343 - Heterogeneous electron transfer kinetics of sperm whale myoglobin

Bioenergetics

BioeZectrochemistrystry ad J_

EZectroanaZ.

Ciicm.

116

(1980)

7 (rgS0)

447-457

447-457

Elsevier Sequoia S.A., Lausanne - Printed in Itaiy

Electron Transfer

343 - Heterogeneous

Myoglobin * 5y

and H.

EDMOND

F_ BOWDENO,

FRED

M_

Kinetics

of Sperm Whale

HAWKRIDGEO,**

N_ BLOUNT’~**

0 Department of Chemistry. Virginia Commonwealth University. Richmond, Virginia 23aSa USA a Brown Chemical Laboratory. The U.aiversity of Delaware. Newark, De!aware rg/rr USA Manwzcript received October

10th rg7g

Very little information has been reported describing the heterogeneous electron transfer kinetics of biological molecules at electrodes. We report here the first application of a recently developed spectroelectrochemical technique to the measurement of the heterogeneous electron transfer kinetics of a biological molecule, sperm whale myoglobin, at a methyl viologen modified gold miuigrid electrode_ The overpotential dependence of the heterogeneous electron transfer rate constant for the reduction of myoglobin at this surface gives rise to values of the formal heterogeneous electron transfer rate constant [/@>A = 3.SS ( f 0-07) x IO-” cm/s] and the transfer coefficient [a = o.SS (k O.OI)] for this electrocatalyzed process_ The importance of studying the heterogeneous electron transfer kinetics of biological molecules lies in the fact that many physiological electron transfer reactions occur heterogeneously_ -Though myoglobin does not function in this manner physiologically, our initial study has been directed at this molecule owing to its stability and ready availability_ It is expected that this technique will be applied to other optically transparent electrodes and other biological molecules thereby providing new insights into understanding biological redox reactions_

The kinetics of biological electron transfer reactions have been widely studied [r-3] and have been largely directed at elucidating the kinetics of homogeneous electron transfer reactions between two biolo&zxl

3-8

* Presented at the 5th International September Igig. Weimar (D-D-R). ** Authors to whom correspondence

03oz-45gS/go/o447-0457

@

Elsevier

Sequoia

Symposium should

S-A.

on Bioelectrochemistry,

be addressed_

448

Bowden.

Hawkridge

and Blount

molecules or between a biological molecule and an exogenous redox specks_ Questions remain regarding the unusually fast rates and selectivities of these reactions_ Little information is available which describes heterogeneous electron transfer kinetics of biological molecules_ Examples of such heterogeneous processes are the redo-u reactions between membrane bound species and diffusing species in photosynthesis [I, 33 and o_xidative phosphorylation [z]_ We have, therefore, developed a technique and an electrode system which permits the spectroelectrochemical measurement of the kinetics of heterogeneous electron transfer reactions of biological molecules_ Electrochemical techniques, often coupled to spectroscopic measurements, have been shown to be particularly suited to the study of biological electron transfer kinetics [4. =J_ Electrochemical techniques provide control of electron transfer reactions through the ability to control the potential of the working electrode_ An important problem in the application of electrochemical techniques to the study of biological redox systems is the general lack of thermodynamically reversible responses Exogenous molecules, mediators, of electrodes to biological molecules_ have been used to couple biological molecules and electrodes [s. 63 just as chemical titrations of biological molecules have required the use of mediators to obtain valid potentiometric measurements [_il_ Recently, there have been reports of direct electron transfer reactions between electrodes and biological molecules which proceed at measurable rates obviating the need for mediators_ Cytochrome c has been shown to undergo heterogeneous electron transfer reactions at optically transparent electrodes (OTB’s) of indium o_xide [SJ. at gold electrodes on which a.a’-dipyridyl is adsorbed [g, IO], at electrochemically modified gold minigrid OTB’s [XI], and at mercury electrodes [~a-I+]_ Cytochrome c, has been shown to be electroactive at mercury [IS-~] and gold [IS], and ferredoxin [q], myoglobin [zo], and hemoglobin [ZI] have been shown to be electroactive at electrochemicahy modified gold electrodes_ Measurement of the heterogeneous electron transfer kinetics of biological molecules at these electrodes was not possible with any e_xisting A spectroelectrochemical species-selective electrochemical technique. method for evaluating the heterogeneous electron transfer rate parameters at OTE’s was recently described and e_xperimentally verified for the model system potassium ferrocyanide at an antimony doped tin o_xide OTE [zz]_ This method is not subject to the interferences which impact the results of either the analogous chronocoulometric method which had been previously described [23] or other voltammetric techniques_ The species selectivity of the spectroelectrochemkal technique in the study of a variety of electron transfer mechanisms is well known [s. 6). Because the spectroscopic change arising from a single species undergoing an electron transfer reaction at an OTE can be selectively monitored, the results This factor is are not affected by other charge consuming processes_ important when considering the unknown impurities which are present in even the most highly purified biological preparations_

Electron

Transfer

Kinetics

of Sperm

Whale

Myo@obin

449

The present study describes the application of the spectroelectrochemical technique to the determination of the heterogeneous electron transfer parameters for the reduction of sperm whale myoglobin at the electrochemically modified gold minigrid electrode [rg. zo]_ Though myoglobin does not function physiologically as an electron transfer agent, our initial investigations have been directed at this molecule owing to its stability and ready availabilityThe results presented here show the applicability of using the spectroelectrochemicl method of determining the heterogeneous electron transfer kinetic parameters of biological molecules at OTE’s_ The extension of the present study to biological molecules which do function physiologically as electron transfer molecu!es is in progress in these laboratories_

Apparattts The potentiostat used in this work consisted of a MCKEE-PEDERSOX 1os6A configured so that current was measured across a load resistor in the auxiliary electrode lead with a unity gain differential input operational amphfierThe step potentials and bias potentials were applied to the input of the potentiostat via a conventional operational amplifier adder circuit_ PHILBRICK Nexus Ioog or 1026 operational amplifiers were used for these functions. The optical data were obtained with a HARRICK SCIEXTIFIC CORPORATION Rapid Scanning Spectrometer operating in the fixed wavelength mode at 434 nm_ The absorbance signal was measured by biasing the output of the log amplifier of the spectrometer to zero with a conventional adder/bias operational amplifier circuit- The absorbance-time transients were recorded on a HEWLETT PACKARD 70x5 X-Y recorder in the time base mode_ Electrochemical and spectroscopic signals were monitored with a KE~~HLEY zoz. a DIGII-EC 262, or a TEKTRONIS DBIjoz digital voltmeter_ The gold minigrid electrodes were ZOO lines per i&h, 67 oh transmittant, 0.1 mil nominal thickness. from BUCKBEE-MEARS, St. Paul, Xinnesota. The spectroelectrochemical cell used for electrode modification and for potential step chronoabsorptiometric experiments has been described [z4]_ An internal platinum wire coil au_xiliary electrode, used instead of an isolated auxiliary electrode, and the minigrid working electrode were mounted in the cell. The working electrode was taped to a quartz plate along with an aluminum foil contact. A 6 mm diameter This tape, hole was punched in the tape to permit optical measurements_ obtained from DIELECTRIX CORPORATION (Farmingdale, NY) was z mil thick Teflon with adhesive on one side. Taping the minigrid electrode to the quartz plate in this fashion placed the O-ring seal against the tape and not against the electrode to prevent tearing of the minigrid during

Bowden,

4.50

Hawkridge

and

Blount

assembly of the cell_ This method of electrode mounting also served keep the minigrid ekctrode pressed against the quartz cover.

to

Reagents Methyl viologen was obtained from K & K L_U~ORATORIES and was recrystallized three times from ethanol. Sperm whale myoglobin (Type II) was from SIGJL~ Chemical CompanyThe phosphate buffer (Titrisol, pH 7-00) was from E_ MERCK Company_ All other chemicals were reagent grade and all solutions were prepared in water distilled twice in glass_ Procedzwes The gold minigrid electrodes were modified using I-O m.M methyl vioIogen, pH 7-00 phosphate buffer, 0-1: _&l NaCl solutions_ A 3 cm3 portion of this solution was introduced into the vacuum degassing bulb, vacuum degassed. and introduced into the cell under nitrogen pressure as previousIy described [24_ A potential of -o-720 V vs_ N.H.E. was then applied for 5 minutes followed by application of 0-430 V ‘us_ NH-E_ for IO minutes The cell was then rinsed with distilled water and dried under vacuum at room temperature_ All experiments reported employed a Ag/AgCI (r-0 M KCL) reference electrode which was calibrated against saturated quinhydrone solutions of known pH [25-J to be 0.230 V vs. N.H.E. at the temperature of the measurements reported here, 25 (&I) OC. AU potentials reported here have been corrected to the N.H.E. reference scale_ A 3 cm3 volume of a myoglobin solution, prepared in pH 7.00 phosphate buffer, 0-r _iiI NaCI. was then introduced into the vacuum degassing bulb, vacuum degassed. and transferred into the cell [24]_ The cell was then positioned in the spectrometer which had been wavelength calibrated using

a hohnium o-tide filter. The initial and step potentials were then set with the working electrode disconnected_ With the working electrode connected the initial potential, 0.430 V in all experiments. was applied for IO mi-

nutes before initiating the potential step_ The potential step chronoabsorptiometric data were then recorded on the X-Y recorder for the

various values of step potentials- After data were recorded for a 50 second potential step, the potential was then returned to the initial value for IO minutes before conducting the next experiment_ Triplicate ments were performed for each potential step value and the order potential steps was random_ The initial step trial was repeated conclusion of each series of e_xperiments to confirm the long-term ducibility of the chronoabsorptiometric response_

Detailed

measurements

of the heterogeneous

electron

experiof the at the repro-

transfer

rate

parameters for the reduction of myoglobin at the modified gold minigrid

Electron Transfer Kinetics of Sperm WhaIe Nyoglobiu

-152

electrode were conducted with 40 +W myoglobin solutions_ Experiments were also performed using 50 $M and 20 ~31 solutions with the results being in agreement with the 40 @I case. The reduction of myoglobin may be represented as

where Mb, and i\Ibd are the o_Gdized and reduced forms, respectively, and A,-,, is the heterogeneous electron transfer rate constant for a given potential step_ The formal heterogeneous electron transfer rate constant, kojh, is obtained by measuring kf, as a function of overpotential, q, for a series of potential step chronoabsorptiometric e_xperiments [zz]_ The intercept of a plot of log kfbZWSZLSq yields KO'Lh, and the slope, -(a@/ (2.303 RT), affords the transfer coefficient, a [22]. Fig_ I shows representative potential step chronoabsorptiometric results obtained for ten different overpotential values_ These data were

Potential step chronoabsorptiometryof myoglobinat a modifiedgold minigridelectrode_ SolutionT 40 g&f myoglobinin phosphate buffer, pH 7-00, 0.1 1x1 XaCl. Trace, potential step (mV us. N-H-E.). and sequence of eqeriment I (a) -330 mV. S ; (b) -370 mV. 4 ; :{;3go mV. 6 ; (d) -400 mV. g ; (e) -410 mV. I ; (j) -4~0 mV, 7 ; (g) -430 mV, 3 : (h) -450 . I ; (22 -470 mV. 10; Q -4gc mV. 3_

acquired in random order as indicated in the figure legend and the reproducibility of a given potential step experiment is indicated in Fig. 3. These results were obtained at three different modified electrodes and at three different myoglobin concentrations. Series (6) of Fig. 2 includes data from the experiments shown in Fig. I: ; one of the traces is the first experiment run and the two other traces were obtained after all the results shown in Fig_ I: had been obtained_ Series (b) demonstrates the

Bowden,

452

Hawkridge

and Blount

Reproducibility of potential step chronoabsorptiometry of myoglobin at a modified gold minigrid electrode_ -AlI data obtained for a potential step to -470 mV. myoglobin in pH 7-00 phosphate buffer. 0-1 _M ?JaCl. (a) 50 +I myoglobin. (6) 40 p.M myoglobin. and (c) 20 piIf myoglobin,

invariance of the system response during the course of these e_xperiments. This check was performed in each set of experiments. Selected experimental resuk from Fig. I are shown in Fig. 3 together with theoretical absorbance-time transients calculated for various values of kfb_ The points are the theoretical results and the lines are the experimental results The experimental results do not agree with

0

10

20

30

40

50

Fig- 3_ Theor&cal and experimental absorbance OCKSUS time behavior of myoglobin gold minigrid electrode_ Eqerimental results taken from Fig- I ; traces are results and points are theory for : 1. -470 mV. kJ++ = r-78 X10-s Cm/S : &Jz = z-95 X IO-* cmls ; a. -400 mV, kfb = 1.66 x IO-~ cmls ; 0. -370 mV. x x0-5 cm/s_

at a modified experimental 0. -420 mV.

+

=

s-50 X

Electron

Transfer

Kinetics

of

Sperm

Whale

Mq’oglobin

453

theory at shorter times during a given potential step and this will be discussed subsequently_ Data from Fig. I were normalized to the diffusion controlled absorbance values over the period of the e-xperiment The theoretical diffusion controlled absorbance-time behavior is calculated from [s]

co is the bulk molar concentration of myoglobin, ba is the difference molar absorptivity of myoglobin at 434 nm (sdz,,= = 96.700 AI-lcm-l), D is the diffusion coefficient of Mb,, (1.14 x IO-= cm2/s [26]). and t is the time in seconds. The difference molar absorptivity was calculated from the measured difference in absorbance of dithionite reduced and air o_xidized myqglobin solutions at 434 nm together with the known value of urea at thus wavelength. rr4.000 LII-~ cm-l [a7]_ Fig. 4 shows the where

Fig. + Normalized absorbance versus log [(kf#i=)/D’l:] working curve and normalized data for the reduction of myoglobin at the modified gold min$rid electrode_ Calculated from data shown in Fig. I. best fit log k,Jz given in parenthesis ; 0. 3 = 396 (-4-55) : q . q= 416 (-4.16) i A. -q = 436 (-3-93) ; 0. q = 446 (-3-75) ; 0. q =456 (-3.66) ; A. q = 466 (-3-53) : 1. q = 476 (-3-33) : 0. q = 496 (-3-10) ; +. q = 516 (-z-75)-

working curve of normalized absorbance versus log [(k,$~)/D~] as well as the normalized experimental data_ The value of kfJzfor each potential step is obtained by plotting the normalized experimental data versus log (OS/I%) and overlaying these data on the working curve_ After best fit overlay of experimental data on the working curve, kfJ, is calculated from the difference in abscissa log values, namely log (kf&) = log (kf&t”/D”) - log (H/DE) _ These data are summarized in Table I.

Bowden.

454

Table

I-

Heterogeneous

globin

at

methyl

vioiogen

ekctron

Hawkridge

and

Blount

transfer rate constants for the reduction

eIectrochemicaJJy

modified

gold

396 416

Z-83 5-50

(+ 03s) x 10-s (& 0_2~)x10-5

minigrid

436

r-17

&o-06)

446 456 466 476

1.66 2-19 2-95 4-68

(2 O-07) x IO-4 (~0_12)x10~ (& 0_1g)x10-" (2 O_ZI)XIO-*

496 516

7-94

(+ o-56)

1-7s

(fo.21)

of myoekctrodes 4

x IO-~

x IO-* x 10-3

p At 25 ( f I) oC_ See test for solution conditions- Rate constants are mean values of five observations over the 30 to 50 second time domain_ Parentheses contain one standard deviation-

The dependence of the experimentally determined log (I&) on overpotential. q. (q = U,, f UdUO’, where r/‘,, is the experimentally measured step potential, CT,, = 0.330 V ‘JS_ N.H.E., and uo’ is the formal potential of myogIobin. 0.046 V us_ N.H.E. [&I) affords the data shown in Fig_ 5_ The formal heterogeneous electron transfer

Dependence of kf- on overpotential. Linear reamion slope = x_@z~ o.ol2) mV-1; intercept = -1o.q z (& 0.00s) cm/s ; coefficient of correlation = o.ggg2.

X 10-s

Electron Transfer Kinetics of Sperm Whale Myoglobin rate

constant

determined

at r) =

kO’% = 3.88 ($- 0.07) x 10-l~ cm/s_

from

the slope of this plot

is 0.88

o (0.046 V vs_ N.N.E.) The transfer coefficient,

455

evaluates as a, calculated

( f 0.01).

Disedon

The theory which was developed and applied to the measurement of the heterogeneous electron transfer kinetics of the reduction of myoglobin at the modified gold minigrid electrode assumed semi-infinite linear diffusion at a planar electrode [zz]. The minigrid electrode, however, is not planar_ Only after sufficient light-absorbing product has been generated to fill the LwZes in the minigrid is semi-infinite linear diffusion mass transfer realized_ It has been shown [sg] that the time required to achieve chronoabsorptiometric responses which are characteristic of semi-infinite linear diffusion may be estimated from consideration of the minigrid hole size and the spatial distribution of the lightThe time, t, required to f;ll the absorbing electrode reaction product. centers of the grid holes to an absorber concentration equal to one-half its value at the grid surface may be calculated from [zg]

where ;~tis one-half the hole edge length and D is the diffusion coefficient From the hole edge length of the grid used in this work (r-03 x of Mb,.. x0-0 cm) and the known value of D [27] the minimum potential step electrolysis time required for the theoretical e_xpressions derived for semi-infinite linear diffusion to be applicable to this minigrid system is calculated to be ca. 25 seconds. Consequently, only chronoabsorptiometric data acquired at times of observation greater than 25 seconds following application of potential steps to the modified minigrid electrodes could be reliably utilized for kinetic analysis_ The pronounced negative deviations of the A-t transients from those theoretically predicted for planar electrodes at times of observation less than ag seconds (Fig. 3) are a manifestation of this (chole-filling r) induction period. In contrast to the heterogeneous electron transfer rate constants noted for the reduction of myoglobin at the methyl viologen modified gold minigrid electrode described in this work (Table I),the heterogeneous electron transfer rate constants for this reductive process at pristine gold minigrid electrodes are immeasureably small even at the most extreme overpotentials employed here [zI]_ The application of the spectroelectrochemical technique to the measurement of the heterogeneous electron transfer kinetic parameters for the reduction of myoglobin at the methyl viologen modified gold minigrid electrode has been demonstrated. Future work will be directed at determining the effect of solution conditions (e.g. ionic strength and

Bowden,

4.56

Hawkrid~e

and Blount

pH) and of the electrode -material on the kinetic parameters -measured here_ Extension of the present work to inchrde cytochrome c, cytochrome c,, and ferredoxin is in progress. It is anticipated that information regarding the role of protein surface charge in controlling the rate of heterogeneous electron transfer wiJ.l be derived from these studies

AcImowIedgement

dation

We gratefully acknowIedge support by the National Science Foun(PCM 77-ooS67j and the National Institutes of Health (HL22821)_

E-E_ Energy

VAX TAMELES (Editor). Connersion

;

Cofacfors;

Bioorganic Probes.

Cilemislry : Electron Transfer and Academic Press. Inc.. Xew York

(1973) VOL IV RX_ RA~XOSD (Editor), Bioinorganic Chemistry--II, _kdvances in Chemistry Series, 163, American Chemical Society, Washington. DC. (1977) GO~IXDJEE (Editor), Bioenergetics of Photosynthesis, Academic Press. Inc., Xew York (1973) D-T. SAWYER (Editor), EZectrochemicaZ Studies of BioZo=icaZ Systems. American Chemical Society Symposium Series, American Chemical Society, Washin8ton. D-C_ (1977) VoL 38 T_ KVWASA and \V_R HEISEX-AS. ActsChetn_ Rer_ 9 (1976) ,+I R SZEX~RIJIAY. P_ YEH and T_ KUWASA. in EZectrochemicaZ Studies of BioZogkaZ Systems, D-T_ SAWYER (Editor), _.merican Chemical Society Symposium Series, American Chemical Society, \Vashir@on. DC. (1977) Vol. 38. pp_ 143-169. and references therein W.&X_ CLARK. Oxidufiott-Reduction PoteMials of Orgunic Sysfems. Williams and WiIkins Co_. Baltimore (1960) P_ YEH and T_ KWNAXA. Chem. Lett. (Japan) 1977. 1143 M-J. EDDOWES and KA.0. HILL. J. Am. Chem. Sot- 101 (1979) 4-261 Bt J_ EDDOWE~ and H.A_O_ HILL, J_ Chem. Sac-. Chem_ Commun_ 1977. 721 M_ WAXG and F_iK HAWKRIDGE. unpublished results S-R BETSO. M-H_ KLAPPER and L-B_ _.DERSOS. J_ Am. Chem- Sot_ 94 (I%=) 8197 B_A KUZ~ETSOV. G-P_ S~UMAKOVICH and X21_ MESTECHKISA. BioeZecZrothem_ Bioenerg_ 4 (1977) 512 F_ SCRELLER, Bioelectrochem. Bioenerg- 4 (1977) 490 K_ XI=. T_ YAGI. H_ Ixoi;nc~r and K. K~wm_~. J_ EZectrochem. SOL 124 0977)

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J_

Am.

Actu

545

Chem. (x979)

Sot_ 86

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Electron Transfer Kinetics of Sperm [IS]

R_

SIXGLETOX,

(r979) 893 [rg] H-L. LAXDRUM. [20]

99 (1977) 31% J-F_ STARGARDT, ('975)

L.

L.

and

CAMPBELL

R-T. SALMOW

and

FM.

FX.

F.&i. HAWKRIDGE

and

Whale

Myoglobin

HAWKRIDGE. HAWKRIDGE. H-L.

457

J_ BacterioZ.

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Anal

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(1979) 556 [23] J-H_ CHRISTIE.

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