An electrochemical assay for the characterization of redox proteins from biological electron transfer chains

ANALYTICAL

BIOCHEMISTRY

1%.

269-274

( 1991)

An Electrochemical Assay for the Characterization of Redox Proteins from Biological Electron Transfer Chains Frauke

Baymann,

Institut

fiir Biophysik

Received

July

David

A. Moss,l

und Strahlenbiologie,

and Werner Universitiit

Mtintele*

Freiburg,

Albertstrasse

23, 7800 Freiburg,

Federal

Republic

of Germany

1, 1991

A sensitive and quick assay for redox proteins based on electrochemical titrations in a thin-layer electrochemical cell is described. Using a combination of modified-electrode and “mediator-enhanced” electrochemistry, equilibration of the cell volume (4 ~1) with the applied potential allows series of spectra as a function of the potential to be recorded rapidly. A complete redox titration between +500 and -600 mV (vs AgI AgCl I 3 M KCl) in 30-mV intervals takes approximately 2 h. The detection limit of the assay, evaluated for cytochrome c at the a-band absorption, is quoted to approximately 100 pmol. The use of this redox assay for the detection of redox-active contaminants in biochemical preparations, for the determination of midpoint potentials of redox enzymes, and for the characterization of complex membrane-bound or soluble redox systems is described. 0 1991 Academic Press, Inc.

In the analysis of the reaction sequence in biological electron transfer chains, fractionation of the membrane, removal of water-soluble redox proteins, and solubilization of membrane-bound proteins are the steps typically preceding a characterization of the individual components by redox titration. In the course of these purification steps, gel electrophoresis is conveniently used for the analysis of the fractionated protein pattern, but spectroscopic techniques are frequently employed as well, mostly as a quick difference spectrum to compare an oxidized (with FeCy3) with a reduced (ascorbate or dithionite) state of the preparation.

i Present address: IRCH, Kernforschungszentrum Karlsruhe, P.O. Box 3640, 7500 Karlsruhe 1, Germany. * To whom correspondence should be addressed. 3 Abbreviations used: OTTLE cell, optically transparent thin-layer electrochemical cell; E,, midpoint potential (at specified pH) ; FeCy, ferricyanide; PATS 4, pyridine-3carboxaldehydethiosemicarbazone; CV, cyclovoltammogram. 0003-2697/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form

While the assay through a gel allows the detection of very small amounts of contaminating proteins without any information on their redox properties (if they are redox-active at all), the assay through oxidized-minusreduced difference spectroscopy at least yields information on the overall redox processes in the purified and/ or contaminating protein. Stepwise chemical redox titrations with spectrophotometric control are more appropriate, since they yield accurate E, values of contaminating proteins, information which is helpful for contaminant identification and for selection of further purification strategies. A precise stepwise chemical redox titration, however, is too time-consuming to be performed at all individual stages of a preparation. Electrochemical titrations in a spectroelectrochemical cell, where the potential is applied electrically at a transparent electrode, would offer several advantages over such classical chemical titrations. These include rapid equilibration of the solution with the applied potential, monitoring of the current and charge flowing to /from the redox center, and the accessibility of a wider potential range. Furthermore, the speed and convenience of such titrations allow their routine application during a purification. We have recently developed an ultra-thin-layer spectroelectrochemical cell with a minimal path length of approximately 10 pm, this being a requirement for measurements in the mid-infrared in regions of strong water absorbance ( 1) . In this cell, equilibration of the total volume with the applied potential proceeds within seconds to at most some minutes. This allows rapid recording of uv lvis (and ir ) spectra of the redox-active species as a function of the applied potential in order to establish an electrochemical titration over a wide potential range. We have successfully used this OTTLE cell with direct electrochemistry at chemically modified gold electrodes for the study of cytochrome c ( 1) and cs ( 2 )

and to record infrared difference spectra between the redox states of these proteins. For the water-soluble c269

Inc. reserved.

270

BAYMANN,

MOSS,

AND

MANTELE

ness of ca. 10 pm. This thickness is reached when the meniscus of the flattened drop has reached the rim of the window. The volume around the gold grid and counter electrode is then filled with electrolyte by an external port, and contacts are made to the potentiostat. Spectroscopy

FIG. 1. Cutaway diagram of the spectroelectrochemical CaF, window mounted with epoxy on plexiglass ring (b) ; (c) ylene body; (d) platinium counter electrode; (e) gold mesh electrode (mesh pattern not to scale) ; ( f ) flat rubber rings; lary connection to reference electrode. The arrow indicates cal axis of the cell.

cell. (a) polyethworking (g) capilthe opti-

type cytochromes and for the copper protein plastocyanin, suitable surface modifiers have facilitated direct electron transfer at the electrode. Using redox mediators at small concentrations, redox titrations of cofactors in large detergent-solubilized membrane proteins or even intact membranes become possible (3). Having thus established that small water-soluble proteins as well as large detergent-solubilized proteins exhibit rapid and quantitative electrode reactions using a combination of modified-electrode and mediated electrochemistry, we have applied this method as an analytical tool to follow the purification steps of redox enzymes and to study complex redox systems. Here we report this assay, its limits, and its application during the purification procedures of cytochromes c, from photosynthetic bacteria. EXPERIMENTAL

Electrochemical

A single-beam spectrophotometer designedin our laboratory was used to record the spectra as a function of the potential set by the potentiostat. Both the potentiostat and the spectrophotometer drive and detection system were controlled by a common software developed in our laboratory. A redox titration was composed of repeated cycles consisting of (a) stepwise setting of the potential, (b) recording of the oxidative or reductive current as a function of time, (c) further equilibration of the solution /suspension for a preset time, and (d) recording of a spectrum. The spectra were stored in the computer for further processing, as were the traces of current vs time. All potentials quoted are vs Ag/AgC1/3 M KC1 if not otherwise stated; add 208 mV for potentials vs standard hydrogen electrode. Sample Preparation Solutions of solubilized proteins or water-soluble proteins were concentrated in a small-volume ultrafiltration cell with an exclusion limit of 10 kD (Centricon 10 from AMICON, Witten). Small-volume (below 50 ~1) concentration was done in an ultrafiltration cell of local design, with a residual retention volume of approximately 5 ~1. Membraneous protein suspensions were concentrated by ultracentrifugation (2OO,OOOg, 1 h) . Concentration was made to improve sensitivity and proved necessary whenever large volumes of column eluates were to be handled. Electrode

Cell

The ultrathin-layer electrochemical cell used for this redox assay was developed from that described in ( 1) and is shown in Fig. 1. Briefly, a 200~lines/in., 6-pmthick gold grid (Buckbee-Mears Co., St. Paul, MN) serves as a working electrode (70% transmission) in the spectrophotometer beam. A Pt foil counter electrode is placed at the rim of the cell, and a miniaturized Ag/ AgCl / 3 M KC1 reference electrode is connected via a salt bridge to the interior of the cell. Contacts are made via Pt wires pressed radially onto the gold mesh and Pt foil, respectively. Only approximately 4 ~1 of concentrated protein solution /suspension are sufficient to fill the cell, this drop being placed on the gold mesh layered on the bottom window of the disassembled cell and pressed to a thick-

and Electrochemistry

Modification

and Mediators

The surface of the gold electrode was chemically modified according to the procedure described by Hill and co-workers (4) after it was layered onto the lower cell window by adding a drop of a solution of pyridine-3-carboxaldehydethiosemicarbazone ( PATS 4). After 10 min, excess PATS 4 was removed with distilled water and the mesh was dried. The PATS 4 molecules form a monomolecular layer on the gold electrode and prevent proteins from adhering and denaturing on it. Proteins may interact directly with a modifier and perform electron transfer at the electrode. In order to perform electron transfer with redox enzymes that badly interact with the modifier, a cocktail of mediators with E,‘s spaced approximately 40-120 mV was added to shuttle electrons between the electrode and the redox enzymes, with each mediator at a concentration of 20 pM. The

ELECTROCHEMICAL TABLE Redox

Mediators

Used

for

Electrochemical

redox

271

PROTEINS

Titrations

Soluble

f216 +133 +74 -8 -63 -108 -220 -333 -433 -560 -628

chloride

REDOX 0.0061

Eb, (vs Ag/AgCl) (mv)

mediators with their given in Table 1.

FOR

1

Compound Ferricyanide l,l-Dimethylferrocene Tetrachlorobenzoquinone Ruthenium hexamine 1,2-Naphtoquinone Trimethylhydroquinone Menadione 2-OH-1,4-Naphtoquinone Antraquinone-Z-sulfonate Benzyl viologen Methyl viologen

ASSAY

midpoint

in

5 fi P e 4 a

Water EtOH EtOH Water EtOH EtOH EtOH Water Water Water Water

potentials

x

0.002 0.000 -0.002

f/ -0.006

I

I

I

I

400

,

I

1

450

Wavelength

are

,

I

I

,

500

I

I

I

,

600

550

[nm]

FIG. 2. Selected difference spectra from a reductive titration of a crude extract of Rs. rubrum cells at potentials from f250 to -650 mV at a step width of 50 mV. The curves shown were taken at potentials of +200, +150, +lOO, 0, -250, and -650 mV (in the order of increasing absorbance at 550 nm), with the reference spectrum obtained at f300 mV.

Data Analysis A full set of titration data consisted of single-beam spectra at closely (20-50 mV) spaced electrode potentials as well as of the respective currents vs time as a consequence of the potential step. The spectra were analyzed using the program E,-TIT written by D.A.M. Briefly, titration curves were calculated for all wavelength elements in the spectra, with a reference wavelength to be chosen to isosbestic points or regions outside absorbance bands in order to correct for baseline shifts or light scattering. The wavelength under investigation as well as the reference wavelength could be chosen conveniently by moving a cursor. The program allowed titration data to be fitted with a Nernst curve with n and E, as variables.

whereas a broad difference band at 420 nm points to a contamination by an additional compound. Analysis of the full set of difference spectra at 50 mV spacing reveals a total of three redox-active and absorbing compounds present in the “crude extract.” Figure 3 shows the titration curve thus obtained for the absorbance at 563 nm, with the reference cursor set at 579 nm. The three components can be observed throughout a wide wavelength range with their relative amplitudes depending on their absorbance maxima. The line drawn through the data points in Fig. 3 represents a weighted sum of three Nernst curves for an n = 1 electron transfer and for midpoint potentials of approxi-

0.0020,

RESULTS

AND

DISCUSSION

The purification procedure for cytochrome cp from Rhodospirillum rubrum according to the method of Bartsch (5) with slight modifications included breaking of the cells in a French press followed by removal of cell debris by a centrifugation step. The “crude extract” thus obtained is then further purified on a sucrose gradient and a diethylaminoethyl column. Figure 2 shows a selection of redox-induced difference spectra obtained from a reductive titration of the “crude extract” in the potential range from -650 to +300 mV at a step width of 50 mV. The difference spectra at +200, +150, +lOO, 0, -250, and -650 mV (in the order of increasing absorbance at 550 nm) were all calculated with respect to a reference spectrum taken at 300 mV. A strong difference band at 552 nm indicates that a reasonable amount of cytochrome cq is present,

0.0016-j

: 2 P

0.0012

2 $

0.0006

a 0.0004

1 1

o.oooo~j -0.7

-0.5

FIG. 3. Absorbance length of 579 nm (data full line represents a midpoint potentials at

-0.1

-0.3

Potential

0.1

[mV]

at 563 nm with respect to a reference wavetaken from the titration used for Fig. 2). The sum of three Nernst curves with n = 1 and approximately +113, -228, and -558 mV.

272

BAYMANN,

MOSS,

0.0031

0.001

:’

:

0.000

‘\ ! ..__ _____._......\..., _ :..__.s _..-.- A-:..

:

-0.001

8

Q

-0.002

:,’

- -___

__-’ . .._

Sensitivity

---__

1;. I’

ii -0.003 Ii -0.0041

I

400

MANTELE

wavelength. Any traces of contaminants-if present at all-were within our detection limit (see below).

0.002’(’,.d\ ‘I‘L

t3 FE ::

AND

, 450

I

I

I

Wavelength

, 500

r

I

7

*

, 550

r

I 600

[nm]

FIG. 4. Separated difference spectra for the individual compounds of the crude extract of Es. rubrum cells calculated from the Nernst fit in Fig. 3. (-) cytochrome cx, 0 mV minus 200 mV, Em ca. +113 mV; ( - - -) cytochrome cc’, -250 mV minus -100 mV, E, ca. -228 mV, “x” (see text), -650 mV minus -500 mV, E, ca. (4 . *) compound -558 mV.

mately +113, -228, and -558 mV, respectively. This Nernst fit allows us to calculate separate difference spectra of the individual components, which are shown in Fig. 4. The full line represents the redox-induced difference spectrum due to cytochrome c2, identified according to the a-band absorption and the midpoint potential of approximately 113 mV (6). This difference spectrum was calculated between 0 and +200 mV, thus ensuring that no contributions from the contaminants appear in the spectrum. The dashed and dotted lines represent the difference spectra of the contaminants. As for the compound with E, approximately -228 mV, the broad peaks at 550 and 420 nm indicate another c-type cytochrome (7)) presumably the cc’ cytochrome present in Rs. rubrum ( 5). The electrochemical titration provides a convenient, quick, and sensitive assay for following the purification steps for the c, cytochromes. Figure 5 shows a redox titration comparable to that in Fig. 2, but taken in the course of the purification of Rps. uiridis cytochrome c,, after chromatographic purification and chromatofocusing according to Welte et al. (8). The redox-induced difference spectra taken between -650 and +300 mV show clear isosbestic points, indicating the presence of only one species in its reduced and oxidized form, respectively. Analysis of the spectra of the purified component at 550 nm as a function of the potential shows a single Nernst curve (Fig. 5, inset) with E, = +88 mV characteristic for cytochrome c2 ( 6). Nernst curves with the same E, and n values were obtained throughout the spectrum. In addition, the Nernst curves did not depend on the selection of the reference

of the Assay

In order to characterize the sensitivity of the assay, we have recorded redox-induced difference spectra from solutions of cytochrome c (horse heart, from Sigma) at different concentrations. We have been able to detect difference signals due to the cytochrome c a-band with as low as 0.0005 absorbance units. This sensitivity is partly due to our design of the spectrophotometer, but mainly arises from the reaction-induced difference technique, which permits comparison of the same sample. This “redox perturbation technique” eliminates uncertainties due to unequivalent concentrations upon comparison of two different samples. With a cell pathlength of approximately 10 pm and the extinction difference ( reduced-minus-oxidized) at 550 nm of 20 ( mM X cm) -I (7), this corresponds to a 25 PM cytochrome c solution. While this concentration appears to be rather high (a 25 PM cytochrome c solution in a l-cm cuvette has a reduced-minus-oxidized absorbance difference of 0.5 at 550 nm) , it should be considered that the total volume of the electrochemical cell is only about 4 ~1, leading to an absolute quantity of about 100 pmol of cytochrome c that can be detected. This detection limit compares well with the detection limit of standard gel electrophoresis with Coomassie blue staining, where protein quantities between 0.5 and 5 pg (in the case of cytochrome c this corresponds to

I 500

Wavelength

[nm]

FIG. 5. Difference spectra of a solution of cytochrome cg of Rps. uiridis after purification (conditions as described in the legend to Fig. 2) _ (Inset) Data points and Nernst curve (Em = +88 mV, n = 1) for the absorbance observed throughout the spectrum (see text). Correction of the baseline by the selection of a reference wavelength is not necessary.

ELECTROCHEMICAL

ASSAY

FOR

REDOX

273

PROTEINS

b

3 -0.02 2 -0.04-

: 2

-0.06-

-O.OB-0.06 -0.10

;

-10

I

,

0

,

,

10

,

,

20

Time

,

,

30

,

,

,

40

50

,

]

60

-I

-10

I

0

’

1,

10

I

20

Time

[set]

’

II

30

1

40

I

50

‘I

60

[set]

(a) Current vs time trace for a cytochrome ca sample of Rps. virirdis upon a potential step from 150 to 100 mV (around midpoint FIG. 6. notential) at t = 0. Note the three kinetic nhases. (b 1 Current vs time trace for a cytochrome c2 sample of Rps. virirdis upon a potential step from 0 to’ -50 mV (outside redox activity j at t = d.

40-400 pmol) are detectable in a single band (9). However, the assay described here yields additional information on the redox properties of the contamining components, which could not be obtained by a determination of the apparent molecular weight. While strongly pigmented proteins can be easily detected with this spectrophotometric titration, redox proteins with weak extinction coefficients are difficult to assess. In these cases, the current vs time traces recorded for each potential step provided additional information on the presence of redox-active proteins. Figure 6a shows the current vs time trace for a cytochrome c2 sample upon a potential step from 0.15 to 0.10 V at t = 0. Before this step, the cell was equilibrated for 2 min at 0.15 V. Following the step, a current flow that decreases to virtually zero within 1 min is observed. The sign of the current flow corresponds to electron flow from the electrode to the cytochrome. At least three kinetic phases can be discerned in the I(t) curve. On the basis of a simultaneous measurement of the absorbance and the current vs time, we can attribute the slowest phase in Fig. 6a to the cytochrome molecules in the grid holes (the bulk of the cytochrome molecules) . Only this phase is observed in the absorbance vs time trace. The dependence of the intermediate phase on the path length of the cell (data not shown) indicates that it arises from cytochrome molecules within a few micrometers of the electrode (either above or below the gold grid bars). In contrast, the most rapid phase in the I(t) curve is independent of the path length and may thus be attributed to cytochromes adsorbed at the electrode surface. While the I(t) trace in Fig. 6a was recorded close to the midpoint potential (E, = +88 mV vs Ag/AgC1/3 M KCl) , the trace in Fig. 6b is the result of a potential step outside the cytochrome redox activity (0 to -0.05 V).

Clearly, the step amplitude is considerably smaller, and the relaxation time differs from that in Fig. 6a. The residual small step in Fig. 6b is caused by the electrochemic response of the mediator. For the quantitative evaluation of current traces, this overlapping mediator response should be subtracted. Ultimately, recording a cyclovoltammogram ( CV) would represent the ideal electrochemical procedure for detecting contaminants or for characterizing a redox protein. However, although the spectroelectrochemical cell is suitable for recording CV (1) , we have found it extremely difficult to obtain the rapid electrode response necessary for a diagnostically useful CV. In conclusion, we have found this assay to be of great use for the optimization of purification procedures and the analysis of purified redox proteins. It allows a quick characterization of complex redox systems in an intact membrane or a single protein complex as well. We have recently used this assay for a full titration of the redox cofactors of the photosynthetic reaction center from the purple bacterium Rps. uiridis ( 10). In this case, titration extended from +400 to -600 mV (vs Ag/AgCl) and included the four hemes ( 11)) the dimeric bacteriochlorophyll primary electron donor, and the quinones (M. Leonhard, M. Bauscher, D. A. Moss, and W. Mantele, unpublished data).

ACKNOWLEDGMENTS The authors thank M. Bauscher, M. Leonhard, and F. Fritz for help with spectrophotometric titrations and S. Grzybeck for developing computer programs. We are grateful for support by the Deutsche Forschungsgemeinschaft (Grant Ma 1054/2-2). W.M. acknowledges a Heisenberg Fellowship from the Deutsche Forschungsgemeinschaft.

274

BAYMANN,

MOSS,

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Diploma

Thesis,

J., and Miintele, Faculty

of Biology,

W. (1990) Univer-

3. Miintele, W., Leonhard, M., Bauscher, M., Nabedryk, E., Breton, J., and Moss, D. A. (1990) in Reaction Centers of Photosynthetic Bacteria ( Michel-Beyerle, M. E., Ed.), SPRINGER SERIES IN BIOPHYSICS, Vol. 6, pp. 31-44, Springer Verlag, Berlin. 4. Hill, H. A. O., Page, D. J., and Walton, N. J. (1987) J. Ekctroanul. Chem. 217, 120-140. 5. Bartsch, R. G. ( 1971) in Methods in Enzymology (San Pietro, A., Ed.), Vol. 23, pp. 344-363, Academic Press, San Diego. 6. Bartsch, R. G. ( 1978) in The Photosynthetic Bacteria (Clayton,

AND

MANTELE R. K., and Sistrom, New York.

W. R., Eds.),

Chap.

13, pp. 249-274,

7. Dickerson, R. E., and Timkovich, R. (1975) (Boyer, P. D., Ed.), 3rd ed., Vol. 11, pp. 397-547, San Diego. 8. Welte, W., Hiidig, H., Wacker, matogr. 269, 341-346.

T., and Kreutz,

Plenum,

in The Enzymes Academic Press, W. (1983)

J. Chro-

9. Hames, B. D. (1981) in Gel Electrophoresis of Proteins: A Practical Approach (Hames, B. D., and Rickwood, D., Eds.), Chap. 1, pp. 22-50, IRL Press, Oxford. 10. Deisenhofer, J., Epp, O., Miki, (1985) Nature 318,618-624. 11. Fritz, J., Moss, publication.

D. A., and

K.,

Mlntele,

Huber, W.

R., and (1991),

Michel,

submitted

H. for