j. biol. chem.-1979-hendler-11288-99 (malah nemen jaduul)

THE JOURNAL OF BHXLXICAL CHEMISTRY Vol. 254, No. 22, Issue of November 25, pp. 11288-11293, 1379 Printed m USA.

Potentiometric Analysis of Escherichia coli Cytochromes in the Optical Absorbance Range of 500 nm to 700 nm*

(Received for publication, April 2, 1979, and in revised form, May 29, 1979)

Richard W. Hendler From the Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014

Richard I. Shrager From the Laboratory of Statistical and Mathematical Methodology, DCRT, National Institutes of Health, Bethesda, Maryland 20014

The oxidation-reduction potentials of Escherichia coli cytochromes have been studied by a recently described technique for automated electrodic potentiometry (Hendler, R. W., Songco, D., and Clem, T. R. (1977) Anal. Chem 49,1908-1913; Hendler, R. W. (1977) Anal. Chem. 49, 1914-1918), where entire spectra are recorded at a series of solution potentials. New techniques for resolution of the spectra uersus voltage data have been applied. The results indicate that a l-electron transport chain conducts electrons from substrate to cytochrome d, which is the cytochrome oxidase. Cytochrome d contains several components which appear to increase electron transfer first to a a-electron stage and then to a 4-electron stage for the final reduction of a molecule of oxygen to 2 molecules of water.

The oxidation-reduction potentials of respiratory components can reveal the magnitude of energy liberation accom- panying electron transfer, the most likely sequential reaction order of the components, the existence of multiple components having indistinguishable optical properties, the number of electrons transferred, and the possible existence of energized members arising during the electron transfer process. Because of the importance of obtaining accurate oxidation-reduction potential data for respiratory components we have devoted a considerable effort to improving the reliability of the collection of such information. We have found that many of the assumptions used in the past to obtain oxidation-reduction potentials are unjustified and that some of the techniques are prone to yielding unreliable information. This paper presents a new approach to the electrochemistry and data analysis for oxidation-reduction potentials and presents results for the electron transport chain of Escherichia cd.

EXPERIMENTAL PROCEDURES

Materials-The mediators used in this work were: potassium ferricyanide, Merck and Co., Rahway, N. J. (I?,,, = 435 mV); quinhydrone, Fisher Scientific Co., Fair Lawn, N. J. (E,,, = 280 mV); 1,2- naphthoquinone (B,,, = 143 mV) and pyocyanine perchlorate (IX,,, = -34 mV), K and K Laborabories, Plainview, N. J.; phenazine methosulfate, Calbiochem, LaJolla, Calif. (E, = 80 mV); and 2- hydroxy-1,4-naphthoquinone, Eastman Organic Chemicals, Roches- ter, N. Y. (E,n = -145 mV). The mediator solutions were freshly prepared for each experiment in stock solutions of 12 mM for potas-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

sium ferricyanide and 6 !nM for each of the other five. E. coli membranes (T-fraction) were prepared as previously described (1). Protein concentration was determined by the Lowry procedure with bovine serum albumin as standard (2). Mediators f E. coli cell membranes were placed in 125 mM KCl, 62.5 mM potassium phosphate at pH 7.0 for analysis.

Equipment and Procedures-The equipment and procedures have been previously described (3-5). Analog signals for electrode voltages and optical density were passed through 20.Hz low pass active But- terworth filters (Frequency Devices, Inc., Haverhill, Mass.) before amplification to remove unwanted components, particularly at 60 Hz. Additional details relevant to the current studies are as follows. Prior to the experimental titration, the system was cycled through a prelim- inary phase of air oxidation followed by endogenous substrate reduction. Although equilibration can be obtained from either direction (4), the system returns to equilibrium much more rapidly after a reductive pulse than an oxidative pulse. Therefore, all titrations were performed by fist raising the voltage of the suspension to the highest value and then proceeding to a stepwise reduction. Optical transmittances across a spectrum of wavelengths were measured and conversion to absorb- antes was performed outside of the spectrophotometer. In this way, a series of spectra as a function of voltage was obtained. The average titration rate was -30 mV/h and the average evaporation rate was 0.03 ml (1% of total volume)/h. The time to titrate 90% of a single component was 4 h divided by the number of electrons transferred. The pH of the titration mixture was 7.0 at the start and finish of all titrations where the highest voltage obtained did not exceed 300 mV (i.e. for cytochrome bl). When the voltage was raised through the range of cytochrome d, however, an acidification of the medium occurred. The pH of the medium did not change further through the reductive titration. The average pH during the titration of cytochrome d was about 6.3. Current studies to be reported separately indicate that the process of acidification of the external medium is related to the oxidative titration of cytochrome d.

Resolution of Spectra versus Voltage Data into Individual Oxi- dation-Reduction Components-In a previous advancement in the treatment of spectral data, Butler and Hopkins (6, 7), and Shipp (8) used fourth differences to sharpen and separate clusters of peaks. Shipp was thus able to observe several individual peaks in clusters that had looked like one peak. Our aim, in contrast, was the detection of transitions with respect to voltage, which was done more simply by following the height of the composite peaks. Since each experiment involves many complete spectra, any simplification is desirable. It should be noted that a transition may be hidden by simultaneous growth and shrinkage of two peaks in a cluster. Still, no method is without some shortcoming, as emphasized by Butler and Hopkins in their discussion of the fourth difference method.

In order to isolate spectral changes due to the change of oxidation- reduction state of an oxidation-reduction component from those due to changes of background shape in our work, either of two analytic techniques were used. When a peak was isolated in the sense that the second derivative of the peak was of much greater magnitude than those in the nearby background, the second derivative at the wavelength of maximum absorbance was determined by a least squares procedure. A parabola was fit through seven points composed of the peak wavelength and three evenly spaced points on each side close to

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Potentiometric Analysis of E. coli Cytochromes 11289

the midpoint. From principles described in Chapter Six of Ralston (9), one can derive the formula for this approach using a central point and n equally spaced points on each side.

Let

and

xZ = central wavelength

A = x:, - x,

N=2n+- 1

Sums not involving y:

S2 = 2A2 i i2 ,=I

S, = 2A4 C i4

r=l

and

u = (NS, - &)I2

Sums involving y:

S.I = A2 1 j'(y, , + y,+,) ,=I

Then:

d2y/clx2 = (NS., - S&)/u.

In our three-point method, n = 1 was used. When a peak was not isolated but was overlapped by peaks of

similar widths, the spectrum was resolved into individual Gaussian components and a flat base level. It is assumed that peaks may grow or shrink but may not change midpoint or half-width. A good choice of peaks is one with the fewest number of Gaussians and in which the synthesized curve fits the experimental curve throughout the whole voltage titration range.

Once the data have been resolved into values for the second derivative or height of a Gaussian component as a function of voltage, a second procedure of numeric analysis involving computer fitting, is employed. In this stage, the number of components obeying the Nernst law is determined as well as their relative amounts and the number of electrons transferred by each (3-5).

RESULTS

Fig. 1 shows absorbance spectra for the components present in the potentiometric titration mixtures studied in this work. The identification of cytochrome components in the E. coli respiratory chain has been traditionally based on reduced versus oxidized difference spectra as shown in Fig. 2. The points of intersection of the reduced and oxidized spectra are called isosbestic points and are believed to represent reference

wavelengths where the absorbance is constant for reduced and oxidized conditions. Quantitative analysis for individual cytochromes is based on selecting a peak and a nearby reference wavelength, preferably an isosbestic point. For cytochrome bl(cu), the wavelength pair 560 nm and 550 nm are

used. For cytochrome bl (p), 530 nm and 540 nm could be used and for cytochrome a,, 596 nm and 585 nm might be taken. The difference spectrum for cytochrome d presents a different

shape than a true Gaussian but the peak and trough wavelengths at 630 and 650 nm present the most logical choice for a two-point analysis. In the current approach, we have not been limited to only two wavelengths for each oxidizing potential but instead have collected entire spectra. Fig. 3 shows surfaces generated by plotting spectra as a function of voltage for a solution containing six mediators alone and for one containing the mediators plus a suspension of E. coli cell membranes.

2

METHOSULFATE

1 PYOCYANINE

A

FERRICYANIDE 1 QUINHYDRONE

OYl.J 1 t.. 320 510 700 320 510 700

WAVELENGTH (nm)

FIG. 1. Optical components present in titration mixtures. The top six panels show spectra for the mediators used in these studies. The symbol 0 denotes the oxidized spectrum and R the reduced. Oxidation and reduction were accomplished electrically in the case of mediators. For the E. coli membranes, air was the oxidant and sodium dithionite, the reductant. Dithionite contributes to the absorbance at 355 nm. The bottom line in the top six panels shows the absorbance for the assembled cuvette containing clear buffer prior to the addition of mediator. In the panel showing E. coli membranes, the bottom line shows the spectrum for the polypropylene used as an optical reference. Phenazine methosulfate was used at two concentrations, 0.1 miw (0, and R,) and 0.04 mrvr (02 and RJ. Potassium ferricyanide was present at 0.2 mrvr, quinhydrone at 0.6 mrvr, and the other three mediators at 0.1 mrvr each. The E. coli membranes were present at a concentration of 6.2 mg of protein/ml. The buffer was 0.125 M KC1 and 0.063 M potassium phosphate at pH 7.0. The optical reference was water for mediators alone and was polypropylene and frosted glass for light-scattering suspensions analyzed for respiratory components. To convert spectra referenced to plastic and glass to absolute spectra, the absorbances shown in the lower right-hand panel should be added to the appropriate relative spectra. Thus, the solid line spectrum was the reference for the surface shown in Fig. 5, the short dashed line spectrum for the surface in Fig. 6, and the long dashed line for the surface in Fig. 7.

As expected from the spectra shown in Fig. 1, most of the features in the surface obtained with mediators alone are found in the lower wavelength regions. Pyocyanine, however, does create a hump in the higher wavelength, higher voltage


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11290 Potentiometric Analysis of E. coli Cytochromes

ABSOLUTE REDUCED MINUS OXIDIZED [-

FIG. 2. Spectra for E. coli membranes. The a and /3 absorbances for the recognized E. coli cytochromes are most commonly seen in difference spectra from 500 to 700 nm. This figure shows a typical difference spectrum (rightpanel) and the original spectra from which the difference spectrum was derived (leftpanel). Isosbestic points are the cross-over points for the oxidized (0) and reduced (R) spectra which show up as 0 AA in the difference spectrum. The oxidized and reduced spectra shown here are enlargements of a portion of the spectra shown in Fig. 1, lower left panel. The divisions shown on the 0 AA line in the right panel are placed at 5-nm intervals.

part of the surface. This is most evident in Fig. 3, Panels 6, c, and d. The presence of E. coli membranes markedly alters the low wavelength, low voltage region with a massive uni- dentified feature centered at 370 nm and the Soret absorption at 430 nm. What is most apparent in the surface from E. coli membranes is the relative smallness of the absorbances of the (Y and /3 bands of the cytochromes and the fact that they are situated as small features on an extensively slanting and changing background. These facts are lost in difference spectra. Another feature which will become much more obvious in subsequent figures is the curvature or hump in the surface seen from the high wavelength side especially in Panels 6 and c. The analysis of the spectral features in the part of the surface containing the characteristic absorbances of the cytochromes was complicated by the presence of pyocyanine, which is an effective mediator only at relatively high concentrations. Although pyocyanine is commonly used, we found that its replacement by succinate resulted in better mediation and the total elimination of the interfering optical properties. This can be seen in the surfaces of Fig. 4, Panels a to d, compared to the corresponding panels in Fig. 3. Although the hump in the surfaces containing E. coli membranes is also reduced (compare Panels a to d in the two figures), it is still an important feature as will be seen subsequently.

For the analysis of the bl cytochromes, the spectrum was started at 490 nm where, because of the lower levels of

FIG. 3. Spectra-voltage surfaces for six mediators f E. coli membranes. Each surface is presented in four aspects. The a panels show a full face view from a low voltage position. The axis running perpendicularly into the depth of the picture represents increasing voltage and is labeled E. The horizontal axis seen in full view represents wavelength and the vertical axis, absorbance (A). The a panels give scaled information for the surface. For example, Panel a shows that the wavelength range extends from 320 to 695 nm. The divisions are in steps of 5 nm each. The E-axis extends from -203 mV to 349 mV. The voltage steps are uneven and reflect oxidation-reduction buffering activity. Mediators or oxidation-reduction buffers are cho- sen to give closer steps in areas of maximum interest. The optical reference for mediators alone (Panels a to d) was water. The reference for the suspension of membranes (Panels a to d) was polypropylene and frosted glass. Panel a shows the absorbance values for several of the maxima in the surface to indicate the range of absorb- antes in the figure. Panel a shows that the voltage range covered for this surface extends from -214 mV to 377 mV. The absorbance scale shows AA relative to the light-scattering reference which had an

absorbance and light scattering, the concentration of E. coli membranes could be increased from 2 to 3.4 mg/ml and the level of phenazine methosulfate increased from 0.04 to 0.1 InM. Furthermore, the wavelength resolution was improved from 5 nm to 2 nm and much closer voltage steps were taken. By these procedures, the cytochrome bl(a) and bl(/3) features stand out clgarly from the surface in all four views (Fig. 5). In spite of the absence of pyocyanine, pronounced localized curvature in the background surface is seen as indicated by the asterisks in Fig. 5, Panels a, b, and c. This feature which is ever present, appears as perhaps a side of an extremely broad Gaussian component centered in the 700 to 800 nm region. It decreases in absorbance with decreasing voltage with an apparent E, of about 30 mV and an n value of 2 electrons. It was not characterized further. A bracket is shown in Panel a to show the peak and isosbestic point traditionally used for the quantification of reduced cytochrome b,(a). Al- though the completely oxidized and reduced spectra have equal absorbances at the isosbestic point of 550 nm (see Fig. 2), the belief that such a point represents a useful reference wavelength for the assay of reduced cytochrome bl(a) is seen in the figure to be unwarranted.

The kinds of surfaces used for analysis of cytochromes d and a, are shown in Figs. 6 and 7. Because these spectra were started at still higher wavelengths than those in Fig. 5, with consequently less light scattering, the level of E. coli membranes could be increased to 6.9 mg of protein/ml. At the highest voltage, 394 mV, an absorption peak at 650 nm is present (Fig. 6, Panel d). With decreasing voltage, this peak appears to shift its location to lower wavelengths centered at about 635 nm (Fig. 6c, at the arrow). With still lower voltages the peak gradually shifts back to a higher wavelength. An alternative view, which our analysis favors, is that changes in individual peaks centered at 650 and 634 nm account for an apparent shift in location of a single peak. It can also be noticed that in the voltage range covered, no absorbance for cytochrome a, is seen in the wavelength region near 600 nm. The surface in Fig. 7 overlaps the region shown in Fig. 6 and extends into a lower voltage region. A cytochrome al absorbance can be discerned in this surface at the lower voltage end, particularly in Panels a and b. The broad composite cytochrome d feature extends throughout the whole voltage range.

Using the same mediators as previously used in chemical titrations of cytochrome b,(a) (4, 5), electrodic titrations revealed the same components, although the relative percent of each was different in the two preparations (Table I). Because of the changing nature of the general absorbance-light-scattering background as voltage is changed (shown in Fig. 5), the

apparent absorbance of 1.76 at 320 nm and 0.67 at 695 nm. The b panels show the surface viewed from the high-wavelength, low-voltage corner of the surface. The c panels view the surface from a position of high wavelength and high voltage. The d panels present the opposite face from that in the a panels, viewed from a position of relatively high voltage. The irregular or jagged nature of a surface seen at high absorbance values reflects the limitation of the 12-bit A/ D converter which admits 4096 possible transmittance values. A variation in signal of 0.1% or 4 units represents an uncertainty of less than 0.004 A in the absorbance range of 0 to 1.0, of 0.043 A at 2.0 A and greater than 0.3 A at 3.0 A. For this reason, quantitative analysis of spectral surfaces is done with data collected in the lower absorbance ranges. Concentrations for the mediators were those shown in Fig. 1 with phenazine methosulfate at the lower value. E. coli membranes were present at 2 mg of protein/ml. The symbols, ?, S, b,(p), bl(a), aI, and d refer, respectively, to components contributed by the E. coli membranes, namely unknown, Soret, cytochromes bl(/3), bl(cu), al, and d.


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Potentiometric Analysis of E. coli Cytochromes

-Ecoli MEMBRANES + Ecoli MEMBRANES

11291

a

a

-214

WAVELENGTH hn) 695

d


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--E. co/i MEMBRANES i-ho/i MEMBRANES

a a

1.18

.77

ii0 WAVELENGTH (nm) 695 0, '

b

Fro. 4. Spectra-voltage surfaces for five mediators (i.e. minus pyocyanine) f E. coli membranes. Refer to the legend of Fig. 3 for general information and concentrations.

two-point method is not reliable. A three-point method leads ponents are greatly altered. The second derivative method to a dramatic change in the resolution of the E, values of the yields results in close agreement to those of the three-point components. The two higher E,, values are markedly reduced method. When pyocyanine is replaced by succinate as media- and the relative amounts of the highest and lower E,,, com- tor, the electrodic titration, even by the two-point methods,


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01 490 WAVELENGTH Cnm)

630

d

FIG. 5. Spectra-voltage surface for cytochromes bl (a and /3). Wavelength steps were at 2 nm each. The optical reference was Five of the six mediators (--pyocyanine) were present at the polypropylene and frosted glass as shown in Fig. 1. Optical features concentrations shown in Fig. 1 except for quinhydrone which was at for cytochromes b,(a) and b,(P) are indicated by arrows. A prominent 0.4 mM. Phenazine methosulfate was present at 0.1 mM, and E. coli curvature of the surface is indicated by *. Further details are pre- membranes at 3.4 mg of protein/ml. Succinate was present at 0.1 mM. sented in Fig. 3.

b

I 680

WAVELENGTH (nm) 694

FIG. 6. Spectra-voltage surface for cytochromes d. Four of the six mediators (-pyocyanine and 2-hydroxy-1,4-naphthoquinonej -were present. Phenazine methosulfate and 1,2-naphthoquinone were at 0.1 mM, ferricyanide was at 0.2 mM, quinhydrone at 0.6 mM, and E. coli membranes at 6.9 mg of protein/ml. Wavelength steps were at 2 nm. The optical reference was polypropylene and frosted glass as shown in Fig. 1. The arrows indicate features discussed in the text. Further information is provided in Fig. 3.


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576 WAVELENGTH hm) 694

FIG. 7. Spectra-voltage surface for cytochromes d and aI. steps were 2 nm. The optical reference was polypropylene and frosted Five of the six mediators (-pyocyanine) were present at the concen- glass as shown in Fig. 1. The symbols a, and d are used to indicate trations shown in Fig. 1 except for quinhydrone which was at 0.5 mM. the location of absorbances for cytochromes aI and d. Further infor- Phenazine methosulfate was present at 0.1 mrvr and E. coli membranes mation is provided in Fig. 3. at 6.9 mg of protein/ml. Succinate was present at 0.1 mM. Wavelength

TABLE I

Cytochrome bl components

bi (4 2-point fit 3.point fit 2nd derivative fit

Err, % E, B E, % Chemical titration (+ pyocyanine) Refs. 4, 5 No. of experiments (14) Electrodic titration (+ pyocyanine)

No. of experiments (3) Electrodic titration (no pyocyanine)

No. of experiments (3)

222 + 5.3 39 f 1.2 107 + 3.7 33 + 1.9

-47 t 5.1 28 + 1.1

214 f 17.9 23 + 5.8 161 + 15.6 40 +- 7.7 157 + 15.3 41 + 7.8 118 -c 1.5 37 -L 6.2 66 + 9.9 38 -c 0.9 63 f 9.7 38 + 7.5

-39 -e 15.6 40 f 1.7 -37 -e 7.5 21 f 5.7 -35 -e 7.3 22 + 1.3

180 + 3.3 30 + 1.2 182 -I 3.7 25 k 2.0 186 + 3.0 24 +- 1.2 49 -t 4.0 35 + 5.3 54 + 2.8 55 k 3.7 57 c 4.7 60 f 5.7

-88 + 5.7 35 f 4.3 -108 + 14.4 20 + 1.7 -105 f 16 16 + 4.5

Electrodic titration (no pyocyanine)

No. of experiments (3)

Not done ?h 60 +- 6.7 (70-100)

?h - n E, values are in millivolts f SE. For the 2-point fit, the peak at 560 nm and the isosbestic point at 550 nm were used. For the 3-point

fit, 570 nm was also used. The 2nd derivative method was based on seven points as described under Experimental Procedures. b The I? shown above and below the E, of 60 mV for cytochrome b,(P) indicates that there was insufficient absorbance to determine

whether the higher and lower E, components seen for cytochrome bl(a) were present.

yields results more like those of the three-point and second pyocyanine/mg of protein and yielded E, values of -17 mV derivative fits obtained in the presence of pyocyanine. The and -30 mV. The third had 29 nmol of pyocyanine/mg of lowest E, component, however, is very difficult to mediate protein and yielded an E, value of -69 mV. Therefore, the fully. In a previous publication using electrodic titrations (4), apparent difference for the lowest E, component seen in the the lowest E, component found by the two-point method in titrations where pyocyanine was replaced by succinate is due the presence of pyocyanine was -72 mV and in the absence of to the superiority of succinate as a mediator for this compo- pyocyanine, it was -34 mV. The two higher E, components nent. Because of this and because pyocyanine contributes were not significantly affected by omitting pyocyanine. In the appreciable absorbance in the regions of interest, the data current data, two of the three titrations in the presence of obtained in the absence of pyocyanine are deemed more pyocyanine were at a concentration ratio of 8.7 nmol of reliable. Fig. 8a shows b,(a) species for a succinate-mediated


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.001232

- .0002549. _ -190 55

# - .000254-9,a 300 55 300

MILLIVOLTS

CYTOCHROME bl ALPHA .001232

CYTOCHROME bl BETA

b

FIG. 8. Cytochromes bl titrations by second derivative analysis. For each spectrum at a different voltage such as shown in Fig. 5, the second derivative at 560 nm (cytochrome b,(a)) and at 530 nm (cytochrome b,(p)) were determined by the least squares procedure using seven points for each determination. The negative of the second

experiment as extracted from spectra by the second derivative approach and fit by one, two, and three components. In consideration of all of the data obtained for cytochromes bl(o), we believe that three components are present and that within experimental error of about -+lO mV, their E, values are 186, 57, and -105 mV.

The total amount of change of absorbance due to titration of the component known as cytochrome I%(/?) is only 0.009 unit on a starting background absorbance of about 1.45 (Table II). This is in contrast to about 5 times as much absorbance due to cytochromes &(a). Therefore the analysis of cytochrome bl(P) is subject to relatively much more noise. Fig. 8b shows data for cytochrome &(/I) fit to one, two, and three components. We are most certain that the middle E, component seen for cytochrome bl(a) is present and accounts for at least two-thirds of the total signal. Because of the relative smallness in the amounts of signal expected from the highest and lowest E, components seen with cytochrome &(a), the amount of signal expected from these two components in bl(p) is too small to be resolved. However, because of the corre- spondence of the middle E, component in the (Y and /I species and because both the cytochrome bl(a) and cytochrome bl(/?) absorbances were present in isolated, purified cytochrome 61 (lo), we feel that our data further establish that both absorb- antes do originate from cytochrome bl. Another point of interest shown in the data of Table I is the fact that the relative amount of the lowest E, species of cytochrome bl(oc) markedly decreases as the number of points in the analysis increase. This illustrates the contribution of background changes in the two-point analysis.

The major two wavelengths which appear to be associated with cytochrome d in the usual difference spectrum of the respiratory components are 630 nm (peak) and 650 nm (trough) (Fig. 2). When the difference in absorbance for these two wavelengths is plotted against the voltage of the medium, a rapid rise is seen with decreasing voltage from voltages above 390 mV down to about 312 mV (Fig. 9a). The difference in absorbance then drops to a low at about 50 mV and then starts to rise again with further reduction of voltages (Fig. 96). According to traditional considerations, this behavior could be taken to indicate that the midpoint potential of cytochrome d is under the influence of the oxidation-reduction state of other components of the respiratory chain so that upon reduction, the midpoint potential of cytochrome d is lowered sufficiently to cause its subsequent reoxidation and corresponding lessening of the 630 nm minus 650 nm AA.

derivative is plotted as a function of voltage with the points showing the experimentally determined values. The computer best fit is shown for a single component by a long dashed line, for two components by a short dashed line, and for three components by a solid line. The results of such analysis are shown in Tables I and III.

TABLE II

Absorbance values of components

cyto- Mediators Protein Due to cyto-

A Total A chrome chrome A A ma/d AA % total

;ii bl(/3) 0.294 1.16 3.4 1.45 0.009 0.62 560 h(a) 0.240 1.01 3.4 1.25 0.046 3.7 600 a: 0.200 1.19 6.8 1.39 0.006 0.43 634 da4 0.184 1.13 6.8 1.31 0.017 1.3 650 d,so 0.178 1.10 6.8 1.28 0.015 1.2

-l Q

.0X62

.01432 by 97

MILLIVOLTS

FIG. 9. Cytochrome d titration by two-point method. Spectra such as those comprising the surfaces shown in Figs. 6 and 7 were used to determine the AA for 630 nm minus 650 nm and these AA plotted as a function of voltage. The open symbols were obtained from experiments in the absence of pyocyanine. The + symbols are from an experiment in the presence of pyocyanine.

A full resolution of the spectra versus voltage surface into background and Gaussian components, leads to a more complete understanding of the changes in absorbance as influ- enced by changes in the oxidizing potential of the medium. The overall background is fit by two broad Gaussians centered at 500 nm and 700 nm and a flat base level which rises and falls. In addition, two major Gaussian components centered at 634 nm and 650 nm and up to three minor Gaussian components at 590,660, and 680 nm account for the spectra observed from 570 nm to 695 nm. The absorbance at 590 nm accounts for the aI component observed in difference spectra of the E. coli respiratory chain. The ability of these resolved components to account for the spectral features observed in the experimental system is shown in Fig. 10 for several different voltages. The component centered at 634 nm in the absorption spectrum was fit by a 4-electron transfer (Fig. 11). The fits based on l- or 2-electron transfers were quite poor. When the


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311 mV I transition

I 139 mV

WAVELENGTH (nml

FIG. 10. Experimental and reconstituted spectra for cytochrome d analysis. Spectra such as those comprising the surface in Fig. 6 were resolved by Gaussian analysis into a flat base-line level, two broad Gaussian components centered at 500 and 700 nm, one minor component at 680 nm, and the cytochrome d components at 634 and 650 nm. Reconstituted spectra were constructed by recom- bining the absorbances of the resolved components. The figure shows the superposition of experimental and reconstituted curves at four critical voltages. At 383 mV, a component at 650 nm is present but the major increase in absorbance of the component at 634 nm has not occurred (refer to Figs. 11 and 12). At 311 mV the transition from a major absorbance at 650 nm to one at 634 nm is occurring. At 278 mV, there has been a recovery of the absorbance at 650 nm. At 139 mV, still more of the 650 nm component is present.

.03214 7-p b

Ht.

.02081

~ i 2% 325 400 4cxl

MILLIVOLTS

FIG. 11. Cytochrome d (634) titration by Gaussian analysis. Spectra such as those comprising the surface in Fig. 6 were resolved into Gaussian components plus a base level to reconstitute experimentally derived curves (see Fig. 10). The heights of the component centered at 634 nm, in absorbance units, plotted as a function of voltage, are shown as open symbols. The computer best fit for a single component transferring 1 electron is shown in Panel a by a long dashed line, a 2-electron transfer is represented by a short dashed line, and a 4-electron transfer by a solid line. The fit shown in Panel b uses a minor 2-electron component at E, = 282 mV and a major 4. electron component at E, = 310 mV.

computer was asked to fit best the number of electrons transferred with the initialization at n = 2, it selected n = 4.3. The root mean square error was essentially equal for a fit with n equal to either 4.0 or 4.3. In most cases (i.e. 4 out of 5) the fitting of the component at 634 nm was slightly improved by including a minor 2-electron transfer component with an E, value about 20 mV lower than the main 4-electron transfer component (Fig. llb). The component absorbing at 650 nm responded in a very complicated way to changes in voltage (Fig. 12). It was extremely difficult to obtain a convergence for a fit that would accommodate the dip occurring at about 326 mV. The best fit in all cases for the voltage region of about 150 to 400 mV was obtained with four components transferring 1 electron, 2 electrons, 4 electrons, and 4 electrons. The voltage

Hf.

.02728

01301 1 100 250 400 -150 75

MILLIVOLTS

FIG. 12. Cytochromes d (650) titration by Gaussian analysis. By the same procedure described in Fig. 11, the heights of the Gaussian component centered at 650 nm are plotted as a function of voltage. The points in Panel a were obtained from a surface like the one shown in Fig. 6 and those in Panel b from a surface like the one in Fig. 7. The solid line on Panel a was the computer best fit for four components as follows: n = 1, E, = 187; n = 2, E, = 268; n = 4, E,, = 309; n = 4, E, = 311. The dashed line includes four components as follows: n = 1, E, = 189; n = 2, Em = 265; n = 4, E, = 309; n = 8, E, = 317. The b panel extends the analysis to lower voltages where an additional component with n = 1 and E, = 28 is indicated.

.45,

I

I C

WI 6 WAVELENGTH (nm)

i20

15

P

I ID

.05

FIG. 13. Enhancement of apparent cytochrome al absorbance by difference spectroscopy. The curves labeled 0 and R are absorption spectra for oxidized and reduced samples of a suspension of E. coli membranes at 6.8 mg of protein/ml. The large dots represent the derived difference spectrum. The scale for AA is twice that of A.

region from 150 mV down to about -100 mV required an additional l-electron component. These components along with the resolved cytochrome b, components are listed in Table III.

The component known as cytochrome ai from reduced versus oxidized difference spectra contributes only about 0.006 A to a background of about 1.4 units (Table II) and is therefore very difficult to characterize accurately. Resolution of this component by both Gaussian and second derivative techniques was carried out. Greater confidence is attached to the second derivative fits even though the scatter is higher. In two experiments, the Gaussian technique gave an E, of -9 f 20 mV; the second derivative technique gave an E, of 25 + 5 mV. The component called cytochrome aI is artifactually accentuated in a difference spectrum as shown in Fig. 13. The oxidized background spectrum has a concavity at about 588 nm to 602 nm. This augments the initial rise in the cytochrome a, component seen in the difference spectrum. It also adds absorbance to that of the real but smaller component that arises upon reduction. The right side of the difference spec-


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TABLE III

Resolved cytochromes

For the b, cytochromes, the traditional designations of a and p chrome d components as discussed more fully in the text. Columns 3 have been retained. For the d cytochromes, two classes have been and 4 show the number of electrons transferred by the component identified, one absorbing at 634 nm and the other at 650 nm. To and whether the individual absorbance is associated with the reduced distinguish multiple components among the individual cytochrome or oxidized state. Amounts are in units which reflect the resolution classes, superscripts are used with the numbering proceeding from technique used. For the second derivative technique, this is absorb- the highest E,, species to the lowest. The cytochromes listed in Group ante units per nm of wavelength squared at the peak wavelength. For 1 have been resolved from the cytochromes providing the major the Gaussian technique it is absorbance units at the peak wavelength. optical absorbance and represent solutions to which we attach greater The values following -c are standard errors of the mean which were confidence. Group 2 contains components providing a lesser amount obtained for three experiments in the case of cytochrome bl and five of absorbance and which therefore are resolved with somewhat less experiments in the case of d. confidence. Group 3 contains an alternative resolution for the cyto-

Resolution tech- - FOrin nique

Cytochrome lLrz No. of electrons R = reduced Amount 2D = second deriva- 0 = oxidized tive

G = Gaussian

bl(d bl(d bl(d d(634) d(650) d(650) d(650)3 d(650)4

b,(P)

$634) d(650)

d(634) d(634) d(650) d(650) d(650)3 dC65014

186 + 3 57 k 5

-105 + 16 323 f 5 324 + 5 322 f 5 276 -c 5 196 & 7

60 rf: 6.7 25 f 5

302 + 10 24 f 4

329 + 5 313 I!z 5 330 + 5 320 + 5 278 + 5 195 -I 7

Group 1 1 R 0.316 x lo- rt 0.031 x lo- 2D 1 R 0.769 x lo- rt 0.086 x lo- 2D 1 R 0.204 x lo- + 0.060 x lo- 2D 4 R 0.017 f 0.0014 G 4 0 0.132 + 0.009 G 4 R 0.136 -t 0.009 G 2 R 0.0054 * 0.0012 G 1 R 0.0044 f 0.0014 G

Group 2 1 R 0.23 X lo- C 0.025 x lo- 2D 1 R 0.095 x lo- + 0.001 x 1o-3 2D 2 R 0.0024 f 0.0010 G 1 0 0.0084 + 0.0004 G

Group 3

8 R 0.0124 + 0.0011 G 4 R 0.0061 f 0.0015 G 8 0 0.0183 f 0.0008 G 4 R 0.0227 + 0.0018 G 2 R 0.0057 + 0.0012 G 1 R 0.0044 f 0.0014 G

FIG. 14. Possible sequence for E. coli respiratory chain of cytochrome components. The scheme is constructed from the components deduced in the current study assuming a linear order arranged in increasing values of the midpoint potentials. Cytochrome designations are by lower case letters with identifying subscripts, superscripts, and wavelengths where needed. RED and OX designate whether the major absorbance is associated with the reduced or oxidized forms of the cytochrome. Evidence for the branching is based on the fitted n values for number of electrons transferred.

CYTOCHROME OXIDASE SCHEME

*

0, +

4e +

4H I

*ti,o

trum for cytochrome a1 is accentuated both by the concavity AA compared to the individual absorbances, an expanded of the oxidized spectrum and by the appearance of the reduced scale is used, which further magnifies the peak. absorbing component at 634 nm which emphasizes the mini- I f the components of Groups 1 of Table III plus the tradi- mum to the right of the cytochrome ai peak in the difference tionally recognized cytochrome al are arranged in a linear spectrum. Furthermore, because of the small magnitude of sequence of increasing E, values, a possible electron transport


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4e P

RED dt6501 1 le *I REDd,66,,, j ( flEDd(6501 1

+ 320 mv

OX d16501

RED d163-41

+ 330 mv

20, + 8e

+

8

FIG. 15. Alternative cytochromes oxidase scheme. This scheme for cytochrome oxidase is constructed in accord with the finding that an n value of 8 also fits the data for cytochrome d components centered at 634 and 650 nm.

chain for E. coli is constructed as shown in Fig. 14. The components with E,, values below 200 mV form a l-electron transport chain. The components of cytochrome d (i.e. cytochrome oxidase), however, multiplex in two stages up to 4- electron transfers, which is the number required to reduce a molecule of oxygen. Whether all of the recognized components are acting in a single electron transport chain and whether they are arranged strictly in accord with their E, values has not been established in the current work. Group 3 in the table has been arrived at by setting the parameter for number of electrons transferred in the steepest slope regions to eight. The closeness of fits as indicated by root mean square devia- tions was noticeably better in these cases than when n was set to 4. An alternative cytochrome oxidase sequence based on these resolutions is shown in Fig. 15.

DISCUSSION

We have found in these studies that many of the techniques and basic assumptions commonly used to study quantitative changes in cytochromes in membrane suspensions can lead to serious errors. The quantification for individual cytochromes is based on split beam spectrophotometry and dual wavelength spectrophotometry of light-scattering suspensions, using a peak and reference wavelength. We have defined and discussed in detail four situations under normal usage of these techniques which lead to nonlinearity of response (11).

In using the AA between two characteristic wavelengths to quantify a given cytochrome it is assumed that: 1) The background changes between these two wavelengths are negligible compared to the changes for the cytochrome; 2) reference points defined by those wavelengths where a totally reduced spectrum crosses a totally oxidized spectrum represent isosbestic points where the absorbance is constant regardless of the oxidation-reduction state of the system; 3) characteristic optical absorbance peaks for a particular cytochrome always increase on reduction so that a reduced minus oxidized difference spectrum shows the reduced spectrum for all of the cytochromes with the light-scattering background canceled out.

We have found that these assumptions do not necessarily hold. The absorbance background is not relatively flat and

does not remain at a constant height nor retain the same shape throughout a range of different oxidation-reduction potentials. Therefore, following absorbance at a peak wavelength or AA between a peak and reference wavelength does not provide reliable data on changes of oxidation-reduction state of a component of interest. We have found further that the oxidized form of a cytochrome can display prominent absorbance features which occur at different wavelengths and have different widths than the reduced absorbance peaks. Therefore, a reduced minus oxidized difference spectrum shows a composite of new features arising on reduction aug- mented by changes due to the subtraction of peaks and troughs present in the oxidized spectrum.

In our work, the two-point analysis of cytochrome bl led to serious errors in the determination of E, values for the species present. We feel that the current values based on determining the second derivative at the peak, from a seven-point analysis, provide much more dependable data. In the case of cytochrome al, the subtraction of an oxidized spectrum and the magnification of the difference spectrum augment the apparent size of this feature, which upon analysis by either the Gaussian or second derivative approach represents an extremely small absorbance peak. The most interesting findings in the current study apply to the cytochrome oxidase (i.e. cytochrome d) region of the spectrum. The two-point analysis leads to a totally incomprehensible response of the cytochrome to changes in oxidation-reduction potential of the medium.

The cytochrome d absorbance feature in the three-dimen- sional surface contour displayed most unconventional behavior. It seemed to shift its location from 650 nm to about 635 nm and then back again to about 645 nm. In fitting the data to behavior of oxidation-reduction entities we had to consider the possibility of whether a single chromophore could display such erratic behavior as a function of its oxidation-reduction state. We rejected this hypothesis on two counts. Firstly, there should be two separate absorbance peaks for oxidized and reduced heme centers and secondly, when computer fits were obtained based on a one-chromophore model, they were de- cidedly inferior to the fits based on the existence of two fixed chromophore centers at 634 nm and 650 nm. Further detailed


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analyses of the cytochrome oxidase components were then based on the two-chromophore model. An unexpected finding was that the absorbance of the component centered at 650 nm started high in the oxidized state, then sharply dropped, then rose sharply at first and then more gradually with decreasing voltage to about 150 mV, then dropped again at lower voltages. Another unexpected finding was that the steepness of the absorbance versus voltage plots for both the feature absorbing at 634 nm and that at 650 nm could be fit only by Nernstian functions involving more than single electron transfers. In fact, the resolution of components absorbing in the cytochrome oxidase region leads to a representation of a possible branched system which collects electrons from four single electron transfer chains and multiplexes them for the eventual reduction of a molecule of oxygen.

Cytochrome oxidase has not been purified from E. coli. I f it is similar, however, to that of mitochondria which have been rigorously studied (12), it will contain two iron and two copper centers. It has long been appreciated that a sequential single electron transfer to oxygen is thermodynamically un- likely because of the instability of 02- (13). Therefore, either a 2-electron reduction of % O2 or a 4-electron reduction of O2 is anticipated. Early electron paramagnetic resonance studies by van Gelder and Beinert (14) suggested an antiferromag- netic coupling between iron in cytochrome a3 and another oxidation-reduction component in the cytochrome oxidase. Many recent studies employing magnetic susceptibility mea- surements and magnetic circular dichroism in addition to electron paramagnetic resonance support the idea of a magnetic coupling of the iron in cytochrome a3 to a copper, resulting in a functional 2-electron transfer component (15- 18). Lindsay and Wilson have reported that chemical potentiometric titration of cytochrome oxidase liganded to carbon monoxide results in a Nernstian value of n. = 2 electrons transferred (19, 20). The native enzyme was reported earlier to titrate with an n = 1 for each iron center (21). Anderson et al. (22) using a coulometric titration technique disagreed with the results of Lindsay and Wilson indicating the involvement of carbon monoxide with 2-electron acceptors and interpreted their findings in terms of a l-electron process. Heineman et al. have shown that natural cytochrome oxidase requires 4 electrons for complete reduction (23) although their coulometric technique does not tell whether the transfers are for single electrons or multiples. Arguments for a concerted multi- electron transfer from the fully reduced enzyme to oxygen have been presented (24-26). The absorbances we have studied at 634 nm and 650 nm most likely represent iron heme groups which individually can transfer only single electrons. Although we are not proposing a particular mechanism at this point, there would have to be a way in which more than a single cytochrome oxidase molecule could interact in the membrane so that reduction of the multimer could occur in a single concerted step when the required number of electrons was amassed. The model with a final 4-electron transfer was presented over that with an &electron transfer although the data more closely fit the 8-electron case, because it makes more sense in terms of the 4 electrons required to reduce a molecule of oxygen. However, if our analysis is correct and if

a coordination of several molecules of cytochrome oxidase can be accomplished, the possibility that 2 molecules of oxygen can be reduced at one time cannot be completely eliminated.

Whether all of the major components revealed in this work participate in an electron transport chain passing electrons from a substrate to oxygen has not been established nor has the actual biological sequence of their operation. In subsequent work using a rapid scanning spectrometer plus natural biological electron donor substrates these questions will be studied in mitochondrial systems as well as in E. coli.

Acknowledgments-We appreciate and acknowledge the continued support of David Songco of the Computer Systems Laboratory and Thomas R. Clem of the Biomedical Engineering and Instrumentation Branch.

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electron transfer process

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