Mechanism of electron transport and energy conservation in the site I region of the respiratory chain

Biochimica et Biophysica Acta, 301 (1973) 105-128 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 86005

MECHANISM

OF

ELECTRON

TRANSPORT

AND

ENERGY

VATION IN THE SITE I REGION OF THE RESPIRATORY

CONSER-

CHAIN

TOMOKO OHNISHI Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pa. 19174 (U.S.A.) (Received November 27th, 1972)

CONTENTS I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

105

H.

Comparative studies of electron transport and energy coupling in the Site I region among different yeast strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

106

III.

Loss of Site I energy conservation and rotenone sensitivity by iron- or sulfur-limited growth of C. utilis cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

109

IV.

EPR-detectable iron-sulfur proteins as electron carriers in the respiratory chain . . . .

110

V.

Multiple iron-sulfur centers in the Site I region and their characteristics . . . . . . .

112

VI.

Involvement of iron-sulfur center(s) in Site I energy coupling, suggested by in vivo induction studies of Site I in C. utilis cells . . . . . . . . . . . . . . . . . . . .

116

VII. Site I bypass mechanism found in C. utilis mitochondria . . . . . . . . . . . . . .

118

VIII. Iron-sulfur Center la as an energy-transducing component at Site I

120

........

IX.

Site I energy coupling in Saccharomyces mitochondria . . . . . . . . . . . . . . .

122

X.

Inhibition mechanism of piericidin A (or rotenone) . . . . . . . . . . . . . . . .

124

XI.

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

126

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

126

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

127

I. INTRODUCTION T h e m e c h a n i s m o f electron transfer and energy c o n s e r v a t i o n in the Site I region has p r o v e n to be one o f the m o s t difficult p r o b l e m s to solve in the study o f the r e s p i r at o ry chain, m a i n l y due to three reasons: (1) the Site I segment o f the r esp i r at o r y chain c o n t a i n s m a n y i r o n - s u l f u r c o m p o n e n t s (as sh o w n in Scheme I) and its labile structure hindered isolation a n d r e c o n s t i t u t i o n studies o f the respiratory c o m p o n e n t s in this s eg m en t o f the c h a i n S - ~ ° ; (2) because o f the low in situ c o n c e n t r a t i o n o f the N A D H d e h y d r o g e n a s e in c o m p a r i s o n to that o f the c y t o c h r o m e chain, and because o f its lack o f readily m e a s u r a b l e a b s o r p t i o n b an d s in the visible region, optical

106

T. OHNISHI Succinate Succinate dehydrogenase

[FAo ~Fe S)7 I We S)8J

NADH dehydrogenase FMN

. Site I

Rotenone I

/

Antimycin A 1

Eo, e s>°Fo*s),,cytbK,b[] pe S>,,cyt

I

Site II

K('N 1 .....

C o:

Site III

Scheme I, Respiratory chain components, energy coupling sites and the reaction sites of respiratory inhibitors. The iron-sulfur centers (Fe-S) are numbered for identification. The (Fe-S)~_4 and (Fe-S)~,,ib in the NADH dehydrogenase region are designated according to Orme-Johnson et aL and Ohnishi et al. 2'3, respectively. (Fe-S)s and (Fe-S)6 are iron-sulfur centers with the halfreduction potentials around 30 mV2 and 0 mV% respectively. (Fe-S)7 and (Fe-S)8 are iron-sulfur centers associated with the succinate dehydrogenase, the (Fe-S)7 signals are detectable at 77°Ks, while the (Fe-S)8 signals are detectable only below 30°K6. (Fe-S)9 is that originally reported by Rieske et al. 7. techniques which have been useful for the study of Sites II and II111-13 have had rather limited merit for the study of Site I; (3) EPR techniques were also considered to have a limited applicability for studying mechanisms of electron transfer and energy conservation in the Site I region, because it was believed that EPR-detectable iron-sulfur proteins accounted for only 10 to 20 % of the chemically determined total iron-sulfur proteins 5, until more iron-sulfur signals were observed using temperatures below that of liquid nitrogen. On the other hand, yeast has provided a very useful biological system for elucidating the importance of iron-sulfur proteins for both electron transport and energy conservation in the Site I region of the respiratory chain. In this review, attention will be focused on the development along this line of biological approach, and then extended to more recent progress on newly identified multiple iron-sulfur centers and their relationships to the mechanism of energy conservation at Site I.

II. COMPARATIVE STUDIES OF ELECTRON TRANSPORT AND ENERGY COUPLING IN THE SITE I REGION AMONG DIFFERENT YEAST STRAINS Table I lists some characteristics of electron transport and energy coupling at the Site I region in various yeast strains. In studies with isolated, loosely coupled S a c c h a r o m y c e s c e r e v i s i a e (baker's yeast) mitochondria, Vitols and Linnane 14 obtained identical P/O ratios with either succinate or endogenous NAD-linked substrates and attributed this apparent lack of Site I phosphorylation to damage during the preparation of the mitochondria. However, Ohnishi et aL 1s'16 reported the same

--***

+

+

+

S. cerevisiae

C. utilis

E. magnusii, yeast type

A n i m a l tissue

+

+

+

--

--

Rotenone sensitivity*

+

÷

+

--

--

in the N A D H dehydrogenase region*

" g -- 1.94"

FMN34

N.T.

F M N a n d F A D aa

FAD29,a2,aa

N.T.

Flavin

9_103v,as

9 or 1023

7 a n d 936

63s

62a

Q, number o f side-chain isoprenoid units

+ a9

N.T.

~_ 23

26

N.T.

-SH ( NADH-flavin) **

S E G M E N T S O F T H E R E S P I R A T O R Y C H A I N 1N V A R I O U S Y E A S T

* + , present; --, absent. ** ÷ , mercurial sensitive; --, m e r c u r i a l insensitive. *** This point will be discussed in m o r e detail in Section IX.

--***

Site I*

u biq ui none.

S. carlsbergensis

N.T. = not tested. Q -

DIFFERENCES AMONG THE NADH-CYTOCHROME

TABLE I

Z

0

-]

Z O

C~

-]

108

T. OHNISHI

phosphorylation efficiencies even in tightly coupled mitochondria prepared from Saeeharomyces carlsbergensis by the mild procedure of disrupting protoplasts by osmotic shock. The lack of Site I phosphorylation in Saccharomyces mitochondria was subsequently confirmed by many investigators 17-2°. On the other hand, all three phosphorylation sites were reported to be present even in loosely coupled mitochondria isolated from different strains, Candida utilis el and yeast-type Endomyces magnusii z2. Ohnishi et al. 23 succeeded in preparing tightly coupled C. utilis mitochondria which showed respiratory control in the oxidation of both endogenous NAD-linked substrates and exogenous N A D H . They demonstrated the presence of Site I phosphorylation in the oxidation of endogenous N A D H and the absence of Site I phosphorylation in the oxidation of exogenous N A D H . They also showed that the endogenous N A D H oxidation in C. utilis mitochondria was inhibited by rotenone similar to that in mammalian mitochondria 2.,25 while exogenous N A D H was oxidized via a rotenone-insensitive pathway as in the case of either endogenous or exogenous N A D H oxidation in Saecharomyces mitochondria 16. These observations suggested a close correlation between the occurrence of Site I phosphorylation and the presence of the rotenone inhibition site, a correlation which was also suggested in mammalian systems by Ernster et al. z4 and Schatz and Racker 25. A further correlation between these two features (Site I energy coupling and rotenone sensitivity) and the presence of the "g = 1.94" EPR signal arising from iron-sulfur proteins associated with the N A D H dehydrogenase region of the respiratory chain was provided from comparative studies in various strains of yeast. The iron-sulfur protein responsible for the "g = 1.94" EPR signal in the N A D H dehydrogenase region is absent in submitochondrial particles of S. cerevisiae 26'27 or S. carlsbergensis 28. On the other hand, it is present in C. utilis submitochondrial particles 29"3° which in this respect are very similar to mammalian submitochondrial particles. Butow and Racker 3t also proposed that energy coupling at Site I occurs at the level of iron-sulfur protein, based on their observation that inhibition of N A D H oxidation by the iron chelator o-phenanthroline was released by ADP plus Pi or by the uncoupler in mammalian submitochondrial particles. These observations implied a close correlation among the occurrence of Site I energy conservation, rotenone sensitivity, and EPR-detectable iron-sulfur proteins in the Site I region. Thus comparative studies among different yeast strains promised to provide a useful approach to understanding the electron transport and energy conservation in the Site I region of the respiratory chain. However, a more detailed comparison of the electron transport system of Saccharomyces mitochondria and that of C. utilis and mammalian mitochondria showed the additional differences presented in the last three columns of Table I: (a) FAD rather than F M N is the flavin component of the N A D H dehydrogenase of Saccharomyces; (b) the reduction of flavin by N A D H is not inhibited by mercurials in Saceharomyces submitochondrial particles indicating that no mecurial-sensitive - S H groups are involved in the electron transport between N A D H and flavin; (c) the Q component in Saccharomyces is Q6 whereas C. utilis and mammalian systems contain higher homologues.

SITE I REGION OF RESPIRATORY CHAIN

109

II1. LOSS OF SITE I ENERGY CONSERVATION AND ROTENONE SENSITIVITY BY IRON- OR SULFUR-LIMITED GROWTH OF C. UTILIS CELLS This biological a p p r o a c h became more promising when Light et al. 4° and Garland 41 reported the following observations: (1) Both Site I phosphorylation and piericidin A (or rotenone) sensitivity were lost when the growth-limiting c o m p o n e n t o f the continuous culture of C. utilis cells was shifted from the carbon source (glycerol) to iron. (2) These conversions were reversed in vivo by aerating the cells (grown in the iron-limited medium) in the presence o f added iron under non-growing conditions. (3) It was subsequently demonstrated that cells grown in sulfur-limited continuous culture were also deficient in Site I phosphorylation and piericidin A sensitivity 42. This conversion was likewise reversed by in vivo induction procedures. F r o m these findings Garland and co-workers 4°-42 proposed that iron-sulfur proteins m a y play a role in Site I energy conservation as well as in piericidin A sensitivity. Since no change occurred in the flavin and Q species during the Site I "on and off" transition in C. utilis cells, these differences were regarded as a species difference and considered not to be directly related to the presence or absence o f Site I energy conservation or the piericidin A-sensitive site. This dependence of the presence o f Site I in C. utilis cells on the iron concentration o f the medium was confirmed by Ohnishi et al. 43"44 using cells grown in the batch culture. These investigators further suggested a close correlation between Site I phosphorylation and iron-sulfur proteins responsible for the "g ---- 1.94" E P R signal in the N A D H dehydrogenase region, from E P R studies using C. utilis cells grown at varying iron concentration and cells before and after in vivo induction o f Site I phosphorylation (Fig. I). 137c 120"I00-

80f-- -30 L

6020

-I0

0

1,0

2.0

I/:-O 25

Initial Iron Concentration (/~M)

Fig. 1. Correlation between phosphorylation at Site i and the normalized peak-to-peak amplitude of "g = 1.94" EPR signal (measured at 77 °K) of iron-sulfur protein in the NADH dehydrogenase region of the respiratory chain in C. utilis submitochondrial particles. Amplitudes of "g = 1.94" signals are normalized for the same instrumental conditions and protein concentration of the samples. Arrows show simultaneous changes in these two parameters during aeration of resting cells, which have grown in low-iron medium44. The P/O ratios were measured with isolated mitochondria, using 20 mM ethanol plus 15 mM semicarbazide as a substrate.

110

T. OHNISHI

IV. EPR-DETECTABLE IRON-SULFUR PROTEINS AS ELECTRON CARRIERS IN THE RESPIRATORY CHAIN Beinert and Palmer 45 previously showed that in beef heart submitochondrial particles the "g = 1.94" iron-sulfur proteins were reduced or oxidized at a rate not grossly different from that of the cytochromes, suggesting their role as electron carriers in the respiratory chain. This suggested role was questioned by several workers. Yamashita and Racker 46 indicated non-involvement of iron-sulfur proteins in the resporatory chain of beef heart mitochondria from their observation that iron-sulfur protein 7 in the cytochrome b, c l region was not required for the reconstituted succinate oxidase activity. Light e t al. 4° and Garland 41 proposed that iron-sulfur protein is not essential for the electron flow in the region between N A D H and (Q, cytochrorne b) because the mitochondrial respiratory rate with NAD-linked substrates was not altered when the continuous culture was shifted from carbon-limited (iron-excess) to iron-limited medium although the concentration of iron-sulfur proteins in the submitochondrial particles decreased by more than 20-fold. Clegg e t al. 4~ also reported that the presence of EPR signals at 77 °K in the "g = 1.94" region is not obligatory for fully active N A D H or glycerol l-phosphate oxidation activity. These questions were examined by Ohnishi e t al. 48 using submitochondrial particles prepared from C. u t i l i s cells grown under controlled growth conditions. As described by Ohnishi and Chance 4., the intensity of EPR signals arising from iron-sulfur proteins in the N A D H dehydrogenase region varied dramatically as a function of iron concentration in the culture medium when C. u t i l i s cells were grown under a specified low aeration system 49. It was found that phosphate, magnesium and cytochrome c were required for obtaining the maximal rate of N A D H oxidation in C. utilis submitochondrial particles prepared from the cells grown in either low- or high-iron medium. When cells were grown in a high-iron (50 ~xM) medium the maximal rate of N A D H oxidation by submitochondrial particles, 1.2-1.6 ~xatoms O" rain -~ . m g protein -1, was much higher than the maximal respiratory rate with NAD-linked substrates measured in intact mitochondria (0.1-0.2 ~xatom O" m i n - l " m g protein-i). When cells were grown in the low-iron medium (1.1 ~M), the respiratory rate of NAD-linked substrates in mitochondria were not altered, but the maximal rate of N A D H oxidation in submitochondrial particles was about 10 times diminished, in parallel with an approximately 10-fold decrease in the intensity of the "g = 1.94" EPR signal arising from iron-sulfur proteins in the N A D H dehydrogenase region (Table II). Another example of concomitant change in the maximum N A D H oxidation rate and the intensity of "g = 1.94" EPR signal was observed when oxygen tension in the culture medium was changed. With much higher oxygen tension (steady level of 02 concentration; 15-20 ~ of air saturation) using an oxystat 5o, the rate of NA D H oxidation in submitochondrial particles decreased to 0.4 Exatom O • min -1 • mg prorein -a with a parallel decrease of the EPR signal in the N A D H dehydrogenase region even when cells were grown in high-iron medium (50 ~xM). In contrast, the rate of glycerol l-phosphate oxidation and the relative intensity of " g - 1.94" EPR signal associated with this dehydrogenase region were almost unchanged in all submito-

SITE I R E G I O N O F R E S P I R A T O R Y C H A I N

1 11

TABLE II PARALLELS BETWEEN THE M A X I M U M O X I D A T I O N RATE O F N A D H A N D G L Y C E R O L 1-PHOSPHATE, A N D THE RELATIVE INTENSITY OF "g = 1.94" EPR SIGNALS (MEAS U R E D AT 77 OK) ASSOCIATED WITH THE C O R R E S P O N D I N G D E H Y D R O G E N A S E C. utilis submitochondrial particles were isolated from cells grown in low- and high-iron culture

medium, respectively. Preparation and reaction medium contained 0.3 M mannitol, 10 mM Trismaleate buffer (pH 7.2), 1 m M MgCI2 and 5 m M potassium phosphate buffer (pH 7.2). Concentration of the following additions: N A D H , 1.5 raM; glycerol 1-phosphate, 10 raM; cytochrome c, 1/~M 48. Initial iron concn in the culture medium (t~M) : 1.1

50

Oxidation rate (l~atoms 0 • rain -~ "mg protein -1)

N A D H -I- cytochrome c Glycerol l-phosphate ÷ cytochrome c

0.10-0.12 0.17-0.24

1.20-1.55 0.15-0.25

Relative intensity o f "g = 1.94" signal

N A D H dehydrogenase region

1

10-13

Glycerol 1-phosphate dehydrogenase region

1

1-2

chondrial particles prepared from C. utilis cells grown under the above described conditions (cf. Table 1I). Only a further extensive decrease of the iron concentration in the culture medium resulted in the diminished content of iron-sulfur protein in the glycerol 1-phosphate (or succinate) dehydrogenase and cytochromes in C. utilis mitochondria. These observations suggest that the dehydrogenase is the rate-limiting step in either NADH or glycerol 1-phosphate oxidation in submitochondrial particles under conditions presented in Table II and a proportionality exists between the maximum oxidation rate and relative intensity of EPR signals arising from iron-sulfur proteins in the respiratory chain. Thus, it is strongly suggested that "EPR-active" iron-sulfur proteins are required for electron transport in yeast submitochondrial particles. This could not be shown in the mitochondrial respiration with NADlinked substrates, since the rate-limiting step is presumably localized in the region of the NAD-linked substrate dehydrogenases which precede the N A D H dehydrogenase in the mitochondrial respiratory chain. It is interesting to point out that NADH-K3Fe(CN)6 reductase activity of the submitochondrial particles (V) which is almost 20 times higher than the overall NADH oxidation activity also decreased several fold when the iron concentration in the culture medium of C. utilis is lowered from 50 to 1.1 EzM. Clegg 51 recently reported that the isolated NADH-K3Fe(CN)6 reductase from iron-deficient cells showed a specific activity (measured at a fixed KaFe(CN)6 concentration) which is only about 10% of that of the NADH-KzFe(CN)6 reductase of iron-sufficient cells. These observations are in agreement with the previous finding by Beinert et al. 52 who demonstratedthe parallel decrease of NADH-K3 Fe(CN)6 reductase activity(Vmax) and the intensity of "g = 1.94" EPR signal on partial inactivation of the isolated NADH

112

T. OHNISIqI

dehydrogenase, indicating the involvement of iron-sulfur protein responsible for the "g = 1.94" signal in the NADH-K3Fe(CN)6 reductase activity. As will be described in the next section, an increasing number of iron-sulfur proteins (centers) has been recently detected in both yeast and mammalian mitochondria by the measurement of the EPR absorbance of samples at temperatures below 77 °K. This technique greatly increased the sensitivity of the EPR measurement of iron-sulfur proteins and has made possible the measurement of the redox level of individual iron-sulfur centers in different metabolic states, such as in State 4, State 3~ or the uncoupled state. Under these conditions, iron-sulfur centers show a response similar to other well established components of the respiratory chain 53. Information from these studies provides us with further evidence to support the proposition that multiple iron-sulfur centers act as members of the respiratory chain.

V. MULTIPLE IRON-SULFUR CENTERS IN THE SITE I REGION AND THEIR CHARACTERISTICS Some bacterial ferredoxins 54 and other enzymes containing iron-sulfur centers 55 have been known to exhibit measurable EPR absorbance signals only at temperatures close to that of liquid helium. Thus EPR signals of iron-sulfur proteins in C. utilis mitochondria have been examined at temperatures between that of liquid nitrogen (77 °K) and liquid helium (4.2 °K), in order to establish a more definite correlation between the presence or absence of EPR-detectable iron-sulfur proteins and Site I energy conservation. Previously unrecognized multiple EPR signals arising from iron-sulfur centers in the Site I region have been detected in C. utilis submitochondrial particles 27'56 at temperatures below 30 °K. Similar iron-sulfur proteins which show measurable EPR signals only at lower temperatures were subsequently found in mammalian systems in three laboratories, namely, by Ohnishi et al. 27 (see Fig. 2), Orme-Johnson et al. 1 and by Albracht and Slater 57. Orme-Johnson et al. 1 resolved multiple EPR signals and identified four separate iron-sulfur centers (Centers 1-4) in the anaerobic N A D H - Q reductase, by a stepwise reduction with measured quantities of reducing equivalents. They reported g values of the prominent peaks of individual iron-sulfur centers in the N A D H - Q reductase as follows: Center 1 (2.022, 1.938, 1.923), Center 2 (2.054, 1.922), Center 3 (2.101, 1.886, 1.864) and Center 4 (2.103, 1.863). These investigators 1 also showed the relative oxidation-reduction potentials of iron-sulfur centers as in the order of 2 > 3 > 4 > 1. From double intergration of the EPR signals, each iron-sulfur center (probably 2 or 4 iron atoms per center) was estimated to take up a number of electrons approximately equal to the molarity of F M N in the N A D H - Q reductase preparation. Ohnishi et al. 58 resolved EPR signals arising from iron-sulfur centers in C. utilis mitochondria and submitochondrial particles by the anaerobic titration procedure of Dutton 59 and Wilson et al. 6°. The EPR spectra of C. utilis submitochondrial particles at different oxidation-reduction potentials are presented in Fig. 3. Individual EPR signals are

SITE I REGION OF RESPIRATORY CHAIN

1 13

designated according to Orme-Johnson et al. I. The characteristics of the EPR absorption spectra arising from individual iron-sulfur centers in the Site I region of C. utilis, such as line shape, field position of prominent absorption peaks, temperature and power profile, are similar to those observed in beef heart systems. The values of C,utilis SMP 194 NADH ~ Piericidin A

B Heart SMP

202

l

205

205

~ 3'z k gauss

g=L94

/

189

18*K

t

1,89

193

3~

202

2 I0 ,,

•

314

3'z

3'.4

3'.6

k gauss

Fig. 2. EPR spectra of iron-sulfur proteins in the N A D H dehydrogenase region of the respiratory chain in C. utilis and bovine heart submitochondrial particles (SMP) at temperatures below 77 °K. Iron-sulfur proteins in these particles were reduced with N A D H in the presence of piericidin A. Submitochondrial particles (56.1 mg of protein per ml) isolated from C. utilis cells which were grown in a synthetic medium containing 25 tbM FeCI3 were previously incubated with piericidin A (0.5 nmole per mg of protein) for I0 min in an ice bath. Bovine heart submitochondrial particles (38.4 mg of protein per ml) were previously incubated with piericidin A (1.2 nmoles per mg of protein) in a similar fashion. In both cases N A D H oxidase activity was inhibited up to 98 ~ and thus about 1 min was allowed prior to anaerobiosis. Both suspensions were frozen in liquid nitrogen in less than 40 s after the addition of 1.7 mM NADH. EPR operating conditions were: field modulation, 100 kHz; modulation amplitude, 12 gauss; microwave power, 9.2 roW; microwave frequency, 9.02 GHz; time constant, 0.001 s; scanning rate, 400 gauss/rain ; temperature, 77, 43, 27, 23, 18, or 4 °K as indicated. Relative instrument gain is shown in the figure 27.

114

T. OHNISHI C.utili$ SMP (I8°K)

J

Eh (mY) +71

d

[]

P

-45

(E -195

-420

s

3'.o

312

3'4

316

(k gouss)

Fig. 3. EPR spectra of iron-sulfur proteins in C. utilis submitochondrial particles as measured at 18 °K. C. miffs submitochondrial particles were suspended at approximately 25 mg of protein per ml in a medium containing 0.3 M mannitol, 5 m M potassium phosphate buffer (pH 7.2), 1 m M MgClz, and 50 m M morphotinopropane sulfonic acid buffer (pH 7.2). Oxidation-reduction potentials were measured potentiometrically 59. Oxidation-reduction mediators added were 40 # M phenazine methosulfate, 40 # M phenazine ethosulfate, 63 # M duroquinone, 6 # M pyocyanine, 6 # M resorufin, 58 # M phenosafranine, 56 # M benzylviologen, 108 p M methyl viologen, and 25 # M 2-hydroxynaphthoquinone. The oxidation-reduction potential of the suspension system was lowerd by stepwise additions of small aliquots of 0.1 M N A D H solution. Oxidation-reduction potentials shown in the figure are relative to the standard hydrogen electrode. The E P R operating conditions were: modulation amplitude, 12 gauss; microwave power, 20.5 roW; microwave frequency, 9.02 GHz; time constant, 0.001 s; scanning rate, 1 kgauss/min; temperature, 18 °K. The ordinate is the first derivative of the microwave absorption in arbitrary units. Small R o m a n letters are placed along the spectra vertically above or below the field position of the resonances typical for various components according to Orme-Johnson et al.1.

SITE I REGION OF RESPIRATORY CHAIN

l 15

half-reduction potentials of iron-sulfur Centers 1, 3 + 4, and 2 are - - 2 4 0 mV, mV and - - 5 0 mV, respectively. Ohnishi et al. 2 extended these studies to pigeon heart mitochondria and submitochondrial particles. Typical oxidationreduction titration curves are shown in Fig. 4. In both C. utilis and pigeon heart

--210

{Ve-'S)7

I00-

~ ,,,..f

-I00

..I..<" (Fe-S)5

(Fe-SIz

"

T./ (F'e-S)34 1 1

-200

" .. o

-300

/

-400

/

o .."* /

,,/ o

/

./

/

~."

./

/'/~'/ /

.'~t ,/ J

(Fe-S)~,r,

/

-I

0 io.(OX)

!

Fig. 4. The oxidation-reduction potential dependence of EPR signals arising from different ironsulfur centers in pigeon heart mitochondria. Pigeon heart submitochondrial particles were suspended at the protein concentration of 23.1 mg/ml inca medium containing 0.25 M sucrose and 40 mM morpholinopropane sulfonic acid buffer (pH 7.2). Oxidation-reduction mediators added were 25/~M diaminodurene, 48 #M phenazine methosulfate, 48/~M phenazine ethosulfate, 62 #M duroquinone, 6/~M pyocyanine, 6/~M resorufin, 25 #M 2-hydroxynaphthoquinone, 60/~M phenosafranine, 72 k~M benzyl viologen and 130 /~M methyl viologen. EPR spectra were recorded by a Varian X-band spectrometer (V 4502) at 77, 18.8 and 8.8 °K. Microwave power for EPR measurements at 77 and 18.8 °K was 20.5 roW, while it was 120 mW for the measurements at 8.8 °K2.

mitochondria, Center 1 appears to be composed of at least two iron-sulfur centers with similar EPR spectra and half-reduction potentials (n value of the redox titration is considerably below 1). EPR signals arising from Centers 3 and 4 are overlapped and these two centers have slightly different half-reduction potentials, which could not be measured separately by potentiometric titration procedures due to the overlap of their EPR spectra. The thermodynamic profile of iron-sulfur centers in pigeon heart mitochondria is presented in Scheme II. Although Center 2 has a half-reduction potential close to that of Q or cytochrome b, it is located on the substrate side of the rotenone inhibition site 27,57,62. In C. utilis submitochondrial particles, Center 2 is not reducible with succinate or glycerol 1-phosphate 27, while it is partially reducible with succinate in the mammalian system 57. This can be explained by the observation that the respective half-reduction potential values~lare - - 5 0 mV and - - 2 0 mV. There is a large difference between the half-reduction potentials of low-potential

116

T. OHNISHI (SDH FelS) + 30mY ¢, Rotenone

NADH ~

Half -Reduction Potential

-"- (Center la,lb)(Center 3,4) ~----~-(Center 2) --

-305mY

-245mV

-20mV

[

-~ (Center 5) =

+40mY

= (RJeske's F e / S ) ~

...... 0 z

+280mV

Scheme ii. Thermodynamic profile of iron-sulfur centers in pigeon heart mitochondria 2. The half-reduction potential of Rieske's iron-sulfur protein 7 is taken from the titration by Wilson and Leigh61. SDH = succinate dehydrogenase.

iron-sulfur centers (Centers 1, 3 and 4) and a high-potential iron-sulfur center (Center 2). This suggests that the energy transduction at Site I is associated with the transfer of electrons across this potential span. In this connection, Singer and G u t m a n 9 indicated the location of Site I energy coupling between iron-sulfur Center 1 and Center 2 from the following two observations: (1) the occurrence of energy-dependent electron reverse from Center 2 to N A D in the rotenone-inhibited, NADH-pretreated submitochondrial particles 62 and (2) the absence of phosphorylation coupled to electron transfer from N A D H to KaFe(CN)6 via Center 125,52.

VI. INVOLVEMENT OF IRON-SULFUR CENTER(S) IN SITE I ENERGY COUPLING, SUGGESTED BY IN VIVO INDUCTION STUDIES OF SITE I IN C. UTIL1S CELLS As described in Section II, Ohnishi and Chance 44 have suggested a close correlation between Site I phosphorylation and iron-sulfur protein(s) responsible for the "g = 1.94" E P R signal in the N A D H dehydrogenase region. In contrast, Garland and co-workers reported that the iron-sulfur protein responsible for the "g = 1.94" signal is not needed for the occurrence of Site I phosphorylation from the following two observations: (1) when C. utilis cells which have been grown under iron-limited conditions are aerated in the presence of a cytoplasmic protein synthesis inhibitor, cycloheximide, Site I phosphorylation is induced while neither the "g = 1.94" EPR signal nor piericidin A sensitivity ~1 are induced; (2) continuous culture of C. utilis cells at the transition from iron-limited to glycerol-limited growth produces cells where mitochondria possess Site I phosphorylation and lack the "g = 1.94" EPR signal arising from the iron-sulfur protein in the N A D H dehydrogenase region 47 as measured at 77 °K. Thus, Garland and co-workers suggested that a small fraction of the non-heme iron proteins which does not show detectable EPR absorbance may play a role in Site I phosphorylation. Since the existence of at least five iron-sulfur centers (Centers 1a, 1b, 2, 3 and 4) in the Site I region of the respiratory chain in C. utilis mitochondria have been demonstrated to have characteristic E P R absorption spectra at temperatures below

SITE I REGION OF RESPIRATORY CHAIN

117

77 ° K 2 7 ' 56, Ohnishi e t aL 63 examined the induction of iron-sulfur proteins responsible for these multiple E P R signals and correlated with the Site I induction in the presence and absence of cycloheximide using C. utilis cells grown in the batch culture. It was observed that the induction of Site I phosphorylation, the appearance of the i r o n sulfur E P R signals arising from both Center 1 and Center 2, and piericidin A sensitivity were concomitantly inhibited by cycloheximide (Table III), in contrast to the TABLE III I N VIVO I N D U C T I O N O F SITE I PHOSPHORYLATION, I R O N - S U L F U R CENTERS IN THE SITE I REGION, A N D P1ERICIDIN A SENSITIVITY IN C. UTIL1S CELLS W H I C H WERE G R O W N U N D E R IRON 63- A N D SULFUR65-DEFICIENT CONDITIONS, RESPECTIVELY Growth conditions

Iron deficiency Nonaerated Aerated Aerated with added cycloheximide Sulfur deficiency Nonaerated Aerated Aerated with added cycloheximide

Site 1 phosphorylation

Iron-sulfur centers

Piericidin A sensitivity

-÷ -

-+ --

--t--

-+ ~-

-÷ +

-+ --

result reported by Garland 4~. The absence of Site I phosphorylation in the cycloheximide-inhibited system was demonstrated not only by an A D P / O ratio of less than 2 in NAD-linked substrate oxidation in tightly coupled mitochondria, but also by the lack of energy-dependent reversal of electrons from glycerol 1-phosphate dehydrogenase to endogenous NAD. Garland 4~ and Ragan and Garland 64 concluded that the induction of Site I energy conservation occurred in the presence of cycloheximide, based on only a small increase in A D P / O ratio, although they could not demonstrate significant energy-dependent reversal of electron flow. These observations further strengthened the previous hypothesis presented by Ohnishi and Chance 44, namely, that EPR-detectable iron-sulfur protein(s) may play a role in Site I energy conservation. On the other hand, Haddock and Garland 42 demonstrated the induction of Site I phosphorylation from A D P / O ratio close to 3 as well as energy-dependent reduction of endogenous pyridine nucleotide in tightly coupled mitochondria prepared from sulfur-limited C. utilis cells aerated in the presence of cycloheximide. In this case, they also reported that "g = 1.94" EPR signals arising from iron-sulfur proteins tested at 77 °K was not induced in the presence of cycloheximide, thus EPR-detectable iron-sulfur protein was considered to be unnecessary for Site I energy conservation ~1,64. However, more recently at the 8th lZEBS Meeting in Amsterdam (September, 1972) Garland et al. 65 reported the following result: "When the sulfur-limited cells are aerated in the presence of cycloheximide, EPR signals arising from iron-sulfur Center 1 were detected at 21 °K". This shows the

118

T. OHNISHi

induction of Center 1 concomitant with the induction of Site I phosphorylation, although a small contribution from Center 2 signal cannot be excluded until the temperature profile of prominent peaks in this systems is measured. Although Garland and co-workers obtained variable results in these in vivo induction studies, they have most recently concluded that EPR-measurable iron-sulfur protein may play an important role in Site I phosphorylation in agreement with Ohnishi e t a / . 44'63. The approach using phenotypic variants of C.utilis cells thus strongly suggests that at least one of the "EPR-active" iron-sulfur proteins in the Site I region plays a role in the Site [ energy conservation.

VII. SITE I BYPASS MECHANISM FOUND IN C. UTIL1S M1TOCHONDRIA Katz s° and Katz et al. 66 raised a question about the suggested role of ironsulfur proteins in Site I energy coupling from the following observations: (1) C. utilis cells, grown in batch using an oxystat (steady oxygen concentration in the medium; 15-25 ~o of air saturation), acquire both rotenone sensitivity and presumably Site I energy conservation on the transition from logarithmic to stationary growth phase due to the depletion of carbon source, evenin the high-iron medium; (2) in continuous culture, an increasing in the level of the carbon source without changing the iron concentration (50 ~tM) results in the disappearance of rotenone sensitivity. These observations suggested that upon shifting the chemostat growth conditions from carbon-limited to iron-limited condition 41'47, a loss of Site I energy coupling and rotenone sensitivity was caused by the transition from carbon-limited to carbonexcess condition, rather than by iron limitation. Ohnishi 67 extended the studies by Katz et al. and demonstrated the absence of functional Site I energy conservation in log-phase mitochondria from the measured ADP/O ratios coupled with the NAD-linked substrate respiration. Piericidin A sensitivity of NAD-linked substrate oxidation measured in intact mitochondria parallels the presence or absence of Site I phosphorylation, but the oxidation of N A D H by submitochondrial particles was sensitive to piericidin A for both log-phase and stationary-phase cells. This strongly suggests that in mitochondria from both log-phase and stationary-phase cells, the piericidin A-sensitive component and most probably the Site ! phosphorylation machinery are present. However, N A D H generated in the mitochondria from log-phase cells is oxidized via an alternate electron transfer pathway (most probably via the external N A D H oxidation pathway) which is piericidin A insensitive and does not involve Site I. It has been shown that there are two different N A D H dehydrogenases located in the cristae of C. utilis mitochondria23.68: one is located on the inner surface of the cristae and oxidizes N A D H generated in the matrix space (internal pathway); the other is located on the outer surface of the cristae and oxidizes cytoplasmic N A D H (external pathway), q-he internal N A D H oxidation pathway is piericidin A sensitive and associated with phosphorylation at Site I, while the external N A D H oxidation pathway is piericidin

SITE I REGION OF RESPIRATORY CHAIN

1 19

A insensitive and shows no energy coupling at Site I. The Site I bypass mechanism does not involve the direct exchange of the reducing equivalents from internally generated N A D H to extramitochondrial NAD, since pyruvateplus malate or ethanolferricyanide reductase activity in the log-phase mitochondria is completely inhibited by antimycin A. On preparation of submitochondrial particles, N A D H dehydrogenase which was present in the cristae space is shifted to the outer (external space) surface of submitochondrial particles because of the reversal of membrane orientation, and the mitochondrial N A D H dehydrogenase for the oxidation of cytoplasmic N A D H was suggested to be either solubilized or dislocated during the sonication of mitochondria 68. Thus, in submitochondrial particles, N A D H is oxidized solely via Site I of the electron transfer chain. It was further demonstrated that the carbon level of the culture medium is not the parameter which directly controls this bypass mechanism. If the oxygen supply is lowered to slow down the growth rate in the log phase, both piericidin A sensitivity and Site I energy coupling can be seen even in mitochondria from cells growing in the excess of carbon source. Moreover, when C. utilis cells are grown in an acetate medium, exponential growth with a doubling time of 1.5-2.0 h can be achieved only at pH values more alkaline than 5.5. Under this optimal growth condition, both the rotenone-sensitive site and Site I are bypassed, and cells can grow in the culture medium containing rotenone or piericidin A. On the other hand, if the pH of the culture medium is more acid than pH 5, the growth rate of the cells is very slow and the cells always respire via a piericidin A-sensitive and Site I-operative respiratory pathway even when they are growing with excess carbon level and sufficient supply of air. Thus, these cells are not capable of growing in the presence of rotenone in the culture medium although isolated mitochondria show the presence of both external and internal N A D H oxidation pathways, which is contrary to the observation reported by Fukami et aL 69, for continuous culture. These observations indicate that when C. utilis cells are growing exponentially with an optimal growth rate, Site I phosphorylation is not utilized even if the energycoupling machinery is present. Thus, energy coupling at Site I is utilized only when cells are growing under suboptimal growth conditions. By contrast, if iron concentration in the culture medium is lowered below a certain level, mitochondria deficient in both Site I energy coupling and piericidin A sensitivity are obtained. In this case, N A D H oxidation measured in submitochondrial particles is also insensitive to piericidin A, in contrast to N A D H oxidation in submitochondrial particles prepared from exponentially growing cells. Therefore, the intrinsic effect of iron deficiency, which causes the lack of energy-transducing components at Site 1, can be distinguished from the bypass mechanism of Site I phosphorylation functioning in mitochondria. As described in the early part of this section, Katz et al. 66 proposed that a loss of both Site I phosphorylation and piericidin A sensitivity in C. utilis cells grown under iron or sulfur limitation in a chemostat was caused by the carbon excess rather than the iron or sulfur deficiency. This point was tested by Light and Garland TM who examined piericidin A sensitivity and Site I energy coupling in C. utilis cells grown in

120

T. OHNISHI

a chemostat when the growth was limited by components other than iron, sulfur and carbon source, namely, magnesium, phosphate or nitrogen source (all correspond to carbon-excess condition). In all cases, mitoehondrial NAD-linked substrate respiration showed piericidin A sensitivity and Site I energy conservation, suggesting that continuous culture is a "suboptimal" condition for the growth cycle of the cells. Only iron or sulfur limitation caused the loss of Site I energy coupling in C. utilis cells growing in a chemostat. These results further strengthened the possible role of ironsulfur proteins in Site 1 energy conservations.

viii. IRON-SULFUR CENTER la AS AN ENERGY-TRANSDUCING COMPONENT AT SITE I Wilson and Dutton 71'vz provided evidence that cytochrome bx and aa are directly involved in energy transduction at Site II and III, respectively, from the phosphate potential dependence of their half-reduction potentials. This experimental approach has been applied to Site I, in combination with low-temperature ( ~ 77 °K) EPR techniques. Yeast mitochondria were found unsuitable for studying direct interaction between externally added ATP and endogenous respiratory chain carriers, probably due to poor accessibility of added ATP to the mitochondrial respiratory chain. For example, the energy-dependent shift of the half-reduction potential of cytochrome bx and reversed electron transfer from glycerol 1-phosphate dehydrogenase to endogenous N A D were observed in C. utilis mitochondria, but they can be demonstrated only by internally produced high-energy states, but not by added ATP (cf. Sato et al.V3). Thus, this experimental approach was followed using pigeon heart mitochondria. In order to determine which of the multiple iron-sulfur centers in the Site I region has an energy-dependent half-reduction potential, the effect of ATP addition on the oxidation-reduction level of individual iron-sulfur centers was examined on "potential-clamped" pigeon heart mitochondria 3. In a series of experiments, pigeon heart mitochondrial suspensions in the presence of suitable redox mediators were equilibrated at potentials ranging from - - 4 6 0 to + 100 mV and then ATP was added. Under these conditions almost no effect of ATP was observed on the redox level of Centers 3, 4 and 2. When the potential was set at values where Center l (EmT.2 = - 305 mV) was partially or almost completely reduced, ATP addition caused a partial oxidation of Center 1 (cf. Fig. 5) and this oxidation was inhibited by either uncoupler or oligomycin. Experimentally only 50 ± 1 0 ~ of the Center l signal is observed to be affected by ATP addition. This ATP-affected center has been designated as Center la. These observations suggested that iron-sulfur Center la has a half-reduction potential which is dependent on the phosphate potential (lowering of Emv.2 on the addition of ATP). The iron-sulfur Center la is thus proposed to be involved in the energy transduction process at Site I of the respiratory chain 3, corresponding to cytochrome bx and aa in Sites II and III, respectively.

SITE I R E G I O N OF R E S P I R A T O R Y C H A I N

30*KI

~

,

J

121

(-438rnV)

Tv 1.94

Y

B

(-437mV)

J

ATP

1.94

f

(-439mV)

f

ATP FCCP Oligomycin

t 31

313 kgo~s

315

Fig. 5. The effect of ATP addition on the EPR signal of iron-sulfur Center 1 in pigeon heart mitochondria. Pigeon heart mitochondria were suspended at a protein concentration of 10 mg/ml in a medium containing 0.3 M mannitol, 50 m M morpholinopropane sulfonate (pH 7.2). To this suspension, 67 # M phenazine ethosulfate, 33 # M duroquinone, 20 # M pyocyanine, 20 # M 2-hydroxynaphthoquinone, 78 # M phenosafranine, 74 # M benzyl viologen and 133 # M methyl viologen were added to act as oxidation-reduction mediators. The oxidation-reduction potential of the suspension was lowered to --438 mV by the addition of aliquots of freshly prepared dilute solution of dithionite. An aliquot (0.3 ml) of the suspension was transferred anaerobically to an EPR tube and frozen in liquid isopentane at 113 °K (Spectrum A). After addition of 3.7 m M ATP, which was previously bubbled with argon to remove oxygen, the oxidation-reduction potential of the suspension was readjusted to the same potential and an aliquot of the suspension was quickly frozen (Spectrum B). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (0.8 nmole/mg protein) and oligomycin (0.8 #g/mg protein) were further added to the mitochondrial suspension and an aliquot was frozen in the same fashion (Spectrum C). EPR operating conditions were: modulation amplitude, 12.5 gauss; microwave power, 50 roW; microwave frequency, 9.113 GHz; time constant, 0.3 s; scanning rate, 500 gauss/rain; sample temperature, 20 °K3.

122

T. OHNISHI

Although more detailed studies are required, this tentative proposal by Ohnishi et al. is supported by the following independent observations: (1) In State 4 respiration with succinate plus glutamate as substrates, pyridine nucleotide, Centers 3 and 4 are over 90 ~ reduced, but Center 1 is only about 40 reduced 3. (2) Slater and co-workers 74.75 have reported that in the absence of added redox mediators, the addition of ATP to NA DH-reduced phosphorylating submitochondrial particles, causes partial oxidation of all iron-sulfur centers measured, including iron-sulfur Center 1 (Centers 3 and 4 were not measured). (3) The addition of ATP caused partial oxidation of Center 1 in the submitochondrial particles where all iron-sulfur centers in the Site I region were kept reduced either by anaerobiosis with added N A D H or by the addition of N A D H to the rotenone-inhibited particles 65. IX. SITE I ENERGY COUPLING IN S A C C H A R O M Y C E S

MITOCHONDRIA

The existence of Site I energy coupling in Saccharomyces (cerevisiae or carlsbergensis) mitochondria has been a matter of controversy for a long time. As summarized in Table IV, Chance 76 observed a cross-over point at the Site I region by the TABLE IV SITE I E N E R G Y CONSERVATION IN S . C E R E V I S I A E

OR C A R L S B E R G E N S I S

Reference

Site I phosphorylation

Growth conditions

Carbon source

Chance ~6

÷

Stationary phase (18-h-starved cells) Stationary phase

Ohnishi and co-workers 15,16 Schatz and Racker 17

_ _

Early log phase Stationary phase

Balcavage and Mattoon 18 Kov~l~ e t al. 19 Light and Garland 2°

---

Schuurmans-Steckhoven77 Ghosh and Bhattacharyya 7a

÷ +

Ohnishi 2a Ohnishi 28

-÷

Pye e t a l f l °

--

Pye e t al. 8°

-~-

Light and Garland 7°

d-

Mackler and Haynes 81 Mackler and Haynes al

-÷

Early stationary phase Early stationary phase Continuous culture (carbon limiting) Early log phase Early stationary phase (starved for 3 h) Early stationary phase Early stationary phase (starved for 3-5 h) Continuous culture (nitrogen limiting) Continuous culture (carbon limiting) Continuous culture (carbon limiting) Early stationary phase Late stationary phase (7-14 h)

(Commercial pressed yeast) (Commercial pressed yeast) Lactate (Commercial pressed yeast) Glucose Glucose

Vitols and Linnane t4

--

Glucose Lactate Glucose Glucose Glucose Glucose Glucose Ethanol Glucose Glucose

SITE I REGION OF RESPIRATORY CHAIN

123

direct measurement of mitochondrial pyridine nucleotide and flavin fluorescence using 18-h starved S. cerevisiae cells, suggesting the presence of Site I energy conservation in the baker's yeast as in the mammalian systems. Subsequent studies on the phosphorylation efficiencies in the isolated mitochondria or submitochondrial particles showed the absence of Site I phosphorylation when they were prepared from cells either at the log phase or early stationary phase 14-2°. On the other hand, Schuurmans-Steckhoven77 reported the presence of Site I phosphorylation in mitochondria isolated from S. carlsbergensis cells in the early log phase. Similarly, Ghosh and Bhattacharyya78 reported Site I phosphorylation in S. carlsbergensis which was harvested in the early stationary phase and starved after washing the ceils. In order to resolve these conflicts, Ohnishi 28 prepared mitochondria from the cells both before and after aeration of the S. carlsbergensis cells and showed the apparent induction of Site I energy coupling by aerating cells under non-growing conditions. More recently, Mackler and Haynes 79 demonstrated that mitochondria from either S. cerevisiae or carlsbergensis can perform energy coupling at Site I only at the late stationary phase (5-7 h after reaching the stationary phase). These observations strongly suggest the following characteristics of Saccharomyces mitochondria: (l) although Garland 41 previously concluded that Saccharomyces mitochondria are genetically incompetent to perform Site I energy conservation, they seem to be competent; (2) similar to C. utilis systems, a metabolic control mechanism to bypass Site I under the "optimal" growth conditions is also functional in Saccharomyces mitochondria; (3) apparently, Saccharomyces cells show a slow response to the transition from carbon-excess to carbon-depleted condition, probably because of a high endogenous carbon storage in the cells. However, there are still additional unknown parameters which regulate the bypass mechanism in Saccharomyces systems. For example, Pye et aL so obtained preliminary results indicating the presence of three phosphorylation sites in S. carlsbergensis cells when they were grown in glucose-limited conditions and two phosphorylation sites when cells were grown under nitrogen-limited (carbon-excess) conditions. This was reflected in the growth yield of the cells grown in both batch and continuous culture. On the contrary, Light and Garland 2°,T° observed two phosphorylation sites in S. carlsbergensis mitochondria prepared from cells growing in a chemostat with glucose-limited (carbon-limited) medium but three phosphorylation sites from the cells grown in ethanol-limited (carbon-limited) medium. Mackler and Haynes al also observed different Site I behavior among different strains of Saccharomyces. Thus, further investigations are required for establishing more definite and reproducible conditions for the presence or absence of Site I energy conservation in Saccharomyces mitochondria. It should also be pointed out that no one has ever demonstrated energy-dependent reversal of electron transfer from glycerol 1-phosphate or succinate to endogenous pyridine nucleotide in Saccharomyces mitochondria or NADH-Q reduction coupled with Site I phosphorylation in the submitochondrial particle preparation. Saccharomyces systems may provide us with an important line of approach for the mechanism of Site I energy conservation, because the EPR signals arising from low-potential iron-sulfur centers in the NADH dehydrogenase

124

T. OHNISHI

region have not been detected at 77 °K26,28'29 and below 77 °K27'82 in contrast to mammalian 1 or C. utilis 27"5a systems. On the other hand, EPR signals arising from ironsulfur centers in the cytochrome b, c region or iron-sulfur centers associated with the succinate dehydrogenase can be detected at and below 77 °K and show very similar characteristics as those in mammalian or C. utilis systems 82. S a c c h a r o m y c e s mitochondria do not show rotenone sensitivity under any growth conditions so far examined.

X. INHIBITION MECHANISM OF PIERICIDIN A (OR ROTENONE) As described in Sections II and III, piericidin A (or rotenone) sensitivity was suggested to be closely related to the presence of Site I energy conservation and to the iron-sulfur protein(s) which give rise to the E P R signals in the "g = 1.94" region. However, three yeast systems have been obtained where Site I energy conservation is present and the accompanying electron transport is piericidin A insensitive: (l) C. utilis cells growing in a chemostat at the transition from iron limitation to carbon limitation47; (2) C. utilis cells where Site I energy coupling was induced by aerating sulfur-limited cells in the presence of cycloheximide41,42; (3) S. carlsbergensis and cerevisiae under certain growth conditions. These observations provide an evidence that electron transfer through the piericidin A-sensitive site is not obligatory for the occurrence of Site I energy conservation. It should be pointed out, however, that there is no example so far known where rotenone-sensitive respiration does not yield phosphorylation at Site I. Bois and Estabrook 83 proposed that rotenone reacts irreversibly with the reduced form of iron-sulfur proteins and this may be the mechanism of rotenone inhibition of the N A D H oxidation. This mechanism was questioned by G u t m a n et aI. 8., because rotenone can be strongly bound to specific binding sites even in the oxidized state of non-heme iron proteins. Ohnishi et al. 27, G u t m a n et al. 62 and Albracht and Slater 57 reported that a high-potential iron-sulfur Center 2 as well as low-potential iron--sulfur centers can be reduced with N A D H in the submitochondrial particles pretreated with rotenone. Thus, the rotenone inhibition site was suggested to be on the oxygen side of iron-sulfur Center 2. G u t m a n et al. 62 demonstrated that in the submitochondrial particles pretreated with rotenone, Center 2 remains reduced after exhausing added N A D H , although all other iron-sulfur centers are oxidized. This reduced Center 2 can not be oxidized by the forward electron transfer by the electron leak through the rotenone inhibition site, but can be completely oxidized by the energy-dependent reversal of electrons to N A D , on addition of ATP. These observations suggest that the rotenone interaction site is not directly on Center 2, but is in a very close proximity of Center 2, on its oxygen side. Consistent with these results Ohnishi et al. 43 previously reported that iron-sulfur centers in the N A D H dehydrogenase region and piericidin A sensitivity are not directly related from the following observation: the relative intensity of the E P R signals arising from iron-

SITE I REGION OF RESPIRATORY CHAIN

125

sulfur proteins in the N A D H dehydrogenase region and piericidin A sensitivity show different profiles when they are measured as functions of iron concentration in the culture medium of C. utilis cells. Ragan and Garland 64 reported spectrophotometric observations suggesting the presence of low- and high-potential iron-sulfur proteins in the Site I region of the respiratory chain. They proposed that induction of both low- and high-potential iron-sulfur proteins is required for the appearance of piericidin A sensitivity in C. utilis cells. This high-potential iron-sulfur protein, however, was characterized as being located between piericidin A and antimycin A inhibition sites, thus it does not correspond to Center 2. Identification of this high-potential iron-sulfur protein by EPR measurement and demonstration of its direct correlation to piericidin A sensitivity are still lacking. The mechanism of piericidin A or rotenone sensitivity was discussed in great detail by Singer and Gutman 9 in their recent review. It would be summarized as follows: (1) Both lipids and proteins are involved in the strong, noncovalent forces holding these inhibitors at the specific sites. Prior binding of piericidin A at the specific binding sites strongly inhibits the extraction of the N A D H dehydrogenase with phospholipase A. This suggests a close proximity of the specific binding site to the phospholipids, which are involved in binding the dehydrogenase to the mitochondrial membrane. (2) Piericidin A and rotenone act competitively on the same site, the former binding more strongly than the latter. The number of specific inhibitor binding sites is two per one respiratory chain. One of these suggested to be a "Type V" - S H group 9 which is located on the oxygen side of iron-sulfur Center 2. However, this relationship between the "Type V" - S H group and rotenone sensitivity does not hold in the S. cerevisiae system. S. cerevisiae does not show any sensitivity to rotenone inhibition, although the "Type V" - S H group is present in this system. The other inhibitor binding site remains unknown. In order to define the site of rotenone inhibition more closely, the isolation of a rotenone-resistant mutant would be of great value. However, this approach was not feasible in the C. utilis system, because these cells can grow normally even in the presence of high concentration of rotenone +2,as. Fukami et al. 69 found that C. utilis cells show rotenone-sensitive growth when cells are grown in acetate (pH 5.0) medium, and they reported that in mitochondria prepared from acetate-grown cells in continuous culture, external N A D H is oxidized via a rotenone-sensitive pathway. Thus, Garland +1 proposed that the screening for rotenone-resistant mutants poses no difficulty. However, Ohnishi 67 demonstrated that the rotenone-insensitive external N A D H oxidation pathway is still present in mitochondria of acetate-grown cells. Rotenone-sensitive or -insensitive growth of C. utilis cells is only a reflection of the metabolic control mechanism switching between rotenone-sensitive and -insensitive (Site I phosphorylating and nonphosphorylating) electron transfer pathways depending on the "suboptimal" (below pH 5 in acetate medium) or "optimal" growth conditions (see Section VII), respectively. Hence, the isolation of rotenone-resistant mutant of C. utilis cells grown on acetate seems to be less promising.

126

T. OHNISHI

XI. SUMMARY

1. A biological approach using phenotypic variants of yeast cells under deprived conditions (especially iron- or sulfur-deficient C. utilis cells) strongly suggested the important role of iron-sulfur proteins in both electron transfer and energy conservation at Site I. 2. EPR signals arising from many previously unrecognized iron-sulfur centers (Centers la, lb, 3, 4 and 2) associated with the Site I region of the respiratory chain have been characterized using EPR measurements at temperatures between that of liquid nitrogen and liquid helium. 3. The half-reduction potentials of iron-sulfur Centers la + l b, 3 + 4 and 2 have been determined by the potentiometric titration procedure; they are - - 305 mV, - - 2 4 5 mV, and - - 2 0 mV, respectively, in pigeon heart mitochondria, showing a large potential gap between low-potential iron-sulfur centers and high-potential iron-sulfur Center 2. 4. All of these iron-sulfur centers are located on the substrate side of rotenone inhibition site. 5. From the phosphate potential dependence of the half-reduction potential, iron-sulfur Center la has been tentatively assigned as an energy-transducing component at Site I, corresponding to cytochrome br and a 3 at Sites II and III, respectively. 6. The molecular mechanism of piericidin A and rotenone inhibition still remains unsolved. 7. The Site I bypass mechanism has been described in rapidly growing C. utilis cells which can be differentiated from the loss of Site I phosphorylation in iron- or sulfur-limited C. utilis cells. The Site I bypass mechanism also seems to be functional in S. cerevisiae or carlsbergensis mitochondria under similar growth conditions. ACKNOWLEDGEMENTS

The author wants to express her gratitude to Drs. B. Chance, D. F. Wilson and G. D. Case for their valuable criticism of the manuscript. She is also grateful to Drs J. S. Leigh, D. DeVault, M. Tamura, T. Asakura, P. L. Datton and T. Yonetani for helpful advice and discussions. NOTE ADDED IN PROOF (1Received June 5th, 1973. 2Received July 25th, 1973)

(l) Preparations of reconstitutively active N A D H dehydrogenase have been reported by Baugh and King s6 and by Ragan and Racker sv. (2) Further investigation has revealed that the half-reduction potential of iron-sulfur Center 2 becomes more positive upon addition of ATP. Hence it is suggested that both Center la and Center 2 are involved in the energy transducing reaction at Site I ss. REFERENCES 10rme-Johnson, N. R., Orme-Johnson, W. H., Hansen, R. E., Beinert, H. and Hatefi, Y. (1971) Biochem. Biophys. Res. Commun. 44, 446-452

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