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**C. Eichwald and J. Walleczeka)
**

Department of Radiation Oncology, Biolelectromagnetics Laboratory, School of Medicine—AO38, 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 ﬁelds radical pair mechanism . In this theoretical study, the response behavior of the enzyme to pulsed magnetic ﬁelds as well as combinations of static and sinusoidally oscillating magnetic ﬁelds is investigated by using an approach that combines enzyme kinetics with magnetic ﬁeld-sensitive spin kinetics. Calculations show that the enzyme behaves like a frequency sensor that is responsive at lower ﬁeld 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 reﬂects the enzyme’s 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 ﬁelds could be used as a tool to study the response behavior of magnetic ﬁeld-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. 1–3 . 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 ﬁelds.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 ﬁelds for investigating the kinetics of magnetic ﬁeld-sensitive enzymes. The background for this study is based on experimental ﬁndings that revealed static magnetic ﬁeld effects on the in vitro activity of B 12-dependent enzymes.4–8 Speciﬁcally, in the case of ethanolamine ammonia lyase it was shown that the magnetic ﬁeld-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 C–Co bond .6,8 More recently, magnetic ﬁeld effects were reported on the redox cycle of horseradish peroxidase.9 An interpretation of these experimental ﬁndings is based on the radical pair mechanism.7 In this mechanism the magnetic ﬁeld modulates the recombination yield of spin-correlated radical pairs that are formed as intermediates in chemical or biochemical reaction pathways.10–13 The radical pair mechanism is a well-established tool for investigating the kinetics of reactions that proceed via free radical-dependent steps.7,10–15

a

Author to whom correspondence should be addressed.

Based on the experimental ﬁndings a prototypical model of a magnetic ﬁeld-sensitive enzyme system has been developed.16 The model is based on an approach that combines enzyme kinetics with magnetic ﬁeld-dependent spin kinetics. It qualitatively reproduces the reported biphasic magnetic ﬁeld 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 ﬁeld 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 ﬁeld effects on enzyme activity.16 In this contribution the original model is extended to enable studying time-dependent magnetic ﬁeld perturbations, including pulsed ﬁelds as well as combinations of static and sinusoidally oscillating ﬁelds. Motivation for these investigations is based on an earlier hyphothesis that suggested the possibility of low-frequency-dependent magnetic ﬁeld 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 ﬁeld to the enzyme, whereas dependencies on the ﬁeld 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 ﬁeld to the radical pair system. The response behavior of the enzyme to time-dependent magnetic ﬁeld perturbations is studied. Results show that the enzyme behaves like a frequency sensor that is responsive at lower ﬁeld 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 ﬁeld effects

region that reﬂects the enzyme’s relaxation behavior to timedependent external perturbations. Results suggest that timedependent magnetic ﬁelds could be used as a tool for studying the relaxation behavior of magnetic ﬁeld-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

T

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), enzyme–substrate complex prior to radical pair generation, S/T (E •• S), enzyme–substrate 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 ﬁeld-dependent rate constant characterizing singlet–triplet 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 ﬁeld 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 ﬁeld 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 ﬁelddependent spin kinetics. The rate constants k 2 radical pair recombination , k 3 forward reaction coordinate motion , and k isc singlet–triplet mixing represent fast time scales in the 1–100 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 .1–3 The introduction of an effective rate constant, k isc , characterizing singlet–triplet mixing is based on the assumption of steady-state kinetics for describing the radical pair system. Under these conditions singlet–triplet interconversion may be represented by an effective rate constant.18 This phenomenological approach has been utilized before in theoretical

studies for interpreting magnetic ﬁeld effects. Examples include effects on photosynthetic reactions in the D1–D2 reaction center complex, and magnetic ﬁeld inﬂuences on the ﬂuorescence decay induced by ﬂash 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 deﬁning 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

5

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 ﬂux in the enzyme reaction cycle. The magnetic ﬁeld changes the ratio of net forward ﬂux to form a product to the net rate of nonproductive substrate dissociation.7,16 In the case of time-dependent magnetic ﬁelds 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 ﬁeld are much slower than the radical pair lifetime, which typically is in the range of 1–100 ns.10–13,15 Consequently, within this time frame the radical pair senses a quasistatic magnetic ﬁeld.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 ﬁeld 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 ﬁelds. Rather these equations may be solved under static magnetic ﬁeld conditions, and the actual value of the magnetic ﬂux density may be substituted to calculate the effect of a slowly varying magnetic ﬁeld. Under the conditions outlined above, Eqs. 2 – 5 remain appropriate as a model for describing inﬂuences of time-dependent magnetic ﬁelds on enzyme kinetics. To ob-

J. Chem. Phys., Vol. 107, No. 13, 1 October 1997

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

4945

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 ﬁnds 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 singlet–radical pair precursor with only one spin 1/2 magnetic nucleus present and restriction to the high ﬁeld limit B A hﬁ , where B is magnetic ﬂux density and A hﬁ is the hyperﬁne 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

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 inﬂuence of periodic external perturbations.

III. RESULTS AND DISCUSSION A. The

1 k3

2

1

2 2 s

s 2 2

1 2 d

2

2 d

1 2

2,

, 7

g mechanism

1

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

2J

2

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 hyperﬁne 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 ﬁelds 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.1–3 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 ﬁrst 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.10–13 The solution of the model, Eqs. 2 – 5 , in the case of rectangular magnetic ﬁeld 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

y

c5 2

1

1

4

1c 5 1

c5

2 2

,

8

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 ﬁelds 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 ﬁeld pulse initial conditions , whereas x s and y s are the steady-state values for t 1 in the presence of the ﬁeld. 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 insufﬁcient information for an estimate of the relaxation time constant. Experimental data should be ﬁtted 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, deﬁned as 1 x s /x 0 , is given as

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

1

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 ﬁeld-sensitive enzyme under the inﬂuence of pulsed magnetic ﬁelds. Time evolution of the variables x and y as a function of the duration of the magnetic ﬁeld pulse. The variable x solid line is the fraction of enzyme in the (ES) conﬁguration, 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 ﬁeld 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 ﬁnds 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 enzyme’s response behavior to magnetic ﬁeld perturbations, the inﬂuence of sinusoidally oscillating ﬁelds, 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 ﬁeld 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 ﬁeld and the variations induced in the concentrations of the

FIG. 2. Response behavior of the enzyme to sinusoidally oscillating magnetic ﬁelds, B(t) B AC cos( ACt), as a function of ﬁeld frequency. The variable x solid lines is the fraction of enzyme in the (ES) conﬁguration, 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 ﬁeld. The phase shift x ( y ) is deﬁned as the phase difference between the maximal amplitude of the oscillating magnetic ﬁeld 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 ﬁeld 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 ﬁeld effects

4947

intermediate states corresponding to x and y Fig. 2 b . The transition region reﬂects the relaxation behavior of the enzyme to oscillating magnetic ﬁelds. 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 ﬁeld 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 ﬁeld 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 inﬂuence of electric ﬁelds show that inclusion of such processes may lead to large variations, as a function of applied ﬁeld frequency, in the average reaction rate, and even to a possible change in the direction of the ﬂux.22–24 In the magnetic ﬁeld case, reversible steps after separation of the spin-correlated radical pair state are not taken into account because they complicate calculations considerably. Speciﬁcally, 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 enzyme’s response behavior on the frequency of the oscillating magnetic ﬁeld. 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 ﬁeld variations cos( ACt), Eqs. 15 – 16 . The variable x solid lines is the fraction of enzyme in the (ES) conﬁguration, 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 deﬁned 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 ‘‘efﬁciency’’ of the system.25–27 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 ﬁeld oscillations. Therefore, only within the intermediate frequency range corresponding to the transition region the correlation functions exhibit a ﬁnite 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 inﬂuence of ﬂuctuations in the number of enzymes in a speciﬁc 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 reﬂects 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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4948

C. Eichwald and J. Walleczek: Oscillating magnetic ﬁeld 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 ﬁeld variations in phase, whereas dx/dt is in antiphase neglecting for the moment the phase shift at higher ﬁeld frequencies . The situation is opposite in the case of the hyperﬁne interaction mechanism, where the application of magnetic ﬁelds 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 ﬂow in periodically driven systems, for example, to study the bifurcation structure of nonlinear oscillators.28–31 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 ﬁeld frequencies are reﬂected in the Floquet exponents. In particular, a comparison with the results shown in Fig. 2 b shows that the ﬁrst 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 ﬁeld 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 ﬁeld variations appears. The connection to the Floquet exponents shows that these frequency-dependent changes of the response behavior of the system are reﬂected in the properties of the local ﬂow. The results presented in Figs. 2–3 reveal that oscillating magnetic ﬁelds 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 ﬁeld interaction with the enzyme reaction cycle leads to a parametrically driven system.

B. Combined hyperﬁne interaction—and mechanisms

FIG. 4. Oscillation diagrams of the intermediate enzyme–substrate complex corresponding to the variable x under the inﬂuence of combined static and sinusoidally oscillating magnetic ﬁelds, B(t) B DC B AC cos( ACt). The ﬁeld frequency AC is indicated on top of the ﬁgure. Time is scaled in units of T AC 2 / AC . The dashed lines represent the steady-state value of x under static magnetic ﬁelds 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.

g

In the preceding section the g mechanism is the basis for a simple but descriptive model for effects of oscillating magnetic ﬁelds on enzyme activity. In general, evolution of the spin-correlated radical pair occurs under the combined inﬂuence of hyperﬁne couplings to magnetic nuclei and the g mechanism.10–13 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 ﬁeld limit is determined by the exponential model, Eq. 7 , where magnetic ﬂux density is given as B(t) B DC B AC cos( ACt). Inclusion of the static magnetic ﬁeld com-

ponent determines that simulations are performed in the high ﬁeld limit within the entire period of the oscillating magnetic ﬁeld component only singlet↔triplet 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 enzyme–substrate intermediates are considerably greater. Figure 4 depicts a number of representative oscillation diagrams. Model simulations reveal that depending on the speciﬁc combination of static and oscillating magnetic ﬁeld amplitudes the recombination probability either follows the external ﬁeld variations in phase, partially in antiphase, or entirely in antiphase. In Fig. 4 b the static magnetic ﬁeld amplitude is ﬁxed 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 inﬂuence of the hyperﬁne—and the g mechanisms. The hyperﬁne—and the g mechanisms exhibit opposite inﬂuences on the radical pair recombination probability.11,12 As a result of this behavior one typically observes a biphasic behavior of the magnetic ﬁeld effect at increasing magnetic

J. Chem. Phys., Vol. 107, No. 13, 1 October 1997

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

4949

ﬂux densities. In the present case a gradual decrease of the enzyme reaction rate is observed at magnetic ﬂux densities 100 mT, whereas at higher ﬂux densities a return to B DC the zero-ﬁeld rate and an increase results see Ref. 16 for further details . Because the additional oscillating magnetic ﬁeld 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 ﬁelds 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 ﬁeld. The situation is different if the static magnetic ﬁeld 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 ﬁeld is observed. In the ﬁrst case the variable x oscillates in phase with the external ﬁeld 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 ﬁeld. 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 speciﬁc combination of static and oscillating magnetic ﬁeld amplitudes critically determines the temporal variations of the enzyme–substrate 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 ﬁeld, 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 ﬁeld component only, because enzyme kinetics follows the magnetic ﬁeld 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 hyperﬁne 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 ﬁelds leads to changes that may not be predicted by knowledge of the effect of the static magnetic ﬁeld 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 ﬁeld frequency ( AC 1). The resulting behavior deviates signiﬁcantly 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 ﬁeld variations appears. These changes at increasing ﬁeld frequencies closely resemble those that are discussed in Sec. III A. They are independent of the details of the magnetic interactions, leading to an inﬂuence on the spin evolution of the radical pair state. In fact, they reﬂect 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 ﬁeld-dependent spin kinetics. Model calculations show that the magnetic ﬁeld-sensitive enzyme behaves like a frequency sensor that is responsive at low ﬁeld frequencies but less responsive at frequencies that are faster than its response time scale. According to the model, timedependent magnetic ﬁelds could be used as a tool to explore the relaxation behavior of magnetic ﬁeld-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 ﬁelds should, at least in principle, exhibit dependencies on the ﬁeld frequency in response to oscillating magnetic ﬁelds is consistent with the results of this theoretical study.14 Therefore they suggest that time-dependent magnetic ﬁeld 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 ﬁelds 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 ﬁeld-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 ﬁeld-sensitive enzyme system is a part of a biological network of signaling processes, especially oscillatory ones, is in agreement with these requirements.14,17,32–34 Recently, different groups have discussed schemes wherein protein molecules like enzymes function as computational elements in biochemical reaction networks.35–38 The latter are capable of operating as simple

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C. Eichwald and J. Walleczek: Oscillating magnetic ﬁeld effects M. B. Taraban, T. V. Leshina, M. A. Anderson, and C. B. Grissom, J. Am. Chem. Soc. 119, 5768 1997 . 10 N. J. Turro, Proc. Natl. Acad. Sci. USA 80, 609 1983 . 11 K. M. Salikhov, Y. N. Molin, R. Z. Sagdeev, and A. L. Buchachenko, Spin Polarization and Magnetic Field Effects in Radical Reactions Elsevier, Amsterdam, 1984 . 12 U. E. Steiner and T. Ulrich, Chem. Rev. 89, 51 1989 . 13 K. A. McLauchlan and U. E. Steiner, Mol. Phys. 73, 241 1991 . 14 J. Walleczek, in Advances in Chemistry 250, Electromagnetic Fields: Biological Interactions and Mechanisms, edited by M. Blank American Chemical Society, Washington, DC, 1995 , p. 395. 15 B. Brocklehurst and K. A. McLauchlan, Int. J. Radiat. Biol. 69, 3 1996 . 16 C. Eichwald and J. Walleczek, Biophys. J. 71, 623 1996 . 17 C. Eichwald and J. Walleczek, Bioelectromagnetics 17, 427 1996 . 18 R. van der Vos and A. J. Hoff, Biochim. Biophys. Acta 1228, 75 1995 . 19 S. N. Batchelor, C. W. M. Kay, K. A. McLauchlan, and I. A. Shkrob, J. Phys. Chem. 97, 13 250 1993 . 20 J. C. Scaiano, F. L. Cozens, and N. Mohtat, Photochem. Photobiol. 62, 818 1995 . 21 D. W. Jordan and P. Smith, Nonlinear Ordinary Differential Equations Clarendon, Oxford, 1987 . 22 R. D. Astumian, P. B. Chock, T. Y. Tsong, and H. V. Westerhoff, Phys. Rev. A 39, 6416 1989 . 23 R. D. Astumian and B. Robertson, J. Chem. Phys. 91, 4891 1989 . 24 R. D. Astumian and B. Robertson, J. Am. Chem. Soc. 115, 11 063 1993 . 25 P. H. Richter, P. Rehmus, and J. Ross, Prog. Theor. Phys. 66, 385 1981 . 26 J. G. Lazar and J. Ross, J. Chem. Phys. 92, 3579 1990 . 27 F. Kaiser, in Energy Transfer Dynamics, edited by T. W. Barret and H. A. Pohl Springer-Verlag, Berlin, 1987 , p. 224. 28 J. D. Crawford and S. Omohundro, Physica D 13, 161 1984 . 29 U. Parlitz and W. Lauterborn, Z. Naturforsch. 41a, 605 1986 . 30 A. Lambert, R. Lima, and R. Vilela Mendes, Physica D 34, 366 1989 . 31 C. Eichwald and F. Kaiser unpublished . 32 C. Eichwald and F. Kaiser, Biophys. J. 65, 2047 1993 . 33 C. Eichwald and F. Kaiser, Bioelectromagnetics 16, 75 1995 . 34 C. Eichwald, Phys. Lett. A 207, 194 1995 . 35 D. Bray, J. Theor. Biol. 143, 215 1990 . 36 D. Bray, Nature 376, 307 1995 . 37 A. Arkin and J. Ross, Biophys. J. 67, 560 1994 . 38 I. Schreiber, Y.-F. Hung, and J. Ross, J. Phys. Chem. 100, 8556 1996 . 39 J. Walleczek, FASEB J. 6, 3177 1992 . 40 J. Walleczek and T. F. Budinger, FEBS Lett. 314, 351 1992 .

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logical gates ‘‘fuzzy logic gates’’ . If the magnetic ﬁeldsensitive 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 ampliﬁcation 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 ﬁnally result for example, altered concentrations or different inﬂux/ efﬂux of speciﬁc 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 ﬁelds might provide a useful tool to explore speciﬁc 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.

1

G. G. Hammes, Enzyme Catalysis and Regulation Academic, New York, 1982 . K. A. Johnson, in The Enzymes Vol. XX, Mechanisms of Catalysis, edited by D. S. Sigman Academic, San Diego, 1992 , p. 1. 3 C. A. Fierke and G. G. Hammes, in Methods in Enzymology Vol. 249, Enzyme Kinetics and Mechanism Part D: Developments in Enzyme Dynamics, edited by D. L. Purich Academic, San Diego, 1995 , p. 3. 4 A. M. Chagovetz and C. B. Grissom, J. Am. Chem. Soc. 115, 12152 1993 . 5 T. T. Harkins and C. B. Grissom, Science 263, 958 1994 . 6 T. T. Harkins and C. B. Grissom, J. Am. Chem. Soc. 117, 566 1995 . 7 C. B. Grissom, Chem. Rev. 95, 3 1995 . 8 E. Natarajan and C. B. Grissom, Photochem. Photobiol. 64, 286 1996 .

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