Volume 267, number 1, 103-106
FEBS 08564 July 1990
Effect of ATP on the activity of bovine heart mitochondrial kc, complex
Michele Lorusso, Tiziana Cocco, Michele Minuto and Sergio Papa
hutltute of Medical Biochemistry and Chemistry, University of Bari, Barl, Itab
Received 20 April 1990
The effect of ATP on the reductase
absolute specificity for ATP smce other polyvalent
that ATP stimulates
acttvity of purtfied bovine heart &c, complex was studied. ATP stimulates
reduction of b and c1 cytochromes,
amens such as EDTA and EGTA produce
the acttvtty of the &c, complex, chelatmg
of the enzyme. There is no
range. It is proposed
action on the enzyme.
activity and the
pre-steady-state also causing changes of kmetic properties
similar effects in the micromolar
exerting a modulatory Inhibitory cation(s),
Mitochondria; b-c, Complex; Electron transport; ATP: Complexing agent
2. MATERIALS AND METHODS
Electron flow in ubiquinol-cytochrome
(b-cl complex or complex III; EC 18.104.22.168) of the
trogenic proton translocation
The bovine heart b-cl complex shows invariably 11
[ 11, namely 8 supernumerary
subunits in addition to those holding the two b hemes,
heme cl and Rieske 2Fe-2S cluster. Attempts made to
clarify the role of the supernumerary
involvement of some of them in proton translocation
and electron transfer activities [2-51. Our group has
recently shown that the complex exhibits a different
susceptibility towards proteolytic digestion depending
on the redox state of prosthetic groups . These and
related observations  indicate that the activity of the
b-cl complex may be modulated by allosteric interac-
tions of the constituent polypeptides.
It has been reported that anions, and in particular
ATP, modulate the redox activity of mitochondrial
cytochrome c oxidase, apparently by interacting with
two supernumerary subunits (IV and VIII) [7-101.
In this paper we report on the effect of ATP on the
activity of bovine b-cl complex. It is shown that ATP
stimulates the reductase activity of the complex either
in the soluble or in the membrane-reconstituted
reduction route of b and cr cytochromes.
chain is coupled to elec-
across the inner
2.1. Enzyme and chemicals
timycin and soybean
Chemical Co. Duroqumol
was from Boehringer
was from K&K Laboratories.
(type II-b), were from Sigma
from bovine heart
c (type VI), an- as in [ll].
2.2. Preparation of b-c, vesicles
of b-cl complex
(pH 7.4) .
dialysis in 100 mM
2.3. Spectrophotometrrc determmatrons
The reductase activity was measured
trophotometer at 550-540
7.2). The rates were corrected
ed either in the absence of the enzyme or with the enzyme inhibited
Redox changes of b and ci cytochromes
followed at 10°C with an air turbine
wavelength couples of 562-540 nm and 550-540
with a dual-wavelength spec-
nm in 100 mM K-Hepes/lO
for the non-enzymatic
mM KC1 (pH
Correspondence address: S. Papa,
Institute of Medical Biochemistry
G. Cesare, University of Bari, Piazza 70124 Bari,
Fig. 1 shows the influence of various anions on the
reductase activity of cholate-solubilized
ATP and ADP induced a two-fold enhancement, at 10
and 20 mM, respectively,
cytochrome c reduction, with effectiveness appearing
to be related to their charge. Inorganic phosphate also
stimulated the reductase activity, its effect being max-
imal at 100 mM concentration
0.23). Other inorganic anions such as chloride and
sulphate exerted only a minor influence on the reduc-
Kinetic analysis (Table I) showed that ATP increased
by 2-4-fold the apparent Km and Vmax of both duro-
quinol and cytochrome c without causing any deviation
from linearity of the Lineweaver-Burk
of the initial rate of
(ionic strength, I =
Pubhshed by Elsewer Science Publishers B. V. (Biomedical Division)
00145793/90/$3.50 0 1990 Federation of European Biochemical Societies
Volume 267, number 1
Fig. 1. Influence
100 mh? K-Hepes/lO
was started by the addition
added as potassium
ADP; (A) inorganic
mM KCI (pH 7.2), 0.02%
c and 3 #g protein/ml
of 33 PM duroqutnol.
salts in 100 mM Hepes (pH 7.2). (0) ATP; (’ )
( 0) KCI; ( ?
on the abscissa after conversion
of soluble complex.
of h-cl complex.
) KlSOa. Concentrattons
into I (ionic strength).
nally added ATP, ADP and inorganic
pears that, upon
respect to ADP and inorganic
of physiological significance
in the cell is in the millimolar
that the steady-state
cytosol is much higher than in the matrix space .
The stimulatory effect of ATP on the reductase
tivity of the b-cl vesicles was influenced
brane potentiaf, being maximal
Similar results were obtained
of ADP and inorganic
of Fig. 2 shows the effect of extcr-
at the outer surface
in the membrane,
of the vesicles
[ATP]/[ADP]. [P,] ratio
This can be
by the mem-
when the PMF was
c reductase activity
K,,, and V,,, values For duroquinol
and cytochrome c
in soluble b-cl complex;
of ATP on the apparent
3 pg protein/ml
from 5.5 to 90 ,&i in the presence of 6 /rM cytochrome
concentration of cytochrome
was used at 90pM. Rates
b-ct complex were suspended
0.02% Tween 80. Where indicated,
The iu,, and V,,% values were calculated
m the reaction
5 mM ATP was
varied In (a), DQHz coneentratlon
c. In (b), the
c varied from 0.18 to 1.35 FM DQHz
are expressed as rmol.mtn-’
k;, are expressed as pM
Fig. 2. Duroqmnol-cytochrome
protem/ml) were suspended
7.2) also containing
vahnomycin and nigertcin;
control was 1.33 pmol .min”mg
in 100 mhl K-Hepes/lO
6 hhl ferrtcytochrome
DQH?, 33 rhl. The spectfic actirny
(A) morganic phosphate
acttvity rn b-cl
(22 /rg into phorpholiptd vestcles
mM KC1 (pH
c and 0.15 pg/ml each
(e) ATP, (,~,) ADP:
duced by other non-physiological
EDTA and EGTA.
at PM concentrations
of the 6--c, complex to the same extent as mM concen-
trations of ATP. Also the effect of EDTA was larger
when the PMF was collapsed.
observed that additions of ATP
saturating concentrations of EDTA
EDTA and EGTA caused
kinetic properties of the enzyme
produced by ATP (TabIe I).
The reversibility of the ATP and EDTA effect was
demonstrated as follows. 6-cl
or reduced by duroquinol
saturating concentrations of ATP
(5 PM). The suspension was then passed
Sephadex G-25 column. The reductase
resulting b-cl vesicles, which
values, was still two-fold stimulated
Separate controls (see also Fig. 5) showed that Mg-
ATP complex was as able as ATP in stimulating
reductase activity of the complex,
influenced by the presence
1 mM free Mg2+.
Fig. 4 shows the effect of ATP on pre-steady-state
reduction kinetics of b and cl cytochromes
quinol in b-cl vesicles supplemented
In the presence of antimycin
stimulation of the apparent
CI cytochromes. Conversely,
by the addition to the vesicle suspension
and nigericin (Fig. 3A).
stimulatory effect of ATP is also pro-
Fig. 3B shows, in fact, that EDTA
stimulated the reductase
in the presence
the same changes
(not shown) as those
vesicles either oxidized
(3 mM) or EDTA
activity of the
by either ATP or
neither was its effect
in the assay medium of
was a two-fold
of b and
rate of reduction
with myxothiazol present
Volume 267, number 1
ATP (m M )
E D TA (PM )
were 0.30 and 1.45 pmol
3. Effect of ATP and EDTA on reductase
acttvtty of b-cl vesicles. The experimental
state, both valinomycin
I fo r the coupled and uncoupled
symbols to the uncoupled
Open symbols refer to the coupled
are those descrtbed
The specific activittes
in the legend to Fig. 2.
in the control
in the coupled and mgericin
with 5 ,uM EDTA
It has been shown that PM concentrations
ions inhibit the reductase
drial b-cl complex (, see also [15,16]). The effect of
Zn2+ ions on the activity
vesicles is shown in Fig. 5A. Zn2+ at about 4 ,uM con-
centration completely reversed
shown), ATP had no effect
The same results were obtained
instead of ATP.
on the rate of b
activity of bovine mitochon-
of ATP stimulated 6-ci
the stimulation of the
D Q H 2
Fig. 4. Effect of ATP on pre-steady-state
ci cytochromes in b-c,
concentration 1 PM Cyt. cr) in the reaction
legend to Ftg. 2 and supplemented
used at 17 GM. Dotted
reduction kinetics of b and
vesicles. The vesicles were suspended (fmal
in the mixture described
with 5 PM antimycin.
solid line (+ 3 mM ATP).
shows that Mg-ATP
hibition of the reductase
of 1 mM MgC12. Higher
about a definite
induced by 3 mM Mg-ATP
of the reduc-
at higher con-
1 60 -
1 40 -
IO O -
Fig. 5. Effect
b-cl vesicles. b-c,
with 1 mM MgCIZ. In Expt. A, ZnClz was titrated
(open symbols) and in the presence
h4g-ATP was added in the absence
presence of 5.5 ,uM ZnClx. Rates are expressed
of Mg. ATP and ZnClz on the reductase
vesicles (22 pg protein/ml)
mixture described in the legend to Fig. 2 and supplemented
were suspended in the
in the absence
In Expt. B,
symbols) and in the
as percent of control
of 3 mM Mg-ATP.
Volume 267. number 1 Download full-text
FEBS LETTERS July 1990
to that observed
to a stimulation
in the absence
of the activity comparable
88.00376 1 I of Constglio
\\as in part
supported by Grant
that ATP specifically
at the quinol
ATP is more effective
b-cl vesicles in the uncoupled
This would suggest that interaction
6-cl complex affects a redox step different
6562 and cytochrome
to be controlled by PMF .
The stimulatory effect produced
enzyme by mM concentrations
by PM concentration
enhances the reductase
titious metal ions, which exert an inhibitory
the complex. It is, in fact, known that Zn2+ inhibits
[ 15,161 and that this effect can be reversed by ATP [ 161
(cf. Fig. 5).
The results presented would indicate
mutually interacting binding
non-physiological anions) and divalent
particular zinc. Mutually exclusive
nucleotides and Zn2+ ions
modulatory effect on the redox activity
at the quinol oxidation site.
of mM concentrations
affects the activity of the complex
than in coupled
of ATP with the
a marked stimulation
this being accompanied
properties of the enzyme.
inhibitors (Fig. 4) indicate
(1983) Eur. J. Biochem.
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the activity of
of ATP with the
cl which has been shown
on the reconstituted
of ATP is also displayed
of EDTA which is a typical metal
activity by chelating
site of the respiratory chain
D.. Cocco. T and
J.S. (1979) J.
G. and Btsson, R. (1986) Biochem.
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the existence of
site(s) for ATP (as well as
heavy metals, in
of the enzyme
to exert a
T. and Papa, S. (1990) in: Structure,
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