Planta
Planta (1988)176:261-268
9 Springer-Verlag1988
The oxidation of exogenous N A D H by mitochondria of Euglena gracilis Ulrich Walter Kiimmel and Klaus Brinkmann Botanisches Institut der Universit/it Bonn, Kirschallee 1, D-5300 Bonn 1, Federal Republic of Germany
Abstract. A novel oxidase activity of external N A D H was found in mitochondria of a streptomycin-bleached mutant and the wild strain of Euglena gracilis. In contrast to higher plants the oxidation of external N A D H in mitochondria of E. gracilis is sensitive to rotenone and yields the same phosphorylation efficiency as the matrix pool of N A D H . Simulation of this activity by the classic complex I of the matrix side of the mitochondrial membrane, as a result of preparation-generated artefacts, is excluded. The external NADH-dehydrogenase activity is bound to the inner mitochondrial membrane with its active side facing the cytosol. State-4 enzyme activity is only slightly influenced by pH in the physiological range, whereas state-3 oxidation indicates an optimum in the physiological pH, as expected from a limitation by the ATPase. The external redox potential of N A D H does not control enzyme activity. The results are discussed with respect to the metabolic status of the cells at the time of harvesting. Key words: ADP/O - Euglena - Mitochondrion - N A D H oxidase - Respiratory chain
Introduction
Previous analysis of Euglena mitochondria has indicated an electron chain principally resembling that of higher plants (Sharpless and Butow 1970 a). By means of inhibitors and low-temperature spectroscopy, some electron carriers were identified together with an unusual bypass which was stimulated by A M P but was insensitive to cyanide and antimycin A (Sharpless and Butow 1970b). The possibility that Euglena even adapts to uncouplers of oxidative phosphorylation has been examined at the cellular and mitochondrial level (Kahn
1973). However, no ADP/O values were available to clarify the linkage of substrates to the redox chain with the aid of phosphorylation efficiencies until the method of tryptic digestion of the cell envelope was introduced by Tokunaga et al. (1976). Although giant net mitochondria have been described repeatedly (Pellegrini 1980a, b; Calvayrac etal. 1981), indicating major difficulties in isolating a coupled state, intact mitochondria have been isolated after digestion with trypsin (GomezSilva et al. 1985) or by sonification (Moreno-S~nchez and Raya 1987). Starting with the method of Tokunaga et al. (1976), we modified the procedure to achieve coupled mitochondria from a bleached mutant as well as from the wild strain. With these preparations we found that the behavior of the respiratory chain with respect to the oxidation of external N A D H was quite unusual. In contrast to mitochondria in higher plants (Moller 1986; Moller and Lin 1986), which oxidize exogenous N A D H by a rotenoneinsensitive N A D H dehydrogenase located on the outside of the inner membrane, the external N A D H oxidase in mitochondria of Euglena gracilis is sensitive to rotenone, yielding ADP/O ratios comparable to those obtained by the oxidation of matrix N A D H . Material and methods Organisms and culture. Euglena gracilis strain No. 1224-5/9 (Pringsheim, G6ttingen, FRG) and the obligatory heterotrophic mutant K2 (isolated in our laboratory by means of streptomycin bleaching) were used in our experiments. Cells were cultivated in 2-1 Fernbach flasks in 850 ml medium, containing per liter: 15.0g sucrose (43.8mM), 3.8 g Na-L-glutamate (20.3mM), 2.0g DL-malic acid (14.9mM), 2.5g glycine (33.3 raM). 0.176 g succinic acid (1.5 raM), 0.33 g (NH4)2HPO4 (2.5mM), 0.4g MgSO4.7HzO (1.6mM), 0.08g CaCO3 (0.8raM), 0.3 g KHzPO4 (2.2raM), 6.0mg thiamine-HC1 (17.8 ~tM), 2.70 mg FeC13 (10 [xM), 3.72 mg ethylenediamine-
262
U.W. Kfimmel and K. Brinkmann: Oxidation of exogenous NADH by Euglena mitochondria
tetraacetic acid (EDTA; 10 gM), 5.36 mg HaBO4 (86.69 gM), 2.36 mg MnC12-4 H20 (11.92 gM), 0.44 mg ZnSO4.7 H20 (1.531xM), 0.0234mg MoO3 (85%; 0A4p.M), 0.159mg CUSO4.5 H20 (0.63 gM). Stocks were maintained in heterotrophic medium with Na-L-glutamate, oL-malic acid and (NH4)2 HPO~ as carbon and nitrogen sources, respectively. An aliquot of 0.1 ml Iloban (Merck, Darmstadt, FRG) to provide vitamin B supply and trace elements as described for the mass culture were added to 250 ml stock culture medium. Cells were grown at 27~ C with shaking under cycles of light and dark LD: 12,12 (800-1100 lx) until the late exponential phase was reached (95 h).
Preparation of mitochondria. Mitochondria were isolated after controlled tryptic digestion as described by Tokunaga et al. (1976) and Gomez-Silva et al. (1985) but with some modifications. After harvesting at 750.g the algae were washed twice with buffer containing 0.3 M sucrose, 0.05 M KHzPO4, pH 7.0 and then incubated on ice with 12 mg trypsin (type III, Sigma, Mfinchen, FRG) per g wet weight while gently stirring. After 50 min the proteolytic activity was stopped by incubation for 10 min with 16 mg trypsin-inhibitor (type II-O, Sigma) per g wet weight. Cells were carefully washed and resuspended in a medium containing 0.25M sucrose, 0.025M KH2PO4, 0.5 mM EDTA, 0.5% bovine serum albumin (BSA, w/v), pH 7.4. The pellicula of the bleached mutant was disrupted by stirring vigorously for 7 min, whereas cells of the wild strain were agitated with less intensity to avoid contamination from the photosynthetic apparatus. Cell debris was pelleted by centrifugation at 1475.g and mitochondria were sedimented at 12100.g. Mitochondria were washed twice and finally resuspended using a Potter-Elvejhem (Zurich, Switzerland) homogenizer.
Respiration and ADP/O measurements. Oxygen uptake was measured with a Clark-type electrode (Beckmann, Mfinchen, FRG) in buffer containing 0.25 M sucrose, 0.01 M KH2PO4, 5 mM KC1, 1 mM MgC12, pH 6.9 unless otherwise stated. Ratios of ADP/O were evaluated by graphic analysis of oxygen consumption after addition of a definite amount of ADP as described by Estabrook (1967).
Determinations of NADH and NAD +. To investigate the permeability of the inner membrane to NADH, mitochondria were aerated after preparation for 20 min on ice and then incubated with NADH (3.3 mM) for 7 and 15 min. Parallel samples without any addition (control) and with sorbitol (4.1 raM) were taken. Each sample was placed on top of a Eppendorf cap containing either 3 N HC104 for NAD +-, or 1 N HC104 for sorbitol-, or 1 N KOH for NADH-determination covered with 0.4 ml of silicone oil. After centrifugation for 30 s with maximum speed in a Eppendorf microfuge the phase below the silicone-oil layer was examined. The NADH was enzymatically oxidized by glutamate dehydrogenase; NAD + was determined enzymatically according to Klingenberg (1974). Sorbitol was determined with L-Iditol: NAD + 5-oxidoreductase (Sigma).
Ferricyanide reduction was assayed at 420 nm in buffer (pH 6.9) containing 250raM sucrose, 10 mM KH2PO4, 5 mM KCI, 1 mM MgCI2, 5 mM KCN and 1 mM K3Fe(CN)6. The reaction was started by the addition of 0.5 mM NADH.
Cytochrome-c oxidase activity was tested at 550 nm in buffer (pH 6.9) containing 250 mM sucrose, 10 mM KH2PO4, 5 mM KC1, i mM MgC12 and 8.2 p.M reduced cytochrome e (from
horse heart; Boehringer, Mannheim, FRG). The reaction was started by the addition of mitochondria.
Results Physiological state o f mitochondrial preparations. Isolated m i t o c h o n d r i a f r o m b o t h sources showed considerable c o n t r o l o f respiration with respiratory c o n t r o l ratios ( R C R s ) o f up to 3.1 for site-I substrates. These values remained c o n s t a n t for a b o u t 5 h. C h l o r o p h y l l determined in m i t o c h o n d r i a l suspensions o f the wild strain ranged between 0.008 and 0.032 ~tg.mg -1 protein, whereas 10 ~tg.mg -1 cell protein was f o u n d in whole cells. Instead o f the typical peaks o f the chlorophylls the a b s o r p t i o n spectrum o f an acetone extract o f wild-strain mitoc h o n d r i a showed peaks at 438, 458 a n d 488 nm.. The observed m a x i m a are explained by c o n t a m i n a tion with stigma pigments (Batra and Tollin 1964). Electron m i c r o g r a p h s o f o u r p r e p a r a t i o n s which have been m a d e b y Dr. Buchen (Botanisches Institut der Universit/it Bonn) in a n o t h e r context show fairly h o m o g e n e o u s suspensions (data n o t shown). The outer m e m b r a n e s have n o t been detached, thus protecting the inner m i t o c h o n d r i a l m e m b r a n e s during the isolation procedure.
Respiratory activities and inhibitor studies. Table 1 summarizes the substrates tested for oxidation by isolated m i t o c h o n d r i a . The activity o f the classical complexes o f m i t o c h o n d r i a l respiratory chains is indicated by the oxidation o f L-malate, succinate and ascorbate/tetramethyl-p-phenylenediamine. In addition, N A D H , L-lactate, c~-oxoglutarate a n d glutamate are oxidized. Table 2 shows the influence o f the three classical inhibitors rotenone, antimycin A and cyanide. The high s t a n d a r d deviations are partially the result o f p r e p a r a t i o n - d e p e n d e n t differences a n d partially they are caused by low respiratory activities, especially in cases o f inhibition o f residual activities. C o m p l e t e inhibition o f the site-I and site-II region o f the electron chain was reached only with r o t e n o n e (malate, N A D H ) a n d antimycin A (Llactate) for state-3 respiration. Cyanide completely inhibits the residual respiration after addition o f r o t e n o n e a n d antimycin A ; salicylhydroxamic acid ( S H A M ) never affected o x y g e n uptake. We conclude that these m i t o c h o n d r i a do n o t have an alternative terminal oxidase ( D a y et al. 1980). A surprising result o f the inhibitor studies is the fact that the oxidation o f e x t r a m i t o c h o n d r i a l N A D H is fully sensitive to r o t e n o n e ; m o r e o v e r , the inhibition is m o r e intensive t h a n with malate
U.W. K/immel and K. Brinkmann: Oxidation of exogenous N A D H by
Euglena mitochondria
Table 1. Representative values of state-4 respiration (nmol O2" m i n - l . m g 1) of isolated mitochondria from the wild strain and the bleached m u t a n t of Euglena gracilis after addition of various substrates. T M P D = tetramethyl-p-phenylenediamine
so- b I! L0- ? D []
Substrates
Ascorbate/TMPD Citrate Ethanol L-Glutamate Glycine Glycolate DL-Isocitric acid L-Lactate L-Malate NADH NADPH e-Ketoglutarate Pyruvate Sueeinate
Concentration (raM)
10.3/0.5 10.3 178.0 5.5 10.3 10.3 and 5.5 and 25.8 5.5 and 1.0 0.25 5.5 and /0.3 10.3
Respiratory activity
40 10.3 12.9
10.3
Bleached mutant
Wild strain
93.0 ~ b ~ b b b 26.8 15.8 17.0 b r b 26.0
43.0 b b 18.7 b b b 25.9 16.0 23.5 b 18.4 b 30.0
Weak respiration was observed only in two preparations probably as a result of contamination by cytosolic citric acid dehydrogenase u No stimulation of initial respiration rate could be observed after addition of substrate Substrate caused increase of oxygen uptake, but no representative values available since tested only once or twice
which represents a substrate of ~:he classic coupling site I, reducing the matrix pool of NAD. Although an external NADH-oxidase system sensitive to rotenone has been described for pigeon-heart mitochondria by Rasmussen and Rasmussen (1985), the unexpected rotenone sensitivity of exogenous
T 30-
le
263
0.10--
0.52 mM NADH-p
0.080.06-
~ ""#
si\
L~molote
~" 0040.02--
'~ 20-
0-
-2
0
2
Z.
6
10
MM rotenone ......
o
0
2'0
.........
i
6'0
10 100 ~M rotenone
20
i
120
i
140
t;0
Fig. 1. Effect of rotenone on state-3 oxidation of N A D H and L-malate by mitochondria of the bleached m u t a n t of Euglena gracilis. T e m p e r a t u r e = 2 4 . 3 ~ final s u s p e n s i o n = 0 . 9 8 1 m g p r o t e i n . m l - 1 ; Ki(Rot ) N A D H = l . 1 5 gM, Ki(Rot) L-malate= 2.18 g M
N A D H oxidation was further investigated to exclude an unspecific interaction of the inhibitor with mitochondria. As shown with the semi-reciprocal plots of respiration rates with L-malate and N A D H against varying concentrations of rotenone in Fig. I, there are only slight differences in the corresponding Ki values (1.15 gM for N A D H and 2.18 gM for Lmalate). The highest achievable inhibition remains constant up to concentrations of 160 gM rotenone. The concentration of rotenone we used in our experiments was 25 gM.
ADP/O ratios. The inhibitor studies, especially those with rotenone, indicate that the oxidation
Table 2. Inhibition (%) of the respiratory rates of mitochondria isolated from the wild strain and bleached m u t a n t of Euglena gracilis after addition of rotenone (25 gM), antimycin A (0.5/2.5 [xM) or cyanide (2.5 mM). Rates are m e a n s + S D (numbers in ( ) indicate the number of preparations) Substrate
Strain
Rotenone State 3
L-Glutamate
Wild Bleached
27-+ 7.8 (4)
Antimycin
Cyanide
State 4
State 3
State 3
a a
a
a
37+13.2
(5)
70+20.1 (4)
~-Ketoglutarate
Bleached
Wild a
9 2 + 5.6 (7)
55_+41.4 (3)
38+19.5(3)
a
a
a
L-Lactate
Wild Bleached
17 _+11.5 (10) 51__ 7.7 (10)
a 19+ 8.0 (9)
33_+ 6.9 (12) 4 9 • 5.6 (7)
81 +16.0 (7) 82+12.8 (8)
L-Malate
Wild Bleached
85-+ 14.8 (18) 72 + 21.9 (10)
75+19.0 (9)
3 0 + 3.6 (3) 5 0 + 8.6 (13)
62_+ 9.6(4) 79_+ 9.2 (9)
NADH
Wild Bleached
96-+ 5.4 (6) 75 4-17.3 (10)
70+16.0 (5)
54_+ 5.3 (4) 51 +15.0 (6)
89_+11.0(3) 81 +12.6 (6)
Succinate
Wild Bleached
a
77_+18.0 (5) 784- 9.4 (7)
7 5 + 7.8 (2) 8 2 + 1.4 (3)
a Not tested
a
a
U.W. Kfimmel and K. Brinkmann: Oxidation of exogenous N A D H by Euglena mitochondria
264
Table 3. ADP/O ratios for the oxidation of various substrates measured with mitochondria of the wild strain and the bleached mutant of Euglenagracilis. Values are means _ SD (the numbers in ( ) indicate the number of measurements from various preparations)
'T
Substrate
E "7
Strain
ADP/O
L-Glutamate
Wild Bleached
1.20+0.22 not tested
(7)
~-Ketoglutarate
Wild Bleached
1.37 4- 0.07 (10) not tested
L-Lactate
Wild Bleached
0.95 + 0.08 (6) 1.04 • 0.27 (69)
L-Malate
Wild Bleached
1.89 4- 0.27 (22) 1.35 • 0.21 (61)
NADH
Wild Bleached
1.38 + 0.36 (I 2) 1.34 + 0.26 (48)
Succinate
Wild Bleached
1.15 4- 0.15 (9) 1.13 _+0.19 (33)
of both the intra- and extramitochondrial pool of NADH is coupled with the generation of 3 moles of ATP per mole NADH. These predictions are confirmed by the corresponding ADP/O ratios in Table 3. For the bleached mutant it is demonstrated that the oxidation of exogenous NADH is coupled with nearly the same energy conservation as the oxidation of L-malate. Phosphorylation of wild-strain mitochondria is more effective with L-malate than with NADH, but ADP/O ratios of NADH oxidation are higher than ADP/O ratios with succinate. The results are contrary to those obtained with higher-plant mitochondria, which oxidize cytosolic NADH with 1/3 less phosphorylation efficiency than matrix NADH.
The oxidation of external NADH. In higher plants the acceptor site of electrons from external NADH is a separate flavoprotein which is associated with the outer face of the inner mitochondrial membrane. This dehydrogenase feeds electrons directly to ubiquinone, bypassing the rotenone-sensitive first site of energy transformation (Day and Wiskich 1979; M011er 1986). The enzyme of higher plants is further characterized by its sensitivity to ethylene glycol-bis (/?-aminoethyl ether)-N,N,N',N'tetraacetic acid (EGTA). Oxidation of NADH by Jerusalem artichoke (Helianthus tuberosus) is nearly completely inhibited in the presence of I mM EGTA (Moller and Palmer 1982). With Spinach (Spinacia oleracea) leaf mitochondria the effect of EGTA is less marked (only 50%) under high-cation conditions and nearly absent under low-cation conditions (Edman et al. /985).
17-
15-
E o
13-
-6
11-
o
L- malate state & I
I
I
mM
2',o o
I
I
I
EGTA
Fig. 2. Sensitivity to E G T A of N A D H (5.43 ~M) and L-malate (13.6 mM) respiration by freshly prepared mitochondria from the bleached mutant of Euglena under low-cation conditions (cations were omitted from isolation buffers during preparation of mitochondria and from reaction buffer for polarographic measurements; KH2PO4 was replaced in all buffers by equimolar concentrations of 3-(N-morpholino)propanesulfonic acid)
We did not find a similar effect in the presence of cations (for concentrations, see under respiration and ADP/O measurements), even with EGTA concentrations up to 2 mM. Only if cations were omitted was it possible to observe an inhibition of external NADH oxidation by EGTA, reaching 40% for state-4 and 30% for state-3 respiration as shown in Fig. 2. The oxidation of matrix NADH (represented by the oxidation of L-malate) was nearly unaffected by the chelator. The following possible explanations of these results were tested: (i) Our preparations contain inside-out particles in addition to the right-side-out mitochondria; (ii) NADH crosses the inner membrane and is oxidized by the internal NADH dehydrogenase of site I; (iii) a shuttle mechanism equilibrates the transmembrane redox potential of NADH; (iv) a highly active transhydrogenase exists in mitochondria of Euglena gracilis (Day and Wiskich 1978); (v) the oxidation of external NADH is mediated by an independent NADH dehydrogenase, located on the outside of the inner membrane (v. Jagow and Klingenberg 1970). The existence of inside-out particles can be excluded, if the integrity of the outer mitochondrial membrane is guaranteed. We proved the intactness of the outer membrane by measuring the cytochrome-c-oxidase activity before and after addition of Triton X-100 or hypotonic treatment (Fig. 3). As described by Kay et al. (1985), this test is also used to estimate the polarity of submitochondrial particles, yielding results similar to those
U.W. Kfimmel and K. Brinkmann: Oxidation of exogenous N A D H by + Triton 10
::::::::::::::::::::::::::
10
::::::::::::::::::::: [iiiiiiilViiiiiiii!i!iiJi!iiiiIi
8
=========================7=
o
6
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o
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ii /
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+ sucrose
sucrose
(0.25 M)
deleted
Fig. 3. Cytochrome-c oxidase activity of mitochondria from the bleached mutant of Euglena after incubation in sucrose-deleted and sucrose-containing (0.25 M) media (light columns) followed by treatment with 0.05% Triton X-100 (dark columns). Freshly prepared mitochondria were incubated on ice for 1 h, sedimented at 12100.g and resuspended in sucrose-containing (0.25 M) buffer
None
Sorbitol
Compound determined
Time of incubation (min) 7
15
NADH NAD +
2.216 0.863
0.815 2.590
NADH+NAD +
3.079 (1.4%)
3.405 (2.0%)
NADH NAD +
" 1.286
a 1.775
NADH + NAD +
1.286
1.775
Sorbitol
5.947 (2.2%)
5.630 (2.7%)
a Not detectable by the method used
1 4 ~ ~ e _ shown in Fig. 3 for inside-out submitochondrial particles. However, much higher centrifugal forces are necessary to gain submitochondrial particles than those we used in our experiments to sediment mitochondria. Although some preparations not treated with detergent showed low cytochrome-c-oxidase activity (up to 1/5 of the activity reached after solubilization), the oxidation of external N A D H cannot be attributed to inside-out particles, since our twicewashed mitochondria still exhibit high malate-oxidizing activity, which is considered to be extruded from electron-transporting particles. Permeation of the inner mitochondrial membrane by N A D H can also be excluded, since after incubation with N A D H the matrix pool of N A D H and N A D § is not increased. Table 4 summarizes the results obtained by freshly prepared mitochondria which were incubated with N A D H for 7 and 15 min. The additional amount of N A D H and N A D § found after silicone-oil centrifugation (1.4% and 2.0%, respectively, of the initial amount in the incubation buffer) must be localized in the intermembrane space and the sediment volume between the organelles. This we conclude since sorbitol, which cannot permeate the inner mitochondrial membrane, is trapped in the intermembrane and intermitochondrial space to a comparable amount of 2.2% and 2.7%, respectively, of the initially offered amount of sorbitol.
265
Table 4. N A D H , N A D + and sorbitol ( n m o l . m g -1 protein) concentrations in mitochondrial fractions of the bleached Euglena mutant incubated at 25 ~ C with N A D H (217.2 n m o l . m g - l protein for 7 min incubation; 169.2 nmol-mg -1 for 15 rain incubation) or sorbitol (271.4 and 211.5 n m o l . m g - 1 protein for 7 and 15 min incubation, respectively) after separation from the incubation medium by rapid silicone-oil centrifugation (figures in ( ) = percent recovered compared with the initial amount in the incubation buffer) Incubation substance
4
~ ' ~nE~ 127 6 -5 IZ
Euglena mitochondria
._c E 6 E -6
2
M, cyt c
DH
/
I
0
5
1(3 1~5 incubc]tiontime (min}
2'Q
Fig. 4. Oxidation of reduced cytochrome c, external N A D H and succinate by mitochondria from the bleached mutant of Euglena after incubation on ice in sucrose-deleted media for various times (for treatment of mitochondria after incubation see Fig. 3). Cytochrome-c oxidation was tested spectrophotometrically, N A D H and succinate oxidation by oxygen consumption; succinate oxidation after hypotonic incubation for 5 and 10 min was not tested
A shuttle mechanism that mediates the transfer from external reduction capacities to the matrix space seems to be unlikely, because the necessary shuttle intermediates should be washed out during the preparation. Nevertheless we investigated this possibility in our preparations. Figure 4 shows the activities of N A D H dehydrogenase succinate dehydrogenase and cytochrome-c oxidase after incubation of mitochondria in sucrose-depleted buffer for different lengths of time. The amount of both succinate and N A D H oxi-
U.W. Kfimmel and K. Brinkmann: Oxidation of exogenous NADH by Euglena mitochondria
266
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c 10
-100 -60 -20 0 20 Alteration (%) of ferricyanide reduction
dation decreases to almost the same extent with loss of integrity of the outer membrane, possibly as a result of thermic disintegration with time. Within the same experimental set the amount of cytochrome oxidation increases, indicating the hypoosmotic disruption of the outer membrane (i.e. increasing accessibility of cytochrome oxidase to external cytochrome c). Such a rupture followed by a washing procedure should have almost completely extruded the shuttle enzymes from the intermembrane space. From the fact that, under such conditions, N A D H oxidation only slightly decreases (comparable with a simultaneous decrease in the membrane-bound oxidation of succinate) we conclude that a shuttle mechanism is not involved in the oxidation of external N A D H . Oxidation of N A D H is furthermore linked to the inhibitor-insensitive ferricyanide reduction as shown in Fig. 5. Electrons from the matrix compartment are linked to ferricyanide via externally facing cytochrome e of the repiratory chain. This flux is sensitive to the inhibitors rotenone and antimycin A as shown for the oxidation of L-malate. Oxidation of external N A D H was only slightly affected by the inhibitor. Surprisingly with antimycin A, the ferricyanide reduction with N A D H was stimulated up to 127% (data not shown). We cannot explain this effect. From these results we conclude that oxidation of exogenous N A D H by mitochondria of Euglena gracilis is connected to the respiratory chain via an N A D H dehydrogenase on the outside of the inner membrane. This enzyme, in contrast to plant mitochondria, is sensitive to rotenone and yields
NADH
7C "T E "T 5s E
Planta (1988)176:261-268
9 Springer-Verlag1988
The oxidation of exogenous N A D H by mitochondria of Euglena gracilis Ulrich Walter Kiimmel and Klaus Brinkmann Botanisches Institut der Universit/it Bonn, Kirschallee 1, D-5300 Bonn 1, Federal Republic of Germany
Abstract. A novel oxidase activity of external N A D H was found in mitochondria of a streptomycin-bleached mutant and the wild strain of Euglena gracilis. In contrast to higher plants the oxidation of external N A D H in mitochondria of E. gracilis is sensitive to rotenone and yields the same phosphorylation efficiency as the matrix pool of N A D H . Simulation of this activity by the classic complex I of the matrix side of the mitochondrial membrane, as a result of preparation-generated artefacts, is excluded. The external NADH-dehydrogenase activity is bound to the inner mitochondrial membrane with its active side facing the cytosol. State-4 enzyme activity is only slightly influenced by pH in the physiological range, whereas state-3 oxidation indicates an optimum in the physiological pH, as expected from a limitation by the ATPase. The external redox potential of N A D H does not control enzyme activity. The results are discussed with respect to the metabolic status of the cells at the time of harvesting. Key words: ADP/O - Euglena - Mitochondrion - N A D H oxidase - Respiratory chain
Introduction
Previous analysis of Euglena mitochondria has indicated an electron chain principally resembling that of higher plants (Sharpless and Butow 1970 a). By means of inhibitors and low-temperature spectroscopy, some electron carriers were identified together with an unusual bypass which was stimulated by A M P but was insensitive to cyanide and antimycin A (Sharpless and Butow 1970b). The possibility that Euglena even adapts to uncouplers of oxidative phosphorylation has been examined at the cellular and mitochondrial level (Kahn
1973). However, no ADP/O values were available to clarify the linkage of substrates to the redox chain with the aid of phosphorylation efficiencies until the method of tryptic digestion of the cell envelope was introduced by Tokunaga et al. (1976). Although giant net mitochondria have been described repeatedly (Pellegrini 1980a, b; Calvayrac etal. 1981), indicating major difficulties in isolating a coupled state, intact mitochondria have been isolated after digestion with trypsin (GomezSilva et al. 1985) or by sonification (Moreno-S~nchez and Raya 1987). Starting with the method of Tokunaga et al. (1976), we modified the procedure to achieve coupled mitochondria from a bleached mutant as well as from the wild strain. With these preparations we found that the behavior of the respiratory chain with respect to the oxidation of external N A D H was quite unusual. In contrast to mitochondria in higher plants (Moller 1986; Moller and Lin 1986), which oxidize exogenous N A D H by a rotenoneinsensitive N A D H dehydrogenase located on the outside of the inner membrane, the external N A D H oxidase in mitochondria of Euglena gracilis is sensitive to rotenone, yielding ADP/O ratios comparable to those obtained by the oxidation of matrix N A D H . Material and methods Organisms and culture. Euglena gracilis strain No. 1224-5/9 (Pringsheim, G6ttingen, FRG) and the obligatory heterotrophic mutant K2 (isolated in our laboratory by means of streptomycin bleaching) were used in our experiments. Cells were cultivated in 2-1 Fernbach flasks in 850 ml medium, containing per liter: 15.0g sucrose (43.8mM), 3.8 g Na-L-glutamate (20.3mM), 2.0g DL-malic acid (14.9mM), 2.5g glycine (33.3 raM). 0.176 g succinic acid (1.5 raM), 0.33 g (NH4)2HPO4 (2.5mM), 0.4g MgSO4.7HzO (1.6mM), 0.08g CaCO3 (0.8raM), 0.3 g KHzPO4 (2.2raM), 6.0mg thiamine-HC1 (17.8 ~tM), 2.70 mg FeC13 (10 [xM), 3.72 mg ethylenediamine-
262
U.W. Kfimmel and K. Brinkmann: Oxidation of exogenous NADH by Euglena mitochondria
tetraacetic acid (EDTA; 10 gM), 5.36 mg HaBO4 (86.69 gM), 2.36 mg MnC12-4 H20 (11.92 gM), 0.44 mg ZnSO4.7 H20 (1.531xM), 0.0234mg MoO3 (85%; 0A4p.M), 0.159mg CUSO4.5 H20 (0.63 gM). Stocks were maintained in heterotrophic medium with Na-L-glutamate, oL-malic acid and (NH4)2 HPO~ as carbon and nitrogen sources, respectively. An aliquot of 0.1 ml Iloban (Merck, Darmstadt, FRG) to provide vitamin B supply and trace elements as described for the mass culture were added to 250 ml stock culture medium. Cells were grown at 27~ C with shaking under cycles of light and dark LD: 12,12 (800-1100 lx) until the late exponential phase was reached (95 h).
Preparation of mitochondria. Mitochondria were isolated after controlled tryptic digestion as described by Tokunaga et al. (1976) and Gomez-Silva et al. (1985) but with some modifications. After harvesting at 750.g the algae were washed twice with buffer containing 0.3 M sucrose, 0.05 M KHzPO4, pH 7.0 and then incubated on ice with 12 mg trypsin (type III, Sigma, Mfinchen, FRG) per g wet weight while gently stirring. After 50 min the proteolytic activity was stopped by incubation for 10 min with 16 mg trypsin-inhibitor (type II-O, Sigma) per g wet weight. Cells were carefully washed and resuspended in a medium containing 0.25M sucrose, 0.025M KH2PO4, 0.5 mM EDTA, 0.5% bovine serum albumin (BSA, w/v), pH 7.4. The pellicula of the bleached mutant was disrupted by stirring vigorously for 7 min, whereas cells of the wild strain were agitated with less intensity to avoid contamination from the photosynthetic apparatus. Cell debris was pelleted by centrifugation at 1475.g and mitochondria were sedimented at 12100.g. Mitochondria were washed twice and finally resuspended using a Potter-Elvejhem (Zurich, Switzerland) homogenizer.
Respiration and ADP/O measurements. Oxygen uptake was measured with a Clark-type electrode (Beckmann, Mfinchen, FRG) in buffer containing 0.25 M sucrose, 0.01 M KH2PO4, 5 mM KC1, 1 mM MgC12, pH 6.9 unless otherwise stated. Ratios of ADP/O were evaluated by graphic analysis of oxygen consumption after addition of a definite amount of ADP as described by Estabrook (1967).
Determinations of NADH and NAD +. To investigate the permeability of the inner membrane to NADH, mitochondria were aerated after preparation for 20 min on ice and then incubated with NADH (3.3 mM) for 7 and 15 min. Parallel samples without any addition (control) and with sorbitol (4.1 raM) were taken. Each sample was placed on top of a Eppendorf cap containing either 3 N HC104 for NAD +-, or 1 N HC104 for sorbitol-, or 1 N KOH for NADH-determination covered with 0.4 ml of silicone oil. After centrifugation for 30 s with maximum speed in a Eppendorf microfuge the phase below the silicone-oil layer was examined. The NADH was enzymatically oxidized by glutamate dehydrogenase; NAD + was determined enzymatically according to Klingenberg (1974). Sorbitol was determined with L-Iditol: NAD + 5-oxidoreductase (Sigma).
Ferricyanide reduction was assayed at 420 nm in buffer (pH 6.9) containing 250raM sucrose, 10 mM KH2PO4, 5 mM KCI, 1 mM MgCI2, 5 mM KCN and 1 mM K3Fe(CN)6. The reaction was started by the addition of 0.5 mM NADH.
Cytochrome-c oxidase activity was tested at 550 nm in buffer (pH 6.9) containing 250 mM sucrose, 10 mM KH2PO4, 5 mM KC1, i mM MgC12 and 8.2 p.M reduced cytochrome e (from
horse heart; Boehringer, Mannheim, FRG). The reaction was started by the addition of mitochondria.
Results Physiological state o f mitochondrial preparations. Isolated m i t o c h o n d r i a f r o m b o t h sources showed considerable c o n t r o l o f respiration with respiratory c o n t r o l ratios ( R C R s ) o f up to 3.1 for site-I substrates. These values remained c o n s t a n t for a b o u t 5 h. C h l o r o p h y l l determined in m i t o c h o n d r i a l suspensions o f the wild strain ranged between 0.008 and 0.032 ~tg.mg -1 protein, whereas 10 ~tg.mg -1 cell protein was f o u n d in whole cells. Instead o f the typical peaks o f the chlorophylls the a b s o r p t i o n spectrum o f an acetone extract o f wild-strain mitoc h o n d r i a showed peaks at 438, 458 a n d 488 nm.. The observed m a x i m a are explained by c o n t a m i n a tion with stigma pigments (Batra and Tollin 1964). Electron m i c r o g r a p h s o f o u r p r e p a r a t i o n s which have been m a d e b y Dr. Buchen (Botanisches Institut der Universit/it Bonn) in a n o t h e r context show fairly h o m o g e n e o u s suspensions (data n o t shown). The outer m e m b r a n e s have n o t been detached, thus protecting the inner m i t o c h o n d r i a l m e m b r a n e s during the isolation procedure.
Respiratory activities and inhibitor studies. Table 1 summarizes the substrates tested for oxidation by isolated m i t o c h o n d r i a . The activity o f the classical complexes o f m i t o c h o n d r i a l respiratory chains is indicated by the oxidation o f L-malate, succinate and ascorbate/tetramethyl-p-phenylenediamine. In addition, N A D H , L-lactate, c~-oxoglutarate a n d glutamate are oxidized. Table 2 shows the influence o f the three classical inhibitors rotenone, antimycin A and cyanide. The high s t a n d a r d deviations are partially the result o f p r e p a r a t i o n - d e p e n d e n t differences a n d partially they are caused by low respiratory activities, especially in cases o f inhibition o f residual activities. C o m p l e t e inhibition o f the site-I and site-II region o f the electron chain was reached only with r o t e n o n e (malate, N A D H ) a n d antimycin A (Llactate) for state-3 respiration. Cyanide completely inhibits the residual respiration after addition o f r o t e n o n e a n d antimycin A ; salicylhydroxamic acid ( S H A M ) never affected o x y g e n uptake. We conclude that these m i t o c h o n d r i a do n o t have an alternative terminal oxidase ( D a y et al. 1980). A surprising result o f the inhibitor studies is the fact that the oxidation o f e x t r a m i t o c h o n d r i a l N A D H is fully sensitive to r o t e n o n e ; m o r e o v e r , the inhibition is m o r e intensive t h a n with malate
U.W. K/immel and K. Brinkmann: Oxidation of exogenous N A D H by
Euglena mitochondria
Table 1. Representative values of state-4 respiration (nmol O2" m i n - l . m g 1) of isolated mitochondria from the wild strain and the bleached m u t a n t of Euglena gracilis after addition of various substrates. T M P D = tetramethyl-p-phenylenediamine
so- b I! L0- ? D []
Substrates
Ascorbate/TMPD Citrate Ethanol L-Glutamate Glycine Glycolate DL-Isocitric acid L-Lactate L-Malate NADH NADPH e-Ketoglutarate Pyruvate Sueeinate
Concentration (raM)
10.3/0.5 10.3 178.0 5.5 10.3 10.3 and 5.5 and 25.8 5.5 and 1.0 0.25 5.5 and /0.3 10.3
Respiratory activity
40 10.3 12.9
10.3
Bleached mutant
Wild strain
93.0 ~ b ~ b b b 26.8 15.8 17.0 b r b 26.0
43.0 b b 18.7 b b b 25.9 16.0 23.5 b 18.4 b 30.0
Weak respiration was observed only in two preparations probably as a result of contamination by cytosolic citric acid dehydrogenase u No stimulation of initial respiration rate could be observed after addition of substrate Substrate caused increase of oxygen uptake, but no representative values available since tested only once or twice
which represents a substrate of ~:he classic coupling site I, reducing the matrix pool of NAD. Although an external NADH-oxidase system sensitive to rotenone has been described for pigeon-heart mitochondria by Rasmussen and Rasmussen (1985), the unexpected rotenone sensitivity of exogenous
T 30-
le
263
0.10--
0.52 mM NADH-p
0.080.06-
~ ""#
si\
L~molote
~" 0040.02--
'~ 20-
0-
-2
0
2
Z.
6
10
MM rotenone ......
o
0
2'0
.........
i
6'0
10 100 ~M rotenone
20
i
120
i
140
t;0
Fig. 1. Effect of rotenone on state-3 oxidation of N A D H and L-malate by mitochondria of the bleached m u t a n t of Euglena gracilis. T e m p e r a t u r e = 2 4 . 3 ~ final s u s p e n s i o n = 0 . 9 8 1 m g p r o t e i n . m l - 1 ; Ki(Rot ) N A D H = l . 1 5 gM, Ki(Rot) L-malate= 2.18 g M
N A D H oxidation was further investigated to exclude an unspecific interaction of the inhibitor with mitochondria. As shown with the semi-reciprocal plots of respiration rates with L-malate and N A D H against varying concentrations of rotenone in Fig. I, there are only slight differences in the corresponding Ki values (1.15 gM for N A D H and 2.18 gM for Lmalate). The highest achievable inhibition remains constant up to concentrations of 160 gM rotenone. The concentration of rotenone we used in our experiments was 25 gM.
ADP/O ratios. The inhibitor studies, especially those with rotenone, indicate that the oxidation
Table 2. Inhibition (%) of the respiratory rates of mitochondria isolated from the wild strain and bleached m u t a n t of Euglena gracilis after addition of rotenone (25 gM), antimycin A (0.5/2.5 [xM) or cyanide (2.5 mM). Rates are m e a n s + S D (numbers in ( ) indicate the number of preparations) Substrate
Strain
Rotenone State 3
L-Glutamate
Wild Bleached
27-+ 7.8 (4)
Antimycin
Cyanide
State 4
State 3
State 3
a a
a
a
37+13.2
(5)
70+20.1 (4)
~-Ketoglutarate
Bleached
Wild a
9 2 + 5.6 (7)
55_+41.4 (3)
38+19.5(3)
a
a
a
L-Lactate
Wild Bleached
17 _+11.5 (10) 51__ 7.7 (10)
a 19+ 8.0 (9)
33_+ 6.9 (12) 4 9 • 5.6 (7)
81 +16.0 (7) 82+12.8 (8)
L-Malate
Wild Bleached
85-+ 14.8 (18) 72 + 21.9 (10)
75+19.0 (9)
3 0 + 3.6 (3) 5 0 + 8.6 (13)
62_+ 9.6(4) 79_+ 9.2 (9)
NADH
Wild Bleached
96-+ 5.4 (6) 75 4-17.3 (10)
70+16.0 (5)
54_+ 5.3 (4) 51 +15.0 (6)
89_+11.0(3) 81 +12.6 (6)
Succinate
Wild Bleached
a
77_+18.0 (5) 784- 9.4 (7)
7 5 + 7.8 (2) 8 2 + 1.4 (3)
a Not tested
a
a
U.W. Kfimmel and K. Brinkmann: Oxidation of exogenous N A D H by Euglena mitochondria
264
Table 3. ADP/O ratios for the oxidation of various substrates measured with mitochondria of the wild strain and the bleached mutant of Euglenagracilis. Values are means _ SD (the numbers in ( ) indicate the number of measurements from various preparations)
'T
Substrate
E "7
Strain
ADP/O
L-Glutamate
Wild Bleached
1.20+0.22 not tested
(7)
~-Ketoglutarate
Wild Bleached
1.37 4- 0.07 (10) not tested
L-Lactate
Wild Bleached
0.95 + 0.08 (6) 1.04 • 0.27 (69)
L-Malate
Wild Bleached
1.89 4- 0.27 (22) 1.35 • 0.21 (61)
NADH
Wild Bleached
1.38 + 0.36 (I 2) 1.34 + 0.26 (48)
Succinate
Wild Bleached
1.15 4- 0.15 (9) 1.13 _+0.19 (33)
of both the intra- and extramitochondrial pool of NADH is coupled with the generation of 3 moles of ATP per mole NADH. These predictions are confirmed by the corresponding ADP/O ratios in Table 3. For the bleached mutant it is demonstrated that the oxidation of exogenous NADH is coupled with nearly the same energy conservation as the oxidation of L-malate. Phosphorylation of wild-strain mitochondria is more effective with L-malate than with NADH, but ADP/O ratios of NADH oxidation are higher than ADP/O ratios with succinate. The results are contrary to those obtained with higher-plant mitochondria, which oxidize cytosolic NADH with 1/3 less phosphorylation efficiency than matrix NADH.
The oxidation of external NADH. In higher plants the acceptor site of electrons from external NADH is a separate flavoprotein which is associated with the outer face of the inner mitochondrial membrane. This dehydrogenase feeds electrons directly to ubiquinone, bypassing the rotenone-sensitive first site of energy transformation (Day and Wiskich 1979; M011er 1986). The enzyme of higher plants is further characterized by its sensitivity to ethylene glycol-bis (/?-aminoethyl ether)-N,N,N',N'tetraacetic acid (EGTA). Oxidation of NADH by Jerusalem artichoke (Helianthus tuberosus) is nearly completely inhibited in the presence of I mM EGTA (Moller and Palmer 1982). With Spinach (Spinacia oleracea) leaf mitochondria the effect of EGTA is less marked (only 50%) under high-cation conditions and nearly absent under low-cation conditions (Edman et al. /985).
17-
15-
E o
13-
-6
11-
o
L- malate state & I
I
I
mM
2',o o
I
I
I
EGTA
Fig. 2. Sensitivity to E G T A of N A D H (5.43 ~M) and L-malate (13.6 mM) respiration by freshly prepared mitochondria from the bleached mutant of Euglena under low-cation conditions (cations were omitted from isolation buffers during preparation of mitochondria and from reaction buffer for polarographic measurements; KH2PO4 was replaced in all buffers by equimolar concentrations of 3-(N-morpholino)propanesulfonic acid)
We did not find a similar effect in the presence of cations (for concentrations, see under respiration and ADP/O measurements), even with EGTA concentrations up to 2 mM. Only if cations were omitted was it possible to observe an inhibition of external NADH oxidation by EGTA, reaching 40% for state-4 and 30% for state-3 respiration as shown in Fig. 2. The oxidation of matrix NADH (represented by the oxidation of L-malate) was nearly unaffected by the chelator. The following possible explanations of these results were tested: (i) Our preparations contain inside-out particles in addition to the right-side-out mitochondria; (ii) NADH crosses the inner membrane and is oxidized by the internal NADH dehydrogenase of site I; (iii) a shuttle mechanism equilibrates the transmembrane redox potential of NADH; (iv) a highly active transhydrogenase exists in mitochondria of Euglena gracilis (Day and Wiskich 1978); (v) the oxidation of external NADH is mediated by an independent NADH dehydrogenase, located on the outside of the inner membrane (v. Jagow and Klingenberg 1970). The existence of inside-out particles can be excluded, if the integrity of the outer mitochondrial membrane is guaranteed. We proved the intactness of the outer membrane by measuring the cytochrome-c-oxidase activity before and after addition of Triton X-100 or hypotonic treatment (Fig. 3). As described by Kay et al. (1985), this test is also used to estimate the polarity of submitochondrial particles, yielding results similar to those
U.W. Kfimmel and K. Brinkmann: Oxidation of exogenous N A D H by + Triton 10
::::::::::::::::::::::::::
10
::::::::::::::::::::: [iiiiiiilViiiiiiii!i!iiJi!iiiiIi
8
=========================7=
o
6
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o
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ililiTii!iiiiJ"iliSiiii!iiiiii] ii!i!iiiii!i!iii!iiiiiiii --i
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ii /
/
/
+ sucrose
sucrose
(0.25 M)
deleted
Fig. 3. Cytochrome-c oxidase activity of mitochondria from the bleached mutant of Euglena after incubation in sucrose-deleted and sucrose-containing (0.25 M) media (light columns) followed by treatment with 0.05% Triton X-100 (dark columns). Freshly prepared mitochondria were incubated on ice for 1 h, sedimented at 12100.g and resuspended in sucrose-containing (0.25 M) buffer
None
Sorbitol
Compound determined
Time of incubation (min) 7
15
NADH NAD +
2.216 0.863
0.815 2.590
NADH+NAD +
3.079 (1.4%)
3.405 (2.0%)
NADH NAD +
" 1.286
a 1.775
NADH + NAD +
1.286
1.775
Sorbitol
5.947 (2.2%)
5.630 (2.7%)
a Not detectable by the method used
1 4 ~ ~ e _ shown in Fig. 3 for inside-out submitochondrial particles. However, much higher centrifugal forces are necessary to gain submitochondrial particles than those we used in our experiments to sediment mitochondria. Although some preparations not treated with detergent showed low cytochrome-c-oxidase activity (up to 1/5 of the activity reached after solubilization), the oxidation of external N A D H cannot be attributed to inside-out particles, since our twicewashed mitochondria still exhibit high malate-oxidizing activity, which is considered to be extruded from electron-transporting particles. Permeation of the inner mitochondrial membrane by N A D H can also be excluded, since after incubation with N A D H the matrix pool of N A D H and N A D § is not increased. Table 4 summarizes the results obtained by freshly prepared mitochondria which were incubated with N A D H for 7 and 15 min. The additional amount of N A D H and N A D § found after silicone-oil centrifugation (1.4% and 2.0%, respectively, of the initial amount in the incubation buffer) must be localized in the intermembrane space and the sediment volume between the organelles. This we conclude since sorbitol, which cannot permeate the inner mitochondrial membrane, is trapped in the intermembrane and intermitochondrial space to a comparable amount of 2.2% and 2.7%, respectively, of the initially offered amount of sorbitol.
265
Table 4. N A D H , N A D + and sorbitol ( n m o l . m g -1 protein) concentrations in mitochondrial fractions of the bleached Euglena mutant incubated at 25 ~ C with N A D H (217.2 n m o l . m g - l protein for 7 min incubation; 169.2 nmol-mg -1 for 15 rain incubation) or sorbitol (271.4 and 211.5 n m o l . m g - 1 protein for 7 and 15 min incubation, respectively) after separation from the incubation medium by rapid silicone-oil centrifugation (figures in ( ) = percent recovered compared with the initial amount in the incubation buffer) Incubation substance
4
~ ' ~nE~ 127 6 -5 IZ
Euglena mitochondria
._c E 6 E -6
2
M, cyt c
DH
/
I
0
5
1(3 1~5 incubc]tiontime (min}
2'Q
Fig. 4. Oxidation of reduced cytochrome c, external N A D H and succinate by mitochondria from the bleached mutant of Euglena after incubation on ice in sucrose-deleted media for various times (for treatment of mitochondria after incubation see Fig. 3). Cytochrome-c oxidation was tested spectrophotometrically, N A D H and succinate oxidation by oxygen consumption; succinate oxidation after hypotonic incubation for 5 and 10 min was not tested
A shuttle mechanism that mediates the transfer from external reduction capacities to the matrix space seems to be unlikely, because the necessary shuttle intermediates should be washed out during the preparation. Nevertheless we investigated this possibility in our preparations. Figure 4 shows the activities of N A D H dehydrogenase succinate dehydrogenase and cytochrome-c oxidase after incubation of mitochondria in sucrose-depleted buffer for different lengths of time. The amount of both succinate and N A D H oxi-
U.W. Kfimmel and K. Brinkmann: Oxidation of exogenous NADH by Euglena mitochondria
266
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r _ ontimycin A
-22 t 6
+
9o-
o
NADH +
0,1 0
30
36 IJM NADH n._.. n__ O_ O... n.~12,D~ 13
c 10
-100 -60 -20 0 20 Alteration (%) of ferricyanide reduction
dation decreases to almost the same extent with loss of integrity of the outer membrane, possibly as a result of thermic disintegration with time. Within the same experimental set the amount of cytochrome oxidation increases, indicating the hypoosmotic disruption of the outer membrane (i.e. increasing accessibility of cytochrome oxidase to external cytochrome c). Such a rupture followed by a washing procedure should have almost completely extruded the shuttle enzymes from the intermembrane space. From the fact that, under such conditions, N A D H oxidation only slightly decreases (comparable with a simultaneous decrease in the membrane-bound oxidation of succinate) we conclude that a shuttle mechanism is not involved in the oxidation of external N A D H . Oxidation of N A D H is furthermore linked to the inhibitor-insensitive ferricyanide reduction as shown in Fig. 5. Electrons from the matrix compartment are linked to ferricyanide via externally facing cytochrome e of the repiratory chain. This flux is sensitive to the inhibitors rotenone and antimycin A as shown for the oxidation of L-malate. Oxidation of external N A D H was only slightly affected by the inhibitor. Surprisingly with antimycin A, the ferricyanide reduction with N A D H was stimulated up to 127% (data not shown). We cannot explain this effect. From these results we conclude that oxidation of exogenous N A D H by mitochondria of Euglena gracilis is connected to the respiratory chain via an N A D H dehydrogenase on the outside of the inner membrane. This enzyme, in contrast to plant mitochondria, is sensitive to rotenone and yields
NADH
7C "T E "T 5s E
