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1997-Eichwald-Low Freq MF Radicals EnzymesRatings: (0)|Views: 2|Likes: 1

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01/15/2013

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Low-frequency-dependent effects of oscillating magnetic ﬁelds on radicalpair recombination in enzyme kinetics

C. Eichwald and J. Walleczek

a)

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-correlatedradical pair state is discussed. The recombination yield of the radical pair state is altered by externalmagnetic ﬁelds

radical pair mechanism

. In this theoretical study, the response behavior of theenzyme to pulsed magnetic ﬁelds as well as combinations of static and sinusoidally oscillatingmagnetic ﬁelds is investigated by using an approach that combines enzyme kinetics with magneticﬁeld-sensitive spin kinetics. Calculations show that the enzyme behaves like a frequency sensor thatis responsive at lower ﬁeld frequencies but less responsive at frequencies that are faster than thetime scales inherent to the kinetic properties of the reaction cycle. There is a characteristic transitionregion in the frequency domain that reﬂects the enzyme’s relaxation behavior to time-dependentexternal perturbations. The transition region is characterized by using methods based on the theoryof 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 theresponse 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 forstudying enzymes

for an overview see, for example, Refs.1–3

. Chemical relaxation methods make it possible to de-termine important information regarding enzyme mecha-nisms and the underlying kinetics. In relaxation techniquesexternal perturbations are applied to the system, includingvariations 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 asingle step impulse.In this contribution a method is discussed that utilizestime-dependent magnetic ﬁelds for investigating the kineticsof magnetic ﬁeld-sensitive enzymes. The background for thisstudy is based on experimental ﬁndings that revealed staticmagnetic ﬁeld effects on the

in vitro

activity of

B

12

-dependent enzymes.

4–8

Speciﬁcally, in the case of etha-nolamine ammonia lyase it was shown that the magneticﬁeld-dependent step is recombination of a transient spin-correlated radical pair that is formed in the reaction cycle of the enzyme

5

-deoxyadenosyl, cob

II

alamin

- radical pairproduced by enzyme-induced homolysis of the C–Cobond

.

6,8

More recently, magnetic ﬁeld effects were reportedon the redox cycle of horseradish peroxidase.

9

An interpre-tation of these experimental ﬁndings is based on the radicalpair mechanism.

7

In this mechanism the magnetic ﬁeldmodulates the recombination yield of spin-correlated radicalpairs that are formed as intermediates in chemical or bio-chemical reaction pathways.

10–13

The radical pair mechanismis a well-established tool for investigating the kinetics of reactions that proceed via free radical-dependent steps.

7,10–15

Based on the experimental ﬁndings a prototypical modelof a magnetic ﬁeld-sensitive enzyme system has beendeveloped.

16

The model is based on an approach that com-bines enzyme kinetics with magnetic ﬁeld-dependent spinkinetics. It qualitatively reproduces the reported biphasicmagnetic ﬁeld effect on the

in vitro

activity of

B

12

-dependentethanolamine ammonia lyase.

16

Calculations show that thecombination of physical and biochemical mechanisms

mag-netic ﬁeld modulation of radical pair recombination probabil-ity and kinetic processes within the enzyme reaction cycle,respectively

is a necessary prerequisite for a qualitative aswell as a quantitative interpretation of magnetic ﬁeld effectson enzyme activity.

16

In this contribution the original model is extended toenable studying time-dependent magnetic ﬁeld perturbations,including pulsed ﬁelds as well as combinations of static andsinusoidally oscillating ﬁelds. Motivation for these investiga-tions is based on an earlier hyphothesis that suggested thepossibility of low-frequency-dependent magnetic ﬁeld ef-fects in biological systems via radical pair recombinationprocesses in enzyme kinetics.

14,17

In this case the radical pairmechanism serves as the primary coupling mechanism of themagnetic ﬁeld to the enzyme, whereas dependencies on theﬁeld frequency are related to the kinetic properties of theenzyme reaction cycle. These interactions are nonresonant innature, meaning that there is no resonant coupling of theoscillating magnetic ﬁeld to the radical pair system.The response behavior of the enzyme to time-dependentmagnetic ﬁeld perturbations is studied. Results show that theenzyme behaves like a frequency sensor that is responsive atlower ﬁeld frequencies but less responsive at frequencies thatare faster than the time scales inherent to the kinetic proper-ties of the reaction cycle. There is a characteristic transition

a

Author to whom correspondence should be addressed.

4943J. Chem. Phys.

107

(13), 1 October 1997 0021-9606/97/107(13)/4943/8/$10.00 © 1997 American Institute of Physics

Copyright ©2001. All Rights Reserved.

region that reﬂects the enzyme’s relaxation behavior to time-dependent external perturbations. Results suggest that time-dependent magnetic ﬁelds could be used as a tool for study-ing the relaxation behavior of magnetic ﬁeld-sensitiveenzyme systems.

II. OUTLINE OF THE MODEL AND CALCULATIONS

The following enzyme reaction scheme is investigated:

16

E

S

k

1

k

1

ES

k

2

k

2

S

E

••

S

k

3

↑↓

k

isc

ES

*

→

k

4

E

P

T

E

••

S

k

3

,

1

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 radicalpair generation,

S

/

T

(

E

••

S

), enzyme–substrate complex wherea spin-correlated radical pair exists, (

ES

)

*

, enzyme–substrate complex prior to product release,

S

, substrate, and

P

, product. The superscript denotes spin multiplicity

S

, sin-glet,

T

, triplet

, and

k

isc

is a magnetic ﬁeld-dependent rateconstant characterizing singlet–triplet mixing. A shorthandnotation,

T

(

E

••

S

), representing all three triplet states

T

→

T

1

,

T

0

,

T

1

is used

the index refers to the magneticquantum number

.The enzyme reaction scheme, Eq.

1

, represents a pro-totypical approach for describing magnetic ﬁeld effects onenzyme activity.

7,16

Radical pair generation is via

k

2

, andrecombination via

k

2

. The rate constant

k

3

characterizesspin-independent forward reaction coordinate motion. It isused here to represent the lifetime of the spin-correlated radi-cal pair state. It is assumed that the magnetic ﬁeld sensitivityarises solely from the modulation of the population of thesinglet and triplet states. All subsequent processes precedingproduct release via

k

4

are not considered to exhibit a require-ment for spin correlation.

16

The enzyme reaction scheme ex-hibits transitions between different states that operate onvery different time scales. This is a result of combining pro-cesses related to chemical kinetics and to magnetic ﬁeld-dependent spin kinetics. The rate constants

k

2

radical pairrecombination

,

k

3

forward reaction coordinate motion

, and

k

isc

singlet–triplet mixing

represent fast time scales in the1–100 ns time domain.

16

For example, for the

B

12

-dependentsystems a rate constant for radical pair recombination in theorder of

k

rec

10

9

s

1

was estimated.

4,8

The remaining rateconstants are related to the much slower processes within theenzyme reaction cycle

typical catalytic rate constants are inthe range 10–10

5

s

1

.

1–3

The introduction of an effective rate constant,

k

isc

, char-acterizing singlet–triplet mixing is based on the assumptionof steady-state kinetics for describing the radical pair system.Under these conditions singlet–triplet interconversion maybe represented by an effective rate constant.

18

This phenom-enological approach has been utilized before in theoreticalstudies for interpreting magnetic ﬁeld effects. Examples in-clude effects on photosynthetic reactions in the D1–D2 re-action center complex, and magnetic ﬁeld inﬂuences on theﬂuorescence decay induced by ﬂash photolysis in the reac-tion of pyrene and dicyanobenzene.

18,19

To simplify calculations in the following, we assumethat substrate concentration is buffered at a constant level,

S

S

0

. Alternatively one may assume that substrateconcentration is much greater than enzyme concentration.The concentrations of the radical pair states,

S

/

T

(

E

••

S

), aresmall and rapidly converge to the steady-state value, becausethe rate constants

k

2

,

k

3

, and

k

isc

are much greater thanthe other rate constants. Therefore it is possible to eliminatethese variables adiabatically. Taking into account these as-sumptions and deﬁning

x

(

ES

)

/

E

total

,

y

(

ES

)

*

/

E

total

,

z

(

S

(

E

••

S

)

T

(

E

••

S

)

)/

E

total

,

t

→

(

k

1

S

0

)

t

,one obtains the following set of equations from the enzymereaction scheme, Eq.

1

:

dxdt

1

1

c

1

c

2

c

3

f

k

isc

x

y

,

2

dydt

c

2

f

k

isc

x

c

5

y

,

3

z

c

3

f

k

isc

x

,

4

where

f

k

isc

1

2

k

isc

/

k

3

1

c

4

2

c

4

/

k

isc

/

k

3

,

5

and the following parameters are used:

c

1

k

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 ef-fective rate constant,

k

2,eff

k

2

f

(

k

isc

), that is related to theforward ﬂux in the enzyme reaction cycle. The magneticﬁeld changes the ratio of net forward ﬂux to form a productto the net rate of nonproductive substrate dissociation.

7,16

In the case of time-dependent magnetic ﬁelds the effec-tive rate constant

k

2,eff

becomes time dependent via the timedependency of

k

isc

. In the following it is assumed that thetemporal variations of the magnetic ﬁeld are much slowerthan the radical pair lifetime, which typically is in the rangeof 1–100 ns.

10–13,15

Consequently, within this time frame theradical pair senses a quasistatic magnetic ﬁeld.

15,20

There-fore, spin evolution of the radical pair may be regarded as aquasi-steady-state process on the time scale of the magneticﬁeld variations. In this special case it is not necessary toexplicitly solve the quantum-statistical equations describingspin evolution of the radical pair by including time-dependent magnetic ﬁelds. Rather these equations may besolved under static magnetic ﬁeld conditions, and the actualvalue of the magnetic ﬂux density may be substituted tocalculate the effect of a slowly varying magnetic ﬁeld.Under the conditions outlined above, Eqs.

2

–

5

re-main appropriate as a model for describing inﬂuences of time-dependent magnetic ﬁelds on enzyme kinetics. To ob-

4944 C. Eichwald and J. Walleczek: Oscillating magnetic ﬁeld effectsJ. Chem. Phys., Vol. 107, No. 13, 1 October 1997

Copyright ©2001. All Rights Reserved.

tain an explicit expression for the function

f

(

k

isc

), Eq.

5

,the effective rate constant

k

2,eff

may be related to the radicalpair recombination probability:

16

k

2,eff

k

2

1

P

R

.

6

In this relation (1

P

R

) represents the probability that theradical pair does not recombine. Consequently, the enzymereaction cycle advances into the forward direction. By com-parison one ﬁnds that

f

(

k

isc

)

(1

P

R

).The approach, Eq.

6

, enables the calculation of

P

R

byusing well-established models known from the theory of radical pair recombination. One possibility is to use the ex-ponential model, which assumes that the radicals separateafter a certain period of time and subsequent re-encountersare neglected

in this case 1/

k

3

is the mean frequency of inter-radical distance changes

.

11,12

In the simplest case of asinglet–radical pair precursor with only one spin 1/2 mag-netic nucleus present and restriction to the high ﬁeld limit

B

A

hﬁ

, where

B

is magnetic ﬂux density and

A

hﬁ

is thehyperﬁne interaction constant

this model yields

11

P

R

B

k

2

k

2

k

3

1

1

2

s

1

2

d

2

1

2

2

s

22

d

2

,

7

1

1

k

3

2

k

3

k

2

k

3

k

2

/2

2

2

J

2

,

2

2

k

3

k

2

k

3

k

2

1

,where

s

,

d

(

)

2

(

a

/4)

2

. In this relation 2

J

correspondsto the energy splitting induced by the exchange interaction,

a

is the hyperﬁne interaction constant of the spin 1/2 magneticnucleus, and

(

B

/

)

B

g

/2 refers to the

g

mecha-nism. In the case of oscillating magnetic ﬁelds varying at afrequency that is much smaller than the reciprocal of theradical 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 investi-gated with pulsed or sinusoidally oscillating externalperturbations.

1–3

The application of pulsed perturbations en-ables an explicit solution of the model, which yields the re-laxation time constants of the enzyme

pulses of a rectangu-lar shape are applied

. The latter are given by the reciprocalof the characteristic exponents:

1,2

1

c

5

2

1

1

4

1

c

5

2

1

c

5

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 casesinvolving sinusoidally oscillating magnetic ﬁelds because of the complex time dependence of the function

f

(

k

isc

). How-ever, 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 matrixfunction exhibiting the period of

f

(

k

isc

). The homogenouspart of Eq.

9

represents a Mathieu type of differentialequation.

21

Floquet theory shows that it has normal solutionsof the form

21

x

i

p

i

t

exp

i

F

t

,

10

where

p

i

are functions with the same period as

f

(

k

isc

), and

i

F

are the characteristic exponents of the system, Eq.

9

Floquet exponents,

i

1,2

. The sum of the Floquet expo-nents is given by the following theorem:

21

1F

2F

1

T

0

T

Trace

P

t

dt

1

c

1

c

5

c

2

c

3

f

k

isc

T

,

11

where

T

is the period of

P

(

t

). The expression on the rightside 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 characterizethe behavior of the system under the inﬂuence of periodicexternal perturbations.

III. RESULTS AND DISCUSSIONA. The

g

mechanism

In this section a ﬁrst example is used to investigate thebasic response behavior of the enzyme system. Calculationsare restricted to the

g

mechanism, leading to coherent spinmixing 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

x

s

x

0

x

s

cos

t

x

sin

t

exp

t

,

12

y

y

s

y

0

y

s

cos

t

y

sin

t

exp

t

,

13

where

(

1

c

5

)/2 and

denote the real part and theimaginary part of the characteristic exponents, Eq.

8

, and

x

(

x

0

x

s

)(

1

c

5

)/2

(

y

0

y

s

)/

,

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 themagnetic ﬁeld pulse

initial conditions

, whereas

x

s

and

y

s

are the steady-state values for

t

1 in the presence of theﬁeld.Figure 1 depicts the analytical solution shown abovewith

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 accountthat the characteristic exponents are conjugate complex, andthe system settles into the steady-state exhibiting dampedoscillation. Therefore a simple measurement of the responseamplitudes contains insufﬁcient information for an estimateof the relaxation time constant. Experimental data should beﬁtted to the model to yield an accurate estimate of the relax-ation time constant.The relative change in the amplitudes may be estimatedin certain limiting cases. Taking into account that

c

3

1and assuming that

c

1

1, the relative change in the ampli-tudes, deﬁned as

1

x

s

/

x

0

, is given as

4945C. Eichwald and J. Walleczek: Oscillating magnetic ﬁeld effectsJ. Chem. Phys., Vol. 107, No. 13, 1 October 1997

Copyright ©2001. All Rights Reserved.

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