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1997-Eichwald-Low Freq MF Radicals Enzymes

1997-Eichwald-Low Freq MF Radicals Enzymes

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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
1
1
 ES
2
2
S
 E 
••
S
3
isc
 ES
*
4
 E 
P
 E 
••
S
3
,
1
where the forward
backward
rate constants are denoted as
i
(
i
), and the other symbols represent the following:
,enzyme, (
 ES
), enzyme–substrate complex prior to radicalpair generation,
S
 / 
(
 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,
, triplet
, and
isc
is a magnetic field-dependent rateconstant characterizing singlet–triplet mixing. A shorthandnotation,
(
 E 
••
S
), representing all three triplet states
1
,
0
,
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
2
, andrecombination via
2
. The rate constant
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
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
2
radical pairrecombination
,
3
forward reaction coordinate motion
, and
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 
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,
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
 / 
(
 E 
••
S
), aresmall and rapidly converge to the steady-state value, becausethe rate constants
2
,
3
, and
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
)
(
 E 
••
S
)
)/ 
 E 
total
,
(
1
S
0
)
,one obtains the following set of equations from the enzymereaction scheme, Eq.
1
:
dxdt 
1
1
c
1
c
2
c
3
 f 
isc
 x
 y
,
2
dydt 
c
2
 f 
isc
 x
c
5
 y
,
3
 z
c
3
 f 
isc
 x
,
4
where
 f 
isc
1
2
isc
3
1
c
4
2
c
4
 / 
isc
3
,
5
and the following parameters are used:
c
1
1
(
1
S
0
),
c
2
2
/(
1
S
0
),
c
3
2
3
,
c
4
2
3
,
c
5
4
/(
1
S
0
).The relation
c
2
 f 
(
isc
) in Eqs.
2
3
represents an ef-fective rate constant,
2,eff 
2
 f 
(
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
2,eff 
becomes time dependent via the timedependency of 
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
(
isc
), Eq.
5
,the effective rate constant
2,eff 
may be related to the radicalpair recombination probability:
16
2,eff 
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
(
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/ 
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
2
2
3
1
 
1
 
2
s
 
1
 
2
2
1
2
 
2
s
 
22
2
,
7
 
1
1
3
2
3
2
3
2
 /2
2
2
 J 
2
,
 
2
2
3
2
3
2
 
1
,where
s
,
(
 
)
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
(
) 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 
(
isc
) and
2
c
2
 f 
(
isc
).To obtain an explicit solution is not possible in casesinvolving sinusoidally oscillating magnetic fields because of the complex time dependence of the function
(
isc
). How-ever, Eqs.
2
3
may be cast into the following form:
dx
   
dt 
e
   
P
 x
   
,
9
where
e
   
(1,0),
x
   
(
 x
,
 y
), and
P
(
) is a periodic matrixfunction exhibiting the period of 
(
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
exp
i
F
,
10
where
p
   
i
are functions with the same period as
(
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
0
Trace
P
dt 
1
c
1
c
5
c
2
c
3
 f 
isc
,
11
where
is the period of 
P
(
). 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
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
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
 
 x
sin
exp
 
,
12
 y
 y
s
 y
0
 y
s
cos
 
 y
sin
exp
 
,
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
 
1 in the presence of thefield.Figure 1 depicts the analytical solution shown abovewith
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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