High and Low Energy States of Cytochromes · Vol. 241, No. 20, Issue of October 25, pp. 4577-4587, 1966 Printed in U.S.A. High and Low Energy States of Cytochromes III. IN REACTIONS

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 241, No. 20, Issue of October 25, pp. 4577-4587, 1966

Printed in U.S.A.

High and Low Energy States of Cytochromes

III. IN REACTIONS WITH CATIONS

(Received for publication, May 7, 1965)

BRITTON CHANCE AND BRIGITTE SCHOENER

From the Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19104

SUMMARY

Upon addition of low concentrations of calcium or manganese to pigeon heart mitochondria in a phosphate-free medium, a characteristic pattern of steady state changes ensues which leads to a high absorbance at 560 mp (at 300’ K) characteristic of cytochrome b. Other components of the respiratory chain are highly oxidized; electron transport from succinate and glutamate is inhibited, as is energy transfer into the pathway for diphosphopyridine nucleotide reduction. The spectroscopic changes and the inhibition of electron transfer represent State 6.

Low concentrations of cations are required for half-maximal change in cytochrome b; about 15 pM calcium gives half- maximal effect. The calcium concentration required is a function of pH; the response of cytochrome b of the largest magnitude is observed at pH 8. High concentrations of calcium (50 to 150 PM) decrease this effect.

The inhibited state is readily reversed by phosphate, leading to reduction of all the components of the respiratory chain except cytochrome b, which proceeds to a less absorbing state. Under these conditions, the functions of electron transport, energy-linked pyridine nucleotide reduction, and response to adenosine diphosphate are largely restored. The inhibited state of electron transfer is readily reactivated by uncoupling agents, but energy-linked pyridine nucleotide reduction is not reactivated.

Examination of cytochrome b spectra at low temperature (77’ K) indicates the accumulation of a compound absorbing at 555 rnp in the presence of low concentrations of cations and its transformation to a compound absorbing at 565 rnp upon addition of phosphate.

Cytochrome c responds most rapidly to cations; cytochrome b and reduced pyridine nucleotide respond less rapidly, but at low concentrations of cations. The alkalinization of the cristal membrane (29) is a slower reaction that requires higher cation concentrations.

Both the reaction of cytochromes with cations and the alkalinization of the cristal membrane are considered as factors in causing the inhibited state of electron transfer, State 6.

In previous papers, we described the kinetics of respiratory carriers in calcium-stimulated respiration (1, 2). At that time,

we observed anomalous oxidation-reduction states of the re spiratory carriers caused by adding low concentrations of calcium in the absence of permeant anions such as phosphate. In spectroscopic studies of difference spectra of mitochondria and submitochondrial fragments in high and low energy states, distinctive forms of cytochromes absorbing in the region of 555 rnp (b& (3-5) were identified. A related compound forms in the cation-supplemented mitochondria, and this paper reports the detailed study of the kinetics of this intermediate (6). The data also show that cation interaction with the respiratory chain leads to inhibited states of electron transport and energy transfer which respond in a novel fashion to phosphate and uncoupling agents.

EXPERIMENTAL PROCEDURE

The experimental methods here consist of fluorometric recordings of the oxidation-reduction state of reduced pyridine nucleotide, spectrophotometric recordings of the oxidation-reduction state of cytochrome b or cytochrome c, and simultaneous measurements of the respiratory activity (7, 8). In this series of experiments, where recordings of both cytochromes c and b are desired, spectroscopic changes are measured in the region of the (Y bands rather than in that of the Soret bands, since cytochrome c is poorly separated from cytochrome b in the Soret region. Under these conditions, possible interference between the spectroscopic measurement of cytochromes and the fluorometric measurements of pyridine nucleotide is easily avoided by a Wratten 47 filter, which protects the fluorometer from the light used to measure cytochrome changes. Either a Wratten 16 or 21 filter transmitting wave lengths longer than 540 rnp or an interference filter transmitting between 540 and 560 rnp is employed to protect the spectrophotometric measurements from interference by fluorometer light. The interference filter is preferable to the color filter since the former fluoresces less brightly.

Pigeon heart mitochondria were prepared according to the method of Chance and Hagihara (S)r and protein was determined by the biuret method (9).

RESULTS

Cytmhrome and Pyridine Nucleotide Kinetics and Spectra

E$ect of Manganese-Fig. 1A illustrates a typical response of the respiratory carriers of pigeon heart mitochondria to the addition of manganese. The pigeon heart mitochondria in State 1

1 Thanks are due to Tami Yoshioka for many preparations.

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4578

PH.

1.4mg

PN

High and Low Energy Rates of Cytochromes. III Vol. 241, No. 20

+-5osec4 PN Reduction

1 366 - 45Omp

r-n PN Reduction 1

Mbu PrImI

I

DPNH

I PN

DPN Reduction--, k-50sec4 ,;pM DPNH

Aerobic Pigeon Heart M,

2.6mg PrImI ,

4, State 4g(-Pi) -

Cytochrome b Reduction

560-575mu Cytochrome b Reduction

560 - 575mp I 1

log I./I = 0.03 log I./I = 0.004

m II3 f

FIG. 1. A, an illustration of the effects of manganese on cytochrome b and pyridine nucleotide (PN) in glutamate-supplemented pigeon heart mitochondria (&). Medium A (4), 0.01 M Tris-Cl, pH 7.6; 1.3 mg of protein per ml; l-cm optical path; 26”. (Ex- periment 912-1 III), B, an illustration of the effects of calcium upon cytochrome b and pyridine nucleotide in glutamate-supple-

.~ I~. . 1 L .I 1 IL:- %6--7~-- 1 I”\ ,?,I, __ -TT er c. ------I-~-L~l-- * c -~.~ -L- ---L-I.. -̂ - . ..l , --. ..-LL n,-o IT?-.

periment 928-13 III).

are supplemented with 1.4 mM glutamate, and cytochrome b shows an immediate but very small reduction. Pyridine nucleotide shows no immediate reduction, but after a clear cut delay of 5 set, reduction occurs and continues until the plateau is reached approximately half a minute later to establish the State 4 level. The delay in reduction has been interpreted as being due to the time for the concentration of high energy intermediates to rise sufficiently to activate the energy-linked pathway for DPN reduction (10). During this interval, the reduction of cytochrome b diminishes, and the trace returns to very nearly the initial level.

An addition of 370 pM manganese is then made, resulting in an increased absorbance at 560 mp relative to that at 575 rnp (as determined in independent controls with the split beam spectro- photometer) which proceeds in an approximately zero order reaction for over 4. min. Thereafter, a plateau is reached. The absorbance change is interpreted at this point as a reduction of cytochrome b, and the metabolic state is termed State 6 (see “Discussion”). DPNH oxidation starts simultaneously and proceeds in an approximately zero order reaction as in the case of cytochrome b. After 20 set a slower rate is recorded. With the assumption that the increased absorbance at 560 rnp is due to the reduction of cytochrome b, a value of 0.01 PM per set is obtained, a very slow rate compared to the diminution of fluorescence, which, calculated as an oxidation of DPNH, corresponds to a rate 10 times more rapid.

As indicated in the diagram, the highly absorbing state of cytochrome b is not stable, and the initial state may be re-established in approximately 5 min; DPN, however, remains oxidized.

Fig. 1B illustrates the related effect of calcium. Addition of glutamate reduces DPN as in Fig. lA, but cytochrome b shows a more rapid and more extensive reduction than it does in Fig. 1B. The larger concentration of glutamate employed in the experiment of Fig. 1B is not the cause of the greater reduction of cytochrome a, as is shown by independent controls. The preparation used apparently has less endogenous substrate than that of Fig. 1A ; thus glutamate addition has a larger effect. Cytochrome b reaches its steady state before reduced pyridine nucleotide (11).

As soon as pyridine nucleotide reaches its steady state reduction level, 150 PM calcium is added, causing a relatively rapid increase in absorbance at 560 rnp and a decrease in fluorescence. The decrease of fluorescence is, however, cyclic in nature and recovery is observed about 2 min later. Cytochrome b remains in its new steady state. It is characteristic of manganese to give larger changes in cytochrome b than calcium. The cause of the cyclic response of DPNH in the presence of calcium is discussed below.

Titrations with Calcium-Titrations of the extent of cytochrome b reduction and reduced pyridine nucleotide oxidation can be carried out with single additions of the cation to a fresh suspension of the mitochondria (see also Fig. 1); in addition, sequential additions may be made to the same suspension. Both these techniques are employed to obtain the titration curves of Fig. 2. Fig. 2A illustrates the unusually high sensitivity of a particular pigeon heart mitochondrial preparation to calcium at pH 7.6 (1). The addition of only 7.4 pM calcium caused nearly as great an effect on cytochrome b as 28 pM calcium, while a relatively greater response of pyridine nucleotide is obtained with the higher concentration of calcium. If we assume that a true plateau is reached in the calcium titration of cytochrome b, then the half-maximal effect of DPNH is obtained with 15 PM calcium. The plateaus in the cytochrome b and DPNH titrations are reached at approximately 7 and 30 ml.cmoles of Ca++ per mg of protein. These are highly sensitive calcium responses, particulsrly in view of the fact that the total respiratory carrier concentrations (8) are in the same range as these calcium concentrations. It is to be noted, however, that larger calcium concentrations cause a diminution in the change of cytochrome b but no diminution in the change of reduced pyridine nucleotide. The pH dependence of the titration curve is illustrated in Fig. 2B. The response to calcium is greater at higher pH; at approximately 20 pM calcium, the effect at pH 7.6 is 3 times that at pH 6.8; at pH 8.0, 1.5 times that at pH 7.6. It is difficult to compute a calcium concentration for half-maximal effect at pH 7.6 and 8.0. The responses of the particular pigeon


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Issue of October 25, 1966 B. Chance and B. Schoener 4579

- 0 o~“‘o~ 200

Added [Co++] pLM [Calcium]pM

A B FIG. 2. A, illustration of titrations of cytochrome b reduction and DPNH oxidation with calcium. Titration of pigeon heart mito-

chondria (1.4 mg of protein per ml) with low concentrations of calcium in the presence of 1.4 mM glutamate; Medium A (4) (49 mM Tris-chloride), pH 7.6; 26”. (Experiment 911-A III). B, illustration of the el%‘ect of pH on the calcium titration of pyridine nucleotide and cytochrome b in pigeon heart mitochondria (1.4 mg of protein per ml) supplemented with 1.4 mM glutamate. 6.8; l , pH 7.6; +, pH 8.0; for cytochrome b: 0, pH 6.8; A, pH 7.6; 0, pH 8.0 (Experiment 914-4,6 III).

For DPNH: w, pH

PN Reduction+ 366 -+450mp PN Reduction4 366 ---*45Omp

380pM Mn++ log I./I= O.OOScm-1 -f

Cytochrome - c Reduction4 _c

log I./I = 0.005

A IB -f

FIG. 3. A, illustration of the response of cytochrome c to the additions of calcium and manganese. Pigeon heart mitochondria (P.H.M,), 1.6 mg of protein per ml. The sensitivities and wave lengths involved in the measurement are indicated in the figure. (Experiment 914-5 III). B, illustration of the effects of calcium upon the steady state of cytochrome c in glutamate-supplemented mitochondria (1.4 mg of protein per ml). PN, pyridine nucleotide (Experiment 914-A-1 III).

heart preparation used in the experiment of Fig. 2B are less than those of Fig. 2A. In Fig. 2B a plateau value in the titration of cytochrome b and DPNH with Ca* corresponds to 30 and 100 mpmoles of Ca* per mg of protein at pH 7.6. It is possible that these differences reflect differences in levels of endogenous anions or high energy intermediates (1, 12, 13). In summary, only very low calcium concentrations are required for these spectroscopic and fluorometric effects in the glutamate-supplemented pigeon heart mitochondria in phosphate-free medium.

Titrations with manganese do not show a comparable affinity; amounts of manganese required for half-maximal effect for cytochrome b and DPNH are of the order of 300 PM. It is probable

that the dissociation constant for Mn* is at least 100 PM, and it is apparent from the reaction kinetics illustrated by Fig. 1, A and B, that not only is more manganese required but the reaction proceeds at a slower pace by a factor of 5.

Response of DPNH and Cytochrome c-The oxidation of DPNH and the reduction of cytochrome b suggest a crossover point between these two components in the calcium and manganese effects. We have, therefore, recorded the kinetics of other components of the chain, particularly cytochrome c. Fig. 3A illus trates the effect of addition of 380 PM manganese to pigeon heart mitochondria supplemented with 3.6 mM glutamate. Cyto- chrome c reduction is recorded at 550 - 540 rnp, other condi-


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4580 High and Low Energy States of Cytochromes. III

TABLE I Crossover points for metabolic states of pigeon heart mitochondria

Addition State” Respiration rat@ -

_-

Cytochrom&

aa I a

-

-. c

Glutamate, 3.6 NIL. ‘%T (-Pi) 100 Mn++, 380 PM.. ‘%z 43 Succinate, 3.6 mu.. 6gs 51 Phosphate, 3.6 mM 4gs 500 ADP, 120 PM.. 33 860

-

+ $1

a g, with glutamate as substrate; s, with succinate as substrate. b As percentage of glutamate rate. c -, oxidized; +, reduced. d Observed only at 77” K. B Transient change. J When observed at 77“ K, this value becomes negative.

565 565

:I::cjj+g

5:5 T fl

+40mr+ 548 j55 , ?T 605

-+40m$--

I -4 I-

40mp

i 548

B c FIG. 4. A, spectrum representing the difference between the

states of cytochromes prior to and following the addition of manganese to glutamate-supplemented pigeon heart mitochondria. Reference base-line is the glutamate-treated material, and the curve represents the difference between the manganese-treated material and the reference. Optical path, 2 mm; protein concentration, approximately 5 mg per ml. B, the effect of phosphate addition upon the spectra of cytochromes in the glutamate- and manganese-treated mitochondria; low temperature difference spectrum; conditions as in A. C, the effect of Dicumarol (Die) upon the absorbance of cytochromes in glutamate- and manganese- treated pigeon heart mitochondria; other conditions as in A. Glut, Glutamate. (Experiment 910 III).

tions being the same as in the experiments shown in Figs. 1 and 2. Addition of glutamate causes reduction of cytochrome c with exponential kinetics and a delayed reduction of pyridine nucleotide. When the reduction of DPN and cytochrome c has come to a steady state, the addition of 380 PM manganese causes oxidation of DPNH as before, but the response of cytochrome c is very different from that of cytochrome b. A small but abrupt reduction of cytochrome c is followed by an exponential rise to a more highly oxidized steady state. This reductionof cytochrome c occurs in less than 1 set, essentially in the mixing time. Thus, the behavior of cytochrome c involves a rapid reduction and slow oxidation while that of cytochrome b involves only a slow reduction.

In Fig. 3B, the calcium response is illustrated; again, on addition of glutamate, typically asynchronous responses of cyto-

- -a

0

+

c+; - - + -

Vol. 241, No. 20

Quinone Flavoprotein

+ - c+; -

0

+ + -

DPN

+ -

0

+ -

chrome c and pyridine nucleotide are recorded. When a steady state is reached, 76 I.~M calcium is added, and reduced pyridine nucleotide is oxidized as usual. Again, cytochrome c shows a rapid reduction followed by an exponential rise in the direction of oxidation, which clearly overshoots the initial level, leading to a net change of cytochrome c in the direction of oxidation. It is of considerable interest that the rapid drop in the cytochrome c trace is observed with both manganese and calcium even though times for completion of the calcium and manganese response differ appreciably. It is possible that a primary event in the cation reaction is revealed by this initial response of cytochrome c.

Crossover Points-The crossover points for a variety of transitions are indicated in Table I. Glutamate addition causes cytochromes c and b, quinone, flavoprotein, and DPN to become more reduced (+) while cytochrome u3 + a appears to become less reduced. As shown in Fig. 4A and in other experiments* addition of 380 PM manganese causes DPNH and quinone to be less reduced (-) and cytochrome c to be initially reduced ( +). Two components, cytochrome c and flavin, show “cyclic” responses, and both indicate a transient reduction (+) followed by a slower oxidation (-). Cytochrome a shows a net change similar to that of cytochrome c, but a transient reduction preceding the oxidation of cytochrome a has not yet been observed. Other data in this table are discussed below.

Low Temperature Spectra-In order to identify the components affected in the changes described so far and to clarify the anomalous behavior of cytochrome b, low temperature spectra representing the difference between the glutamate- and the glutamate plus manganese-supplemented pigeon heart mitochondria are shown in Fig. 4A. The preparation of the samples is identical with that shown in Figs. 1, 2, and 3; namely, they are supplemented with approximately 1 mM glutamate and allowed to stand until the reduction of the DPNH has been established. One-half of the sample was rapidly frozen, and the remainder was treated with 500 j&M manganese for 60 set and then also rapidly frozen. The spectra thus obtained represent the difference between States 6 and 4 (see “Discussion”). Most noticeable is the fact that cytochrome c (548 rnp) and cytochrome a (603 mp) are largely oxidized in accordance with the data of Fig. 3A. The spectroscopic changes in the region of cytochrome b are complex. A band has appeared at 556 rnp, while the band at 562 ml disap- pears.

2 B. Chance, unpublished experiments.


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Issue of October 25, 1966 B. Chance and B. Xchoener 4581

0.27pM O,/sec

Oxygen -+ 0.25pM O,/sec

I

0.7pM O,/sec

Pigeon Heart M, b

+

4mM Glutamate 625pM Mn++

I

6;5pM Mn++ 1.4mM Pi

+-- 5Osec -4

A

log [pi JpM

5 FIG. 5. Illustration of the change of steady state of cytochrome b and DPN caused by the addition of 1.4 nnvr phosphate to pigeon

heart mitochondria (M,) supplemented with 625 PM manganese and 4 mM glutamate (the respiration rate is indicated in micromoles per liter of oxygen per see). Pigeon heart Preparation 928, dilution, 0.2/l.G; 5-mm path. A, experimental record of the effect of phos- ahate (Exneriment. 909-E III). B. diagram of the effect of nhosahate concentration on the rate of oxidation of reduced cytochrome b. IExperiment 909-E III). ’ ’ -

It is apparent that the component which increases its absorption upon addition of calcium or manganese absorbs at a shorter wave length than cytochrome b. It is further apparent that the component that we measure at 430 rnp and recognize as cytochrome b decreases its absorption in the cation reaction at room temperature (2). We have not as yet observed the separated absorption bands of Fig. 4A at room temperature; the absorbance increase at 556 rnp predominates, and this band is expected to shift several millimicrons to longer wave lengths at room temperature.

Effect of Phosphate at Room Temperature-The reactions described so far have been carried out in a phosphate-deficient reaction medium, and the effect of added phosphate upon cytochromes 6 and c is remarkable. Fig. 54 illustrates the typical spectroscopic and fluorometric responses to 625 PM manganese observed with pigeon heart mitochondria supplemented with 4 mM glutamate. Cytochrome b proceeds to a highly reduced state as measured at 560 - 575 rnp, and DPNH is highly oxidized. Upon addition of 1.4 mM Pi, cytochrome b is rapidly oxidized, and DPN is reduced. Generally, this response suggests a reversal of the cation effect, completely in the case of cytochrome b and partially in the case of reduced pyridine nucleotide. In Fig. 5B, the values on the abscissa are plotted in logarithmic coordinates. With no added phosphate, there is a measurable rate of oxidation of cytochrome b of approximately 0.006 pM per sec. Half-maximal effect is obtained with 550 ,u~ Pi; this value approximates the concentration of manganese added (625 KM) and suggests that the phosphate is removing the manganese from an inhibitory site. The phosphate concentration is larger than that required for the stimulation of respiration in the ADP- and succinate-supplemented pigeon heart mitochondria (8) and less than that required for the stimulation of /3-hydroxybutyrate oxidation in rat liver mitochondria (14).

Effect of Phosphate at Low Temperature-The spectroscopic effect of phosphate upon the mitochondria supplemented with glutamate and manganese is striking, and it is represented in the

low temperature difference spectrum in Fig. 4B. Here the procedure is identical with that already described for Fig. 5A where mitochondria supplemented with glutamate and manganese are rapidly frozen. The remainder of the sample is supplemented with 1 mM phosphate and is frozen 1 min thereafter. Thus, this sample corresponds to that of the experiment of Fig. 5A. The absorption band of the cytochrome having a peak at 555 mp has disappeared, and one absorbing near 565 rnp has appeared. The position of the 565 rnp peak at low temperature is somewhat affected by the large change at 555 mp and could be at a shorter wave length (562 rnp, in accordance with Fig. 4-4). At room temperature we associate the major portion of the change in Fig. 4B with the disappearance of absorption at 560 rnp; the appearance of the absorption band corresponding to that at 565 rnp at low temperatures is not observed. The disappearance of absorption at 600 rnp is attributed to cytochrome a3 + a. However, there appears only a small shoulder on the 555 mp band to indicate that the cytochrome c has become more oxidized; the cytochrome c change is indicated more clearly after addition of succinate (see Fig. 8 and Table I).

E$ect of Dicumarol at Room Temperature-The response of the highly absorbing form of cytochrome b to the addition of Dicu- marol is indicated in Fig. 6. The initial state is established by the addition of glutamate and calcium to pigeon heart mitochondria. As recorded in Fig. 1B the oxidation of reduced pyridine nucleotide is not stable but cycles back toward the initial base-line. At that time, addition of 13 pM Dicumarol causes a high degree of oxidation of cytochrome b and pyridine nucleotide. These components, particularly cytochrome b, respond first rapidly and then slowly. However, neither a second addition of 13 PM Dicumarol nor the addition of 6.7 mM phosphate causes any further change of cytochrome b. Titration data show that the system has a high sensitivity to Dicumarol, but it is not so sensitive as the energy-linked pathway for DPN

reduction (15).


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4582 High and Lou Energy States of Cytochromes. III Vol. 241, No. 20

-f 5pM DPN

DPN Reduction-y PN Reduction

1 366 - 450mp 1.

-̂- t -t- CBLI

Aerobic Pigeon Heart M,

2.6mg PrImI

4 Cvtnehromc! b-+-. - , . - -. . . - - -

Reduction

FIG. 6. Illustration of the effect of Dicumarol (Die) upon calcium- and manganese-supplemented pigeon heart, mitochondria ($f,) (2.6 mg of protein per ml). The figure illustrates the effect of Dicumarol on the respiratory activity, pyridine nucleotide (PN), and cytochrome b. Other conditions as in Fig. 1 (Experiment 928-13 IV).

+ 5Osec 4 . -,-,~,~,~-,,-.,-,~.~,,~,,,~,,~-,-~---,-~-,l-.-;-.-,-,,~,-,~~,,-.-~-,~,, - -,,-,.-Y-F ,

r ?r\ PN Red&on 1 36&k 4501& f

Aerobic PH. Mw-

(1.5mg Pr/ml) - I 3.6mM Phosphate

I kz-” 3.6mM Glutamate t

Cytochrome b Reduction1 560-575mp \

1. log L/I = 0.002 cm-l

-f

FIG. 7. Illustration of the inhibited state of respiration in manganese- and succinate-supplemented mitochondria. The figure pigeon heart, mitochondria (P.H.M,) supplemented with glutamate and manganese and succinate to which 3.6 mM phosphate is (1.5 mg of protein per ml). PN, pyridine nucleotide. Other conditions as in Fig. 1 (Experiment 914-A-10 III).

shows added

The respiratory activity under these circumstances is indicated by the platinum electrode trace but will be considered with the general inhibitory effects of Mn++ on respiration.

Effect of Dicumarol--Low temperature spectra: The difference spectrum corresponding to Dicumarol treatment of the mitochondria in this state is indicated in Fig. 4C. The mitochondria are supplemented with glutamate and manganese. One-half of the sample is frozen as in previous experiments. The remainder of the sample is treated with Dicumarol as in Fig. 6 and is frozen 30 set thereafter. Here we find a most interesting complex of absorption bands. The large absorbance band at 555 rnK has disappeared and one at 565 rnp has appeared. There are sug- gestions of subsidiary bands between these two wave lengths. In addition to these two components, cytochrome c at 548 rnp and cytochrome a at 605 rnp are oxidized. The separation of the peaks at 548 rnh and 555 rnp is sufficient to identify clearly two components: cytochrome c and cytochrome b (555 mp). The change of cytochromes c and a observed with Dicumarol is relatively larger than that with phosphate; this is caused by the greater respiratory stimulation (see below).

Cation Inhibition of Respiration-Effect of phosphate on Mn++ inhibition: Up to this point, data on respiratory activities have generally been de-emphasized in order to focus our attention on the steady states of the respiratory carriers. Important changes in the electron flow occur in three different states so far studied following supplementation with glutamate, cations, and phosphate or Dicumarol. (Figs. 6, 7, 8, and 9 include polarographic recordings of the respiratory rates.)

In the experiments shown in Fig. 7, the highly reduced form of cytochrome b is obtained following the addition of 3.6 mM

glutamate and 380 PM manganese. The respiratory rate is increased to 0.58 PM oxygen per set following the addition of glutamate and decreased to 0.25 PM oxygen per set as manganese caused the reduction of cytochrome 6 and the oxidation of DPNH. The response to added succinate differs in three distinct ways from that to be expected in glutamate-supplemented mitochondria in the absence of manganese (10). Upon addition of succinate in the presence of manganese, various reactions occur: first, the respiratory activity, instead of increasing several fold, showed only a 20% increase to 0.3 pM oxygen


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I+-- 50sec +

Aerobic - PH. M,

(1.5mg/ml)

r

Cytochrome -c Reduction 1

1 log L/I= 0.005 cm-l

o>Ki

f

Fm. 8. Illustration of the inhibited state of respiration in pigeon heart mitochondria (P.H.MWj supplemented with 3.6 mM glutamate 185 FM calcium, and 3.6 mM succinate, and its reactivation by addition of 3.6 mM phosphate. Cytochrome c is recorded; 1.5 mg of protein per ml. PN, pyridine nucleotide (Experiment 914-A-4 III).

PN -- DPN Reduction 1

-f 366mp- 45Omp 5B~

Aerobic PH. mm,

i 2.6mg Pr/ml

FIG. 9. Illustration of respiratory control in mitochondria (P.H.M,) which have been supplemented with sufficient calcium to pro- duce the inhibited state of respiration, followed by reactivation by phosphate. Other conditions as in Fig. 6. PN, pyridine nucleotide (Experiment 928-8 III).

per set; second, the energy-linked reduction of pyridine nucleotide by succinate which is readily observed in glutamate-supplemented mitochondria does not occur (if anything, DPNH becomes slightly more oxidized); third, cytochrome b, which usually shows increased reduction on succinate addition, shows a distinctive oxidation. It is apparent that the system is inhibited in its response to electron donors. Addition of 3.6 mM phosphate reverses the inhibition and increases the respiratory activity by a factor of approximately 10, and DPN reduction and cytochrome b oxidation occur in the course of 1 min. It is noteworthy that the respiration reaches its maximum before the new steady state levels of cytochrome b and pyridine nucleotide are established; this could be explained by the accumulation of high energy intermediates.

Effect of phosphate on Ca++ inhibition: Fig. 8 illustrates a similar state obtained with calcium addition in which cytochrome c instead of cytochrome b is recorded. The inhibited state is achieved by successive additions of 3.6 mM glutamate and 185 pM calcium. After a small stimulation the respiration is inhibited to one-third of the total rate by this calcium concentration. Addition of succinate causes a 3-fold stimulation of respiration and a small but delayed reduction of cytochrome c and

pyridine nucleotide. However, the respiration rate remains at a relatively inhibited level until the addition of 3.6 mM phosphate, which accelerates the respiration rate approximately 5-fold in the course of 10 set and causes DPN reduction and cytochrome c reduction as well.

Effect of Dicumarol on Ca++ inhibition: We may now refer back to the oxygen trace of Fig. 6 and note a similar effect; namely, when the respiratory activity is slowed to 0.15 PM per set by supplements of glutamate and calcium, the addition of 13 PM Dicumarol affords a g-fold increase in the respiratory rate, which, in this case, is again further stimulated by phosphate. As a consequence of these observations, we identify the highly absorbing form of cytochrome b with an intense inhibition of glutamate and succinate oxidation. This inhibition is released either by phosphate or by Dicumarol. It is noteworthy that in the inhibited state the pyridine nucleotide may be reduced as in Fig. 6 or oxidized as in Fig. 7, depending upon whether manganese or Ca++ is added.

Whereas it is unlikely that mitochondria in the inhibited state would respond to ADP, a suitable control to this point is indicated by Fig. 9, which also gives further information on the kinetics of cytochrome b in the presence of both glutamate and


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4584 High and Low Energy States of Cytochromes. III Vol. 241, No. 20

succinate on the addition of 150 PM calcium. After a small burst of respiration, an inhibited state (0.37 PM oxygen per set) is established. Cytochrome 6 is reduced, and pyridine nucleotide passes through a cyclic oxidation and reduction. At this point, 130 PM ADP is added, and, except for the small dis- turbances during the stirring of the suspension, no effects result; the system is unreactive to ADP. The addition of phosphate at this point, however, gives an &fold stimulation of respiration to 3.1 PM per set for the duration of the stimulated respiration interval. DPNH exhibits a cyclic oxidation and a partial reduction. As the ADP is depleted, respiration falls to 2.2 pM

per set, indicating that the calcium treatment has diminished the otherwise high respiratory control ratio of the pigeon heart preparation. The response of cytochrome b is interesting; it is oxidized in a reaction that approximates the zero order. A second addition of 130 pM ADP stimulates respiration to 3.8 PM per set and causes a further oxidation of DPNH. There is no measurable response of cytochrome 6. The period of stimulated respiration is, however, cut short by the exhaustion of oxygen in the solution, and cytochrome b becomes highly reduced while DPNH is partly reduced.

DISCUSSION

Inhibited States of Mitochondria-Electron transport in the respiratory chain can be controlled directly by inhibition of the energy transfer reactions that couple electron transport to energy conservation (14). There are two known regulations involving ADP. First, ADP and phosphate regulate energy transfer reactions which consequently regulate electron flow in the respiratory chain (14) in the active State 3 and the resting or inactive State 4. The respiratory enzymes show clearly defined sets of responses in these two states, in accordance with the crossover theorem (14). A second type of inhibited st,ate is identified in pigeon heart mitochondria in which succinate oxidation, reactivated by a low concentration of ATP, is first stimulated and then highly inhibited by the addition of low concentrations of ADP. This is also termed inverse respiratory control. Under these conditions, DPNH, flavin, cytochrome c (IS), and cytochrome b as we112 were observed to be highly oxidized. Our explanation for this phenomenon is the inhibition of succinate oxidation by the accumulation of oxalacetate (16) ; another explanation involves the assumption of energy requirements for succinate oxidation (17).

The experiments of this paper show a third type of inhibited respiration, one which is induced by the addition of low concentrations of cations in the absence of phosphate. Here the inhibition can be observed with either a DPN-linked or succinate- linked substrate and differs from the case mentioned above (16), and thus does not involve a specific effect of phosphate on succinate oxidation. In addition, the inhibition is reversed not only by permeant anions such as phosphate or acetate but also by uncoupling agents. Since the inhibition of respiration exceeds that observed in State 4 and is reversed upon the addition of phosphate, the inhibited state is identified by a new number, State 6. (For a listing of metabolic states of mitochondria see References 14 and 15.) In the transition induced by the addition of cations in the absence of added phosphate, we will identify the steady state changes of carriers, together with the inhibition of electron transport, as a State 4 to 6 transition. It is convenient to identify the various metabolic states of mitochondria in this way, and

doubtless many more will be discovered as other specific inhibi- tors of energy transfer are found to lead to distinctive oxidation- reduction states of the respiratory carriers. A further state of mitochondria, involving a specific inhibition of the energy-linked pathway of DPN reduction, is obtained with hyperbaric oxygen (18) and is termed State 7 since, in this case, DPNH is oxidized and cytochrome c is relatively reduced, whereas both are oxidized in State 6.

Crossover Pdnts-In the State 3 to 4 transition, the decreased rate of electron transport is accompanied by distinctive accumu- lations of reduced forms and depletions of oxidized forms of the respiratory carriers on each side of the crossover point (19). In the State 4 to 6 transition obtained 2 min after a manganese supplement, we find a highly oxidized state of cytochrome c. Cytochrome a3 + a, quinone, flavoprotein, and pyridine nucleotide are also oxidized, but probably for different reasons. We attribute the highly oxidized state of the cytochromes to a block of the energy transfer reaction, which leads to an inhibition of electron transport (14). In the case of pyridine nucleotide, it is probable that the energy requirements for its reduction (10) are not met in the inhibited State 6 in the presence of cations. Simi- lar considerations may be involved for a portion of the flavin or a majority of the quinone, which is now thought to operate mainly in the energy-linked pathway of pyridine nucleotide reduction

(11). The steady states of cytochrome b can effectively be contrasted

with those of cytochrome c in the respiration-inhibited state (State 6). On the one hand, cytochrome c is almost as highly oxidized as in the absence of substrate (State 2) while, on the other, cytochrome b is in a highly absorbing form. Respiration is stimulated on addition of phosphate, and cytochrome c becomes more reduced while the absorbance of cytochrome b decreases, suggesting a more oxidized state of this carrier (6). These responses of cytochrome b are anomalous and do not fit simply with its role in either electron transport or energy transfer, a topic which warrants a brief review at this point.

Forms of Cytochrome &To provide an understanding of high energy compounds associated with cytochrome b, it is essential to review the nature of the multiple forms of cytochrome 6 that have already been identified and their possible functions. Two types of cytochrome 6 are observed in mitochondria, one associated with respiratory chain and clearly identified by antimycin A treatment at a peak at 560 rnp at room temperature. The other is the microsomal pigment cytochrome bs (14). A more complicated situation exists in disrupted mitochondria; for example, in Keilin and Hartree heart muscle particles three forms of cytochrome b are identified at room temperature (20). The 562 rnp band of cytochrome b is best demonstrated by the spectra in which a succinate supplement specifically reduces cytochrome b in a preparation in which cytochromes c, cr, and a3 are already reduced by ascorbate addition to the cyanide-treated material.

A different absorbance maximum (566 rnp) was observed when cytochrome b was reduced in the presence of antimycin A. This peak is attributed to a compound of cytochrome b with antimycin A (20). A third peak at 557 rnF, broader than the narrow peak of the Dicumarol-sensitive component, is observed upon addition of dithionite to a preparation which had already been treated with succinate and antimycin A. It is apparent from these data that absorbance changes of cytochrome b are observed at long wave lengths and that 566 rnp is not an unusual value.


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Role of Cytochrome b in Electron Transport and Energy Transfer -While observations of cytochrome b in nonphosphorylating succinate and DPNH oxidases clearly show its behavior to be inconsistent with its role in electron transport (21), its responses to ADP in the State 4 to 3 transition and the kinetics of its oxidation and reduction are sufficiently consistent with the responses of cytochrome c to afford a basis for reconsidering its role as a member of the phosphorylating chain (14, 22). The discovery of energy-linked DPN reduction (23-26) has led to the consideration that changes in the steady state levels of cytochromes, flavins, and pyridine nucleotides may also reflect an equilibration with high energy intermediates in the mitochondria. On this basis, it has been suggested that cytochrome b, quinone, and pyridine nucleotide are components of a chain of carriers in the energy-linked pathway of DPN reduction (ll), their steady state levels being responsive to both energy levels and electron flow. Under the conditions of the experiments of this paper, we in- terpret the steady state levels of cytochrome 6 as an indicator of the energy state of the mitochondria. Any other consideration leads to inconsistencies with available data: if cytochrome 6 were to participate exclusively in electron flow, it should be oxidized in State 6, together with cytochrome c. If it were to function only in the energy-linked pathway of DPN reduction, it should again be oxidized in State 6, together with DPN.

The possibility that the spectroscopic changes of cytochrome b are not simply due to oxidation-reduction states, but may be due to the formation of a high energy compound as well, must now be considered. Spectroscopic studies of this paper indicate three forms of cytochrome 6 at low temperatures (77” K); the transition from State 6 to State 4 causes a decrease of absorption at 555 - 556 rnp and the appearance of an absorption band at 565 mp. The usual position of the cytochrome b absorption band is 559 rnp. On this basis, however, we regard the change in the state of cytochrome b not only to include a valency change in an oxidation-reduction reaction, but also to involve transformations in the ligand binding or conformation of cytochrome 6. Thus, there may be a series of reactions of cytochrome b involving high and low energy states and, in addition, interactions with cations and with phosphate.

Possible Chemical Nature of Cytochrome b5s5-While the main purpose of this series of papers is to identify a novel response of cytochrome b, we may consider whether it represents a phospho- rylated or a nonphosphorylated form. In addition to uncouplers, ADP and phosphate cause a similar but less extensive decomposi- tion of the compound, possibly due to a more highly irreversible response to uncouplers than to ADP and phosphate. Phosphate alone caused a small response in mitochondria and a definite response in submitochondrial particles. A definite response to phosphate was also obtained following the addition of phosphate to mitochondria in State 6. In this case, however, electron transport is not inhibited by sulfide, and a number of reactions are ini- tiated by the addition of phosphate. Although our experiments on this point are neither complete nor conclusive, further study of the b5s5 compound as a nonphosphorylated intermediate is warranted.

Biochemical Aspects of State ~-TWO explanations are considered here for State 6, one of them related to the response of cytochrome b as a component of energy transfer rather than electron transport, the other based on the recent identification of pH changes in the crista of the mitochondria caused by cation accumulation in the absence of a permeant anion (27-30). In

chemical terms we may consider State 6 to be a consequence of the direct combination of the cation with a component such as cytochrome b of the energy transfer system or a consequence of cation accumulation due to the alkalinization of the cristal membrane. Essentially, the choice is between the reaction of the energy transfer component directly with the cation or with a hydroxyl ion.

The response to phosphate and other permeant anions would be similar in the two cases; these anions would remove a cation bound to the energy transfer intermediate to form either calcium phosphate or a soluble salt of calcium such as calcium acetate. At the same time a hydroxyl ion is observed to be ejected from the mitochondrial membrane and a reactivation of respiration occurs.

The direct binding of phosphate or anions to cytochrome has clear precedents in the case of other hemoproteins where anion binding has been studied in detail (31) and reasonably small values of dissociation constants are observed. In the case of phosphate, it is probable that the active anion is HzP04 (32).

Activation of the inhibited State 6 by addition of uncoupling agents is also readily explained under both theories. In the case of inhibition by the direct reaction of the cation with the energy transfer intermediate, its hydrolysis could readily be caused by the addition of an uncoupling agent. In the case of the reaction with the hydroxyl ion, neutralization of the intramitochondrial al- kalinity is caused by low concentrations of uncoupling agents (29). While it is probable that a resolution of these two ap- proaches to the biochemistry of the inhibited state will not be achieved until the energy transfer intermediate is isolated, a consideration of the possibilities not only directs further experiments but also puts important limitations on reaction mechanisms which involved appreciable significant pH changes in the cristal membrane; it is apparent that shifts of less than 1 pH unit in the membrane (29) lead to a highly inhibited state of electron transfer.

Comparison of Cytochrome and Bromthymol Blue Kinetics-The experiments of this paper and those on bromthymol blue responses (29, 30) were carried out under very similar conditions; the effects in both cases are caused by accumulation of cations in the absence of permeant anions resulting in an inhibited state of respiration that is reactivated by the addition of anions such as phosphate or acetate. The indicator technique shows the accumulation of hydroxyl ions in the cristal membrane, and this paper shows changes of the steady state levels of the components of the respiratory chain to new values. The correlation of the time course of these two types of changes shows similarities and discrepancies. In the experiments with bromthymol blue, the endogenous substance termed Hi+ greatly diminished the initial response of bromthymol blue to small concentrations of cations. This influence of Hi+ is not observed in the response of the respiratory carriers, a result which suggests that the carriers are pri- marily cation-responsive and secondarily pH-responsive. Sec- ondly, the displacement of the steady states of the respiratory carriers appeared to be maximal at smaller concentrations of cations than those required for maximum response of bromthymol blue (compare Fig. 2A of this paper with Figs. 7, 8, and 9 of Reference 30). Lastly, the rapid change of cytochrome c which occurs immediately upon addition of cations is not observed with the bromthymol blue technique. Generally, the respiratory enzymes behave as intermediates in the cation reaction in rela- tion to the over-all reaction of membrane alkalinization indicated by the bromthymol blue technique.


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4586 High and Low Energy States of Cytochromes. III Vol. 241, I’Jo. 20

TABLE II Low temperature spectra of high and low energy states of mitochondria and submitochondrial particles

Material Type of transition - c b a bsss

_ Pigeon heart mitochondria Low to high ATP - S- + malonate - - -561 +557 Pigeon heart mitochondria Low to high Mn++ - glutamate - - -562 +556 Submitochondrial particles Low to high ATP - S=‘, succinate, Mg++, DPNH - - f562 +556 Submitochondrial particles Low to high ATP - S-, Mg++ - $561 +555

Pigeon heart mitochondria High to low Dicumarol - S- -554 Pigeon heart mitochondria High to low Dicumarol - glutamate, Mn++ +565 -555 Submitochondrial particles High to low Dicumarol - S-, succinate, Mg++, + -555

DPNH, ATP Washed submitochondrial particles High to low Dicumarol - S=, Mg++, ATP -556 Washed submitochondrial particles High to low Pi - S=, Mg++, ATP -555

- - a The first substance named is added to one cuvette only. The others are present in both cuvettes. b + indicates increased absorption at the wave length indicated; - indicates decreased absorption at the wave length indicated.

- Cytochromesb

Summary of Spectroscopic Data-Table II provides a summary of the spectroscopic data on high and low energy states of cytochromes in mitochondria and submitochondrial particles on the basis of the three papers of this series. The first four entries represent low to high energy transitions caused by the addition of ATP to terminally inhibited systems under substrate-sufficient conditions in the case of mitochondria, or with or without substrate supplement in the case of submitochondrial particles. In addition, a low to high energy transition in pigeon heart mitochondria caused by a manganese supplement is included.

Oxidation of cytochrome c is observed uniformly in the four cases, and in three cases, a clearly defined oxidation of cytochrome a is observed. This is a usual response to ATP-activated, reversed electron transfer (20). The changes in cytochrome b are listed under two headings, those at a long wave length (561 to 562 rnp) and those at a short wave length (555 mp). In all four cases the 555 rnl.c absorption increases, while the response at 561 to 562 rnp is variable and probably is dependent upon the initial state of the system. With intact mitochondria in a high energy state, a decreased absorption is observed at the longer wave length corresponding to an oxidation of a cytochrome b

component, while in the submitochondrial particles, where the coupling with ATP may be less adequate, we observe an increased absorption at the longer wave length, corresponding to a reduction of a cytochrome. It is apparent that reversed electron transport operates in such a way as to reduce the component of lower potential which is not already reduced, and the electron flow activated thereby causes oxidation of the components of higher potential. This is evident when crossover points for intact mitochondria and for submitochondrial particles are compared. In intact mitochondria, cytochromes (see Table II) are oxidized as well as flavin and ubiquinone (11, 20). In submitochondrial particles, the crossover point is changed toward the high potential end of the chain so that cytochrome c is oxidized and cytochrome b is reduced. It is implicit in a current formula- tion of the mechanisms of forward and reversed electron transport that the sites of energy conservation are identical, a supposition which is in agreement with the data presented in Table II (11, 14, 20). The fact that the bh55 compound appears in increased concentration in the low to high energy state transition in spite

of the changing nature of the cytochrome b response is consistent with the role of b555 in energy transfer.

REFERENCES

1. CHANCE, B., J. Biol. Chem., 240, 2729 (1965). 2. CHANCE, B., in B. CHANCE (Editor), Energy-linked functions of

mitochondria, Academic Press, Inc., New York, 1963, p. 253. 3. CHANCE, B., Federation Proc., 22, 404 (1963). 4. CHANCE, B., AND SCHOENER, B., J. Biol. Chem., 241, 4567

(1966). 5. CHANCE, B., LEE, C. P., AND SCHOENER, B., J. Biol. Chem., 241,

4574 (1966). 6. CHANCE, B., Federation Proc., 23, 265 (1964). 7. CHANCE, B., AND WILLIAMS, G. R., J. Biol. Chem., 217, 383

(1955). 8. CHANCE, B., AND HAGIHARA, B., in A. N. M. SISSAEIAN (Edi-

tor), Proceedings of the Fifth International Congress of Bio- chemistry, Moscow, 1961, Pergamon Press, New York, 1963, p. 3.

9. GOHNALL, A. G., BARDAWILL, C. S., AND DAVID, M. N., J. Biol. Chem., 177, 751 (1949).

10. CHANCE, B., AND HOLL&N&R, B., J. Biol. Chem., 236, 1534 (1961).

11. CHANCE, B., in R. A. MORTON (Editor), Biochemistry of qui- nones, Pergamon Press, London, 1965, p. 459.

12. CHANCE, B., AND YOSHIOKA, T., Federation Proc., 24, 425 (1965).

13. RASMUSSEN, H., CHANCE, B., AND OGATA, E., Proc. Natl. Acad. Sci. U. S., 63, 1069 (1965).

14. CHANCE, B., AND WILLIAMS, G. R., Advan. Enzymol., 17, 65 (1956).

15. CHANCE, B., AND HOLLUNGER, G., J. Biol. Chem., 238, 445 (1963).

16. CHANCE, B., AND HAGIHARA, B., J. Biol. Chem., 237, 3540 (1962).

17. FUGMANN-RASMUSSEN, U., Biochem. Biophys. Res. Commun., 16, 19 (1964).

18. CHANCE, B., JAMIESON, D., AND COLES, H., Nature, 206, 257 (1965).

19. CHANCE, B., HIGGINS, J. J., HOLMES, W., AND CONNELLY, C. M.. Nature. 182, 1190 11958).

20. CHANCE, B., >. Biol. Chem., 238, 1544 (1961). 21. CHANCE, B., Nature, 169, 215 (1952). 22. CHANCE, B., AND WILLIAMS, G. R., J. Biol. Chem., 217, 429

(1955). 23. CHANCE, B., AND HOLLUNGER, G., Federation Proc., 16, 703

(1957).


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24. ERNSTER, L., in T. W. GOODWIN AND 0. LINDBERG (Editors), 27. CHANCE, B., Abstract M-4, Abstracts of the Third Meeting of the Biological structure and function, Academic Press, Inc., New Federation of European Biochemists Society, Warsaw, April, York, 1961, p. 139. 1966, Academic Press, Inc., New York, 1966, p. 109.

25. KLINGENBERG, M., in T. W. GOODWIN AND 0. LINDBERG (Edi- 28. MELA, L., Federation Proc., 26, 414 (1966). tors), Biological structure and junction, Academic Press, Inc., 29. CHANCE, B., AND MELA, L., Proc. Natl. Acad. Sci. U. S., 66, New York, 1961, p. 227. 1243 (1966).

26. CHANCE, B., in T. W. GOODWIN AND 0. LINDBERG (Editors) 30. CHANCE, B., AND MELA, L., J. Biol. Chem., 241, 45% (1966). Biological structure and function, Academic Press, Inc., New 31. CHANCE, B., J. Biol. Chem., 194, 483 (1952). York, 1961, p. 119. 32. CONNELLY, J. L., Federation Proc., 24, 424 (1965).


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High and Low Energy States of Cytochromes · Vol. 241, No. 20, Issue of October 25, pp. 4577-4587, 1966 Printed in U.S.A. High and Low Energy States of Cytochromes III. IN REACTIONS