on the right side of Eq. 11 represents the time-averaged contraction of phase space, again being related to the relaxation behavior of the enzyme. The relation may thus be used to characterize the behavior of the system under the inuence of periodic external perturbations.

III. RESULTS AND DISCUSSION A. The

1 k3
2

2 2 s

s 2 2

1 2 d

2 d

1 2
2,

, 7

g mechanism

2k 3 k 1 k 3 k 3 k 2 /2 2

2J

2k 3 k k3 k

2 2

1,

) 2 (a/4) 2 . In this relation 2J corresponds where s,d ( to the energy splitting induced by the exchange interaction, a is the hyperne interaction constant of the spin 1/2 magnetic nucleus, and ( B / )B g/2 refers to the g mechanism. In the case of oscillating magnetic elds varying at a frequency that is much smaller than the reciprocal of the radical pair lifetime, one may substitute the actual value of B(t) into Eq. 7 to calculate the recombination probability. The relaxation behavior of the enzyme may be investigated with pulsed or sinusoidally oscillating external perturbations.13 The application of pulsed perturbations enables an explicit solution of the model, which yields the relaxation time constants of the enzyme pulses of a rectangular shape are applied . The latter are given by the reciprocal of the characteristic exponents:
1 1,2

In this section a rst example is used to investigate the basic response behavior of the enzyme system. Calculations are restricted to the g mechanism, leading to coherent spin mixing between the singlet and triplet T 0 states because of different g values of the radicals.1013 The solution of the model, Eqs. 2 5 , in the case of rectangular magnetic eld pulses reads as x xs y ys x 0 x s cos y 0 y s cos t t
x

sin sin

t exp t exp

t , 12 t , 13

c5 2

1c 5 1

c5

2 2

where 1 1 c 1 (c 2 c 3 ) f (k isc ) and 2 c 2 f (k isc ). To obtain an explicit solution is not possible in cases involving sinusoidally oscillating magnetic elds because of the complex time dependence of the function f (k isc ). However, Eqs. 2 3 may be cast into the following form: dx dt e P t x, 9

where e (1,0), x (x,y), and P(t) is a periodic matrix function exhibiting the period of f (k isc ). The homogenous part of Eq. 9 represents a Mathieu type of differential equation.21 Floquet theory shows that it has normal solutions of the form21

( 1 c 5 )/2 and denote the real part and the where imaginary part of the characteristic exponents, Eq. 8 , and (y 0 y s )/ , x (x 0 x s )( 1 c 5 )/2 y (x 0 x s ) ( 1 c 5 ) 2 /4 (y 0 y s )( 1 c 5 )/2 . The parameters x 0 and y 0 represent the steady states before application of the magnetic eld pulse initial conditions , whereas x s and y s are the steady-state values for t 1 in the presence of the eld. Figure 1 depicts the analytical solution shown above with t T pulse . Usually the characteristic relaxation time of the enzyme may be estimated from the response amplitudes.1 However, in the present case one has to take into account that the characteristic exponents are conjugate complex, and the system settles into the steady-state exhibiting damped oscillation. Therefore a simple measurement of the response amplitudes contains insufcient information for an estimate of the relaxation time constant. Experimental data should be tted to the model to yield an accurate estimate of the relaxation time constant. The relative change in the amplitudes may be estimated 1 in certain limiting cases. Taking into account that c 3 1, the relative change in the ampliand assuming that c 1 tudes, dened as 1 x s /x 0 , is given as

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C. Eichwald and J. Walleczek: Oscillating magnetic eld effects

c5 / c2 1 c5 c5 / c2 1 c5

f k isc B 0 f k isc B

14

FIG. 1. Relaxation behavior of the magnetic eld-sensitive enzyme under the inuence of pulsed magnetic elds. Time evolution of the variables x and y as a function of the duration of the magnetic eld pulse. The variable x solid line is the fraction of enzyme in the (ES) conguration, and y dashed line is the fraction in the (ES) * complex see Eq. 1 . Initial conditions are x(0) x 0 , y(0) y 0 , where x 0 and y 0 are the steady-state values before the application of the magnetic eld pulse. Parameters: c 1 0.1, c 2 10, c 3 10 5 , c 4 10, c 5 1.5, k 2 109 s 1, k 3 108 s 1, g 0.25, a 0, 2J 0, B DC 30 mT.

c 5) 1 then 0, and no considerable If c 5 / c 2 (1 changes in the amplitudes may be observed. However, in the opposite case where c 5 / c 2 (1 c 5 ) 1, one nds by applying Eq. 6 that 1 1 P R (B 0) / 1 P R (B) . In this case even small changes in the recombination probability may result in large changes in the amplitudes. For example, a one percent change from P R (B 0) 0.9 to P R (B) 0.91 produces a change of about 10% ( 0.11). To further explore the enzymes response behavior to magnetic eld perturbations, the inuence of sinusoidally oscillating elds, B(t) B AC cos( ACt), is investigated in the following. In Fig. 2 the response behavior of the enzyme is characterized as a function of the applied eld frequency. The diagrams reveal that there is a characteristic transition region near AC 1 where the oscillation amplitudes of the variables x and y drastically decrease Fig. 2 a , and a phase shift appears between the temporal variations of the external eld and the variations induced in the concentrations of the

FIG. 2. Response behavior of the enzyme to sinusoidally oscillating magnetic elds, B(t) B AC cos( ACt), as a function of eld frequency. The variable x solid lines is the fraction of enzyme in the (ES) conguration, and y dashed lines is the fraction in the (ES) * complex see Eq. 1 . a Oscillation amplitudes of the variables x and y. b Phase shift x and y between the oscillations of the variables x and y and the external magnetic eld. The phase shift x ( y ) is dened as the phase difference between the maximal amplitude of the oscillating magnetic eld at t 0 and the minimum maximum of the variable x (y). c Correlation functions of the time derivatives of the variables x and y and the external eld variations cos( ACt), Eqs. 15 16 . d The real part of the Floquet exponents. Parameters are the same as in Fig. 1, except B AC 30 mT. J. Chem. Phys., Vol. 107, No. 13, 1 October 1997

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C. Eichwald and J. Walleczek: Oscillating magnetic eld effects

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intermediate states corresponding to x and y Fig. 2 b . The transition region reects the relaxation behavior of the enzyme to oscillating magnetic elds. At lower frequencies enzyme kinetics follows the imposed oscillations adiabatically. This means that the resulting oscillation patterns may be obtained by setting dx/dt dy/dt 0 and solving for the time-dependent variables x and y in Eqs. 2 3 . In contrast, at temporal variations of the magnetic eld that are faster than the system inherent response time, enzyme kinetics are too slow to follow the rapid changes. This leads to the observed decrease in the oscillation amplitudes and to the appearance of the phase shift. Although the size of the oscillations decreases considerably, the average values of the variables x and y approximately remain the same at increasing eld frequencies. Within this transition region the time average of the variable x exhibits a small decrease in the order of one percent, whereas the time average of y slightly increases by a similar amount. The reason for these small changes is that the enzyme reaction cycle, Eq. 1 , does not include reversible steps after separation of the spin-correlated radical pair state via k 3 . In comparison, results obtained with a model of a transmembrane enzyme under the inuence of electric elds show that inclusion of such processes may lead to large variations, as a function of applied eld frequency, in the average reaction rate, and even to a possible change in the direction of the ux.2224 In the magnetic eld case, reversible steps after separation of the spin-correlated radical pair state are not taken into account because they complicate calculations considerably. Specically, a reversible generation of the radical pair state, that is, the transition (ES) * (E S), resembles a quasirandom radical encounter no spin correlation . A priori it is not clear how such a process could be integrated into the enzyme model adequately. However, the simple reaction scheme, Eq. 1 , already reveals a unique dependence of the enzymes response behavior on the frequency of the oscillating magnetic eld. There are several ways to further characterize the behavior of the enzyme system within the transition region, for example, by calculating the following correlation functions: C x,F dx cos dt dy cos dt
AC t T

FIG. 3. Correlation functions of the time derivatives of the variables x and y and the external eld variations cos( ACt), Eqs. 15 16 . The variable x solid lines is the fraction of enzyme in the (ES) conguration, and y dashed lines is the fraction in the (ES) * complex see Eq. 1 . In i it is assumed that the radical pair generation step via k 2 proceeds at a much slower rate than product release via k 4 , whereas in ii substrate release via k 1 proceeds at a much higher rate than product release. Parameters: i c1 0.1, c 2 1.5, c 3 10 5 , c 4 10; ii c 1 10, c 2 10, c 3 10 5 , c 4 10, c 5 1.5; other parameters are the same as in Fig. 2.

15

C y,F

AC t T

16

where T denotes time averaging over one period of the internal oscillation of the enzyme states, that is, T 1 2 (2 / AC ). The procedure dened by Eqs. 15 16 is related to concepts that have been utilized to characterize oscillatory responses of externally driven systems by means of the dissipation or efciency of the system.2527 Figure 2 c depicts the calculated correlation functions C x,F and C y,F . One observes that the absolute value of these functions is near zero, except within the frequency region, where the changes in the response behavior of the enzyme are observed. At frequencies much below the reciprocal of

the relaxation time, the time derivatives of the variables x and y are small, because enzyme kinetics follows the imposed oscillations adiabatically. In contrast, at frequencies above the reciprocal of the relaxation time the enzyme oscillations gradually become maximally anticorrelated with the external eld oscillations. Therefore, only within the intermediate frequency range corresponding to the transition region the correlation functions exhibit a nite value. In situations where it is experimentally more accessible to measure rate changes rather than absolute concentrations the calculation of the correlation functions, Eqs. 15 16 , might provide a useful tool for studying the response behavior of the enzyme. In particular, the time-averaging procedure should decrease the inuence of uctuations in the number of enzymes in a specic state of the reaction cycle. The peak values of C x,F and C y,F are at slightly different frequencies. The reason for this shift is based on the kinetic properties of the enzyme reaction cycle. The correlation function C x,F reects the characteristics of the substrate binding and subsequent radical pair generation steps, whereas C y,F is representative of the product release step. In the simulations these processes proceed at different rates. Therefore both correlation functions also reveal information regarding kinetic properties of the enzyme reaction cycle. To further illustrate this behavior, Fig. 3 graphs i depicts the results obtained by assuming that product release via k 4 proceeds at a much higher rate than radical pair generation via k 2 . As a result the peak value of correlation function C y,F is shifted toward a higher frequency. Alternatively, one may assume that nonproductive substrate release via k 1 is much faster than product release via k 4 . In this case the peak value of the correlation function C x,F is located at a higher frequency graphs ii . The sign of C x,F and C y,F yields information regarding the underlying interaction mechanism that causes mixing of

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C. Eichwald and J. Walleczek: Oscillating magnetic eld effects

the singlet and triplet radical pair states. The g mechanism increases intersystem crossing between the singlet and triplet T 0 states thus increasing the population of the enzyme substrate complex corresponding to the variable y and decreasing the one corresponding to x. As a result the time derivative dy/dt follows the eld variations in phase, whereas dx/dt is in antiphase neglecting for the moment the phase shift at higher eld frequencies . The situation is opposite in the case of the hyperne interaction mechanism, where the application of magnetic elds results in a decrease of intersystem crossing also see the discussion in Sec. III B .11,12 The Floquet exponents within the frequency region where the changes in the response behavior of the enzyme occur are conjugate complex, except for two intervals located around AC 0.52 and AC 1.05 Fig. 2 d . Floquet exponents and related concepts have routinely been used to characterize the behavior of the local ow in periodically driven systems, for example, to study the bifurcation structure of nonlinear oscillators.2831 The enzyme model represents a parametrically driven linear system wherein the real part of the Floquet exponent is always negative. Therefore the system does not exhibit bifurcations into new states. Nevertheless, the structural changes of the oscillation patterns at increasing eld frequencies are reected in the Floquet exponents. In particular, a comparison with the results shown in Fig. 2 b shows that the rst interval around AC 0.52, where the Floquet exponents are real, is located within the frequency range where the phase shift x between the variable x and the eld variations rapidly increases. In a 1.05 is similar manner, the second interval around AC located at the frequency range where the phase shift y between the variable y and the eld variations appears. The connection to the Floquet exponents shows that these frequency-dependent changes of the response behavior of the system are reected in the properties of the local ow. The results presented in Figs. 23 reveal that oscillating magnetic elds may be utilized as a tool to explore the relaxation behavior of the enzyme system. In contrast to other relaxation methods where the external perturbation is additive, the magnetic eld interaction with the enzyme reaction cycle leads to a parametrically driven system.
B. Combined hyperne interactionand mechanisms

FIG. 4. Oscillation diagrams of the intermediate enzymesubstrate complex corresponding to the variable x under the inuence of combined static and sinusoidally oscillating magnetic elds, B(t) B DC B AC cos( ACt). The eld frequency AC is indicated on top of the gure. Time is scaled in units of T AC 2 / AC . The dashed lines represent the steady-state value of x under static magnetic elds only. Parameters: c 1 0.1, c 2 10, c 3 10 5 , c 4 100, c 5 1.5, k 2 109 s 1, k 3 107 s 1, g 0.05, a 5 mT, 2J 0.5 mT; a B DC 85 mT, i B AC 15 mT, ii B AC 35 mT; b B DC 100 mT, iii B AC 25 mT, iv B AC 50 mT; c B DC 125 mT, v B AC 25 mT, vi B AC 75 mT.

In the preceding section the g mechanism is the basis for a simple but descriptive model for effects of oscillating magnetic elds on enzyme activity. In general, evolution of the spin-correlated radical pair occurs under the combined inuence of hyperne couplings to magnetic nuclei and the g mechanism.1013 Therefore in this section the simple approach discussed in the preceding section is extended by taking into account a single spin 1/2 magnetic nucleus. The radical pair recombination probability, P R , in the high eld limit is determined by the exponential model, Eq. 7 , where magnetic ux density is given as B(t) B DC B AC cos( ACt). Inclusion of the static magnetic eld com-

ponent determines that simulations are performed in the high eld limit within the entire period of the oscillating magnetic eld component only singlettriplet T 0 transitions . Model simulations show that the temporal variations in P R are rather small and are in the order of 0.5% for the parameter combination used. The corresponding variations of the different enzymesubstrate intermediates are considerably greater. Figure 4 depicts a number of representative oscillation diagrams. Model simulations reveal that depending on the specic combination of static and oscillating magnetic eld amplitudes the recombination probability either follows the external eld variations in phase, partially in antiphase, or entirely in antiphase. In Fig. 4 b the static magnetic eld amplitude is xed at a value where one observes the maximal decrease in the enzyme reaction rate for the parameters used (B DC 100 mT). This minimum results because of the combined inuence of the hyperneand the g mechanisms. The hyperneand the g mechanisms exhibit opposite inuences on the radical pair recombination probability.11,12 As a result of this behavior one typically observes a biphasic behavior of the magnetic eld effect at increasing magnetic

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ux densities. In the present case a gradual decrease of the enzyme reaction rate is observed at magnetic ux densities 100 mT, whereas at higher ux densities a return to B DC the zero-eld rate and an increase results see Ref. 16 for further details . Because the additional oscillating magnetic eld decreases, the recombination probability compared to 100 mT, the value of the variable x is its value at B DC below the steady state obtained with static magnetic elds 0.1 the left-hand side diaonly dashed lines . At AC grams in Fig. 4 the steady-state value is reached at time points t 1/4T AC , 3/4T AC . The variables x and the corresponding enzyme reaction rate display an oscillation period that is twice the period of the external oscillating magnetic eld. The situation is different if the static magnetic eld amplitude is either smaller Fig. 4 a or greater Fig. 4 c than B DC 100 mT. At values of B AC where either B DC B AC 100 mT or B DC B AC 100 mT, an oscillation period that is equal to the period of the oscillating magnetic eld is observed. In the rst case the variable x oscillates in phase with the external eld graph i , whereas in the second case it is in antiphase graph v . In both examples the enzyme reaction rate displays an oscillation period that is the same as the one of the oscillating magnetic eld. Increasing B AC beyond the value where the above inequalities hold results in the formation of a second peak in the oscillation diagrams graphs ii and vi . The enzyme reaction cycle now exhibits oscillations involving two time intervals T 1 ,T 2 such that T2 T AC and T 1 T2 . T1 The behavior described above illustrates that the specic combination of static and oscillating magnetic eld amplitudes critically determines the temporal variations of the enzymesubstrate states as well as of the enzyme reaction rate. In particular, different oscillation periods are observed, including those with the same period as the one of the external oscillating eld, others with twice the period, and eventually others exhibiting a combination of two time intervals such that their sum equals the external period. The origin of this complex behavior lies in the contribution of the different magnetic interactions to coherent spin evolution of the radical pair. 0.1, the The results obtained at low frequencies AC left-hand side diagrams in Fig. 4 may, in principle, be predicted by the knowledge of the effect of the static magnetic eld component only, because enzyme kinetics follows the magnetic eld variations adiabatically. However, a detailed interpretation regarding observed changes in the enzyme reaction rate requires a precise theoretical description of the underlying interaction mechanism. The latter has to include both the quantum-statistical properties determining spin evolution of the radical pair hyperne couplings, g mechanism, etc. and the biological context related to enzyme kinetics the site of a radical pair step in the reaction cycle, etc. . At higher frequencies, the application of oscillating magnetic elds leads to changes that may not be predicted by knowledge of the effect of the static magnetic eld component only. On the right-hand side diagrams in Fig. 4 the

temporal variations of the variable x are shown at a ten times higher eld frequency ( AC 1). The resulting behavior deviates signicantly from the one at lower frequencies. The divergence manifests itself in a breaking of symmetry of the oscillations around t T AC /2. In contrast to the lowfrequency case, the variable x does not reach its steady-state value at t 1/4T AC ,3/4T AC . Finally, the oscillation amplitudes decrease and a phase shift to the external eld variations appears. These changes at increasing eld frequencies closely resemble those that are discussed in Sec. III A. They are independent of the details of the magnetic interactions, leading to an inuence on the spin evolution of the radical pair state. In fact, they reect the kinetic properties of the underlying biological system, being related to the characteristic response behavior of the enzyme.
IV. SUMMARY AND OUTLOOK

The simple enzyme reaction cycle used in the simulations serves as a prototypical approach enabling the formulation of a consistent concept that combines enzyme kinetics with magnetic eld-dependent spin kinetics. Model calculations show that the magnetic eld-sensitive enzyme behaves like a frequency sensor that is responsive at low eld frequencies but less responsive at frequencies that are faster than its response time scale. According to the model, timedependent magnetic elds could be used as a tool to explore the relaxation behavior of magnetic eld-sensitive enzymes. This method could be used as a supplementary procedure for the well-established chemical relaxation techniques that are utilized to study enzyme kinetics. The hypothesis that any enzyme reaction that is susceptible to magnetic elds should, at least in principle, exhibit dependencies on the eld frequency in response to oscillating magnetic elds is consistent with the results of this theoretical study.14 Therefore they suggest that time-dependent magnetic eld effects on radical pair recombination steps within biological or biochemical systems might be more complex than previously discussed.15,20 Model simulations with combined static and oscillating magnetic elds reveal a complex variety of oscillatory responses of the enzyme, depending on the combination of magnetic interactions that determine spin evolution of the radical pair. The question arises under which conditions different oscillation patterns in the enzyme reaction rate may produce biologically relevant changes. To establish such a connection the following two requirements have to be met: 1 the existence of a magnetic eld-sensitive enzyme system; and 2 the existence of a biological detection mechanism that is capable of responding to temporal changes in the product formation rate of the enzyme. In principle, an approach wherein the magnetic eld-sensitive enzyme system is a part of a biological network of signaling processes, especially oscillatory ones, is in agreement with these requirements.14,17,3234 Recently, different groups have discussed schemes wherein protein molecules like enzymes function as computational elements in biochemical reaction networks.3538 The latter are capable of operating as simple

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logical gates fuzzy logic gates . If the magnetic eldsensitive enzyme system is integrated into such a scheme of interconnected cyclic elements, then it is theoretically feasible that the periodic modulation of the output the enzyme reaction rate of one of the elements is coherently processed throughout the network including transduction as well as amplication steps . Whether such an enzyme systems exist within biological signaling processes is an open question, although several possibilities have been discussed before.14,17 If the biological network is a part of cellular signaling processes, then a macroscopically detectable change may nally result for example, altered concentrations or different inux/ efux of specic ions into the cell .14,17,39,40 The concrete realization of this proposal awaits future experimental as well as theoretical studies. In this context the application of oscillating magnetic elds might provide a useful tool to explore specic kinds of biological response behavior.
ACKNOWLEDGMENTS

C.E. is a recipient of a Fetzer Institute post-doctoral fellowship. Work at the Bioelectromagnetics Laboratory is supported by the Fetzer Institute and the U.S. Department of Energy.
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J. Chem. Phys., Vol. 107, No. 13, 1 October 1997

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