Inhibition of the bacterial complex I by a specific inhibitor of Q reduction, rotenone, is very different from that of the mitochondrial enzyme.. The inhibitor is capable of suppressing th
Trang 1NADH oxidation and NAD+ reduction catalysed by tightly coupled
Alexander B Kotlyar and Natalia Borovok
Department of Biochemistry, George S Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel
Tightly coupled inside-out vesicles were prepared from
Paracoccus denitrificanscells (SPP, sub-Paracoccus particles)
and characterized kinetically The rate of NADH oxidation,
catalysed by SPP, increases 6–8 times on addition of
gram-icidin The vesicles are capable of catalysing DlH+
-dependent reverse electron transfer from quinol to NAD+
The kinetic parameters of the NADH-oxidase and the
reverse electron transfer carried out by membrane-bound
P denitrificans complex I were estimated and compared
with those of the mitochondrial enzyme The data
demon-strate that catalytic properties of the dinucleotide-binding
site of the bacterial and mitochondrial complex I are almost
identical, pointing out similar organization of the site in
mammals and P denitrificans Inhibition of the bacterial complex I by a specific inhibitor of Q reduction, rotenone, is very different from that of the mitochondrial enzyme The inhibitor is capable of suppressing the NADH oxidation reaction only at micromolar concentrations, while the activity of mitochondrial enzyme is suppressed by nano-molar concentrations of rotenone In contrast to the mito-chondrial enzyme, rotenone, even at concentrations as high
as 10 lM, does not inhibit the reverse, DlH+-dependent NAD+-reductase reaction on SPP
Keywords: NADH:Q oxidoreductase; complex I; reverse electron transfer; Paracoccus denitrificans; rotenone
NADH-ubiquinone reductase (EC 1.6.5.3), commonly
known as complex I, catalyzes electron transfer from
NADH to ubiquinone and couples this process to proton
translocation across the inner mitochondrial membrane
The isolated mitochondrial enzyme is composed of more
than 40 individual subunits [1,2] and contains at least five
iron–sulfur centers, a flavine mononucleotide moiety, and
tightly bound ubiquinone molecules, which participate in
electron transfer from NADH to ubiquinone The
mito-chondrial enzyme is capable of transferring electrons in the
opposite direction, from quinol to NAD+ The electron
transfer from high potential electron donor (quinol) through
complex I to low potential electron acceptor (NAD+)
requires the input of free energy in the form of DlH+ The
latter can be produced by mitochondria or coupled
submitochondrial particles (SMP) either during ATP
hydrolysis or oxidation of succinate The former,
ATP-supported electron transfer reaction is sensitive to
uncou-plers and to excess of oligomycin The latter, succinate
energy-supported reaction is sensitive to inhibitors of
succinate oxidation, i.e antimycin, cyanide and malonate,
and is insensitive to oligomycin (reviewed in [3]) Both the
forward and reverse electron transfer reactions are inhibited
by rotenone, a classical inhibitor of complex I [4–8] The
inhibitor blocks the electron transfer between the
com-plex I-associated iron–sulfur clusters and the ubiquinone
pool [8] The affinity of rotenone to mitochondrial
complex I is extremely high and the inhibitor is capable of suppressing NADH-Q reductase activity in nanomolar concentrations [9–11]
Complex I from P denitrificans was shown to be almost identical to its mitochondrial counterpart in terms of composition and thermodynamic properties of redox active groups and sensitivity to specific inhibitors [12–15] The electron transfer within complex I from P denitrificans is coupled to proton translocation across the bacterial mem-brane The latter was confirmed by the following experi-mental observations The rate of NADH oxidation, catalysed by the inside-out vesicles prepared from P deni-trificans cells increased up to 10 times on addition of uncouplers [12] The electron flow within the bacterial complex I can be reversed by DlH+tightly coupled sub-Paracoccus particles (SPP) were shown to catalyse an efficient DlH+-dependent reverse electron transfer from quinol to NAD+ [16] Energization of tightly coupled membrane vesicles from P denitrificans results in changes
of EPR characteristics of iron–sulfur cluster 2 of complex I [16] All signs of energization of complex I detected by EPR
in SPP [16] were also observed with SMP [17], indicating a similar mechanism of energy conservation in the bacterial and mitochondrial enzymes
The bacterial enzyme can serve as a useful model for studies of the mechanism of complex I The aerobic respiratory chain of P denitrificans is evolutionarily related
to the mitochondrial one [18] The functional properties of bacterial complex I (NDH-1) are almost identical to those
of the mitochondrial enzyme; however, the bacterial enzyme
is structurally simpler [19,20] An additional advantage of using SPP for the study of the coupling mechanism in complex I stems from the ability to genetically manipulate bacteria using molecular biology techniques
Understanding the molecular mechanism of complex I requires knowledge about kinetics of DlH+-dependent
Correspondence to A Kotlyar, Department of Biochemistry,
George S Wise Faculty of Life Sciences, Tel Aviv University,
Ramat Aviv, 69978, Israel.
Fax: + 972 (3) 640 68 34, E-mail: s2shak@post.tau.ac.il
Abbreviations: SMP, submitochondrial particles; SPP, sub-Paracoccus
particles.
(Received 19 March 2002, revised 11 June 2002, accepted 3 July 2002)
Trang 2reactions catalysed by the enzyme Unfortunately this
information is available only for mitochondrial complex I
In this work the kinetic parameters of the direct and reverse
reactions carried out by the membrane-bound P
denitrifi-canscomplex I are estimated and compared with those of
the mitochondrial enzyme The data on inhibition of
NADH-oxidase and DlH+-dependent NAD+-reductase
reactions by specific inhibitors of NADH- and
ubiquinone-binding sites of complex I are presented
M A T E R I A L S A N D M E T H O D S
All chemicals were obtained from the Sigma Chemical
Company
The P denitrificans strain Pd1222 was kindly supplied by
R van Spanning (Free University of Amsterdam, the
Netherlands) Bacteria were grown anaerobically with
succinate as the substrate and nitrate as the added terminal
electron acceptor under growth conditions described by
John and Watley [21] Inside-out vesicles from P
denitrifi-canswere prepared as described by John & Hamilton [22]
except that 1 mgÆmL)1BSA (fatty acid free) was added to
the buffer in which the lysozyme-treated cells were
suspen-ded The vesicles were stored at 4°C for up to 2 weeks
without noticeable reduction of either NADH-oxidase or
reverse electron transfer activities The protein content in
SPP was determined with Biuret reagent
NADH-oxidase and NAD+-reductase activities were
measured at 25°C in 0.7 mL of assay solution containing:
5 mMHepes buffer, pH 7.0 and 1 mMmagnesium acetate
NADH-oxidase reaction was initiated by addition of
10–50 lg of SPP to the assay solution supplemented with
100 lM NADH, 1 lg gramicidin and 15 mM ammonium
acetate The succinate-supported NAD+-reductase reaction
was initiated by addition of 50–100 lg of SPP to the assay
solution supplemented with 2 mM NAD+ and 2.5 mM
succinate-K The initial rates of NADH oxidation or
(e¼ 6.2 mM )1Æcm)1)
Other details of the assays are indicated in the legends to
figures
R E S U L T S
The SPP used in this work are tightly coupled Addition of
gramicidin to SPP, respiring on NADH, results in an
sevenfold increase of the NADH oxidation rate (data not
presented) The SPP are capable of catalyzing the DlH+
-dependent reverse electron transfer from quinol to NAD+,
driven by succinate oxidation The rates of the direct and
reverse reactions depend hyperbolically on concentrations
of NADH and NAD+, respectively (see Fig 1A,B)
The kinetic parameters of the reactions were estimated
from the analysis of dependencies in Lineweaver–Burk
plots (see Fig 1) The Km and Vmaxvalues are equal to
5.1 lMand 1.2 lmolÆmin)1Æmg protein)1and 19.6 lMand
0.1 lmolÆmin)1Æmg protein)1 for NADH-oxidase and
NAD+-reductase reactions, respectively The above values
are not significantly different from those estimated earlier
[23] for SMP-catalyzed reactions (see Table 1)
The bacterial complex I is strongly inhibited by
ADP-ribose, a competitive inhibitor of the mitochondrial enzyme
[24] The K value for competitive inhibition of the bacterial
enzyme by ADP-ribose estimated from the analysis of the data in Dixon plots (Fig 2) is equal to 45 lM This value is similar to that estimated recently for ADP-ribose induced inhibition of mitochondrial complex I [24] ADP-ribose selectively inhibits the direct, NADH-oxidase but not the reverse NAD+-reductase reaction, catalyzed by SPP The data presented in Fig 3 demonstrate that addition of ADP-ribose to the assay has no effect on the initial rate of the reverse electron transfer; furthermore, ADP-ribose stimu-lates accumulation of NADH in time A similar effect of ADP-ribose on the reverse electron transfer reaction catalyzed by SMP has been demonstrated recently by Vinogradov and coworkers [24] Comparison of the kinetic data obtained in the present study with those obtained previously for mitochondrial complex I (Table 1) shows resemblance of the NAD(H) binding sites of P denitrificans and mitochondrial complex I
The bacterial complex I has much lower affinity to rotenone, a specific inhibitor of Q reduction, than the mitochondrial enzyme As seen in Fig 4, the inhibitor is capable of suppressing the rate of NADH oxidation of SPP
Fig 1 Kinetics of NADH oxidation and NAD+reduction by SPP Lineweaver–Burk plots of the initial rates at different concentrations of NADH (A) and NAD+(B) The initial rates (V 0 ) of N ADH oxidation
or NAD+reduction were measured at different dinucleotide concen-trations as described in Materials and methods V 0 is expressed in lmolÆmin)1Æmg protein)1.
Trang 3in micromolar concentrations, while the mitochondrial
enzyme is inhibited by nanomolar concentrations of
rote-none (see Table 1)
Rotenone selectively suppresses direct, NADH-oxidase but not the reverse, NAD+-reductase reaction As seen in Fig 5, rotenone at 5 lMstrongly inhibits NADH-oxidase activity of the enzyme; however, the inhibitor does not affect the initial rate of the reverse electron transfer reaction Moreover, rotenone stimulates the process by increasing the extent of NAD+reduction in the reverse electron transfer reaction As seen in Fig 5B, the rate of NADH accumu-lation is reduced in time (curve 1) The reason for that is the dinucleotide oxidation in the NADH-oxidase reaction
At a certain NADH concentration the rate of NAD+ reduction becomes equal to that of NADH oxidation and the steady state is achieved The ability of rotenone to selectively suppress the direct reaction results in an increase
of steady-state level NADH and in straightening up the curve (curve 2)
D I S C U S S I O N The results of this work clearly show that the affinity of the NADH-binding site of the bacterial complex I to substrates of the direct and the reverse reactions is not greatly different from that estimated for the mitochondrial
Table 1 Catalytic properties of membrane particles from mitochondria (SMP) and P denitrificans (SPP).
Reaction Preparation V max (lmolÆmin)1Æmg)1) K m (l M ) KADPribosei ðlM) K rotenone
i ðlM)
NAD+-reductase SMP 0.29a 37.0a No inhibition 0.03g
NAD + -reductase SPP 0.11 b 19.6 b No inhibition No inhibition a
Data taken from [23];bEstimated from Fig 1;cData taken from [24];dEstimated from Fig 2;eData taken from [10];fEstimated from Fig 4;gData taken from [9].
Fig 2 Competitive inhibition of the NADH-oxidase by ADP-ribose.
The initial rates of NADH oxidation (V 0 ) were measured as described
in Materials and methods in the presence of: 2 (curve 1), 4 (curve 2),
6 (curve 3), and 8 l M NADH (curve 4) The dependencies of initial
rates of the reaction on concentration of ADP-ribose are presented in
Dixon coordinates V 0 is expressed in lmol of NADH oxidized per
min per mg of protein.
Fig 3 The effect of ADP-ribose on the time-course of the
succinate-supported NAD+reduction Traces depict the change of absorbency at
340 nm associated with succinate supported NAD + -reductase
reac-tion The reaction was assayed as described in Materials and methods
in the solution, containing 50 l M NAD+, 2.5 m M succinate-K
(curve 1) and 1 m M ADP-ribose (curve 2).
Fig 4 Dependence of NADH-oxidase activity of SPP on the concen-tration of rotenone SPP were preincubated in assay solution, con-taining 5 m M Hepes, pH 7.0, 1 m M magnesium acetate, 2 l M NADH, and rotenone (concentrations are indicated in the figure) for 2 min prior to simultaneous addition of: 100 l M NADH, 15 m M ammonium acetate and 1 lg of gramicidin to the assay mixture The initial rates (V 0 ) were measured as described in Materials and methods and are expressed in lmol of NADH oxidized per min per mg of protein Solid curve corresponds to a single hyperbolic best fit with the following parameters: K i ¼ 1.0 l M , V max ¼ 1.2 lmol of NADH oxidized per min per mg of protein.
Trang 4enzyme (see Table 1) The NADH-oxidase activity of the
bacterial enzyme is strongly suppressed by ADP-ribose, a
competitive inhibitor of the dinucleotide-binding site of
the mitochondrial enzyme [24] As in the case of
mitochondrial complex I, ADP-ribose is capable of
select-ive suppression only of the NADH-oxidase reaction
catalysed by highly coupled SPP The initial rate of the
energy-dependent NAD+reduction by succinate is
insen-sitive to ADP-ribose (Fig 3) The ability of ADP-ribose
to selectively inhibit only the NADH-oxidase reaction
results in an increase in the steady-state level of NADH,
which was established during aerobic succinate-supported
reverse electron transfer catalysed by tightly coupled SPP
(Fig 3) A simulative effect of ADP-ribose on the reverse
electron transfer activity, similar to that shown in this
work, has been demonstrated by Vinogradov and coworkers on mitochondrial complex I [24] Comparison
of the data presented in this work with those obtained previously on mitochondrial complex I (Table 1) clearly shows that the functional properties of the dinucleotide-binding site of P denitrificans complex I are almost identical to those of the mitochondrial enzyme
Bacterial complex I is much less sensitive to rotenone than the mitochondrial one The NADH-oxidase activity of SPP can be strongly suppressed only at micromolar rotenone concentrations This result is in good agreement with the observation of Mejer and coworkers [25], demon-strating relatively low affinity of the whole cells and membrane particles of P denitrificans to rotenone and complete reversibility of rotenone-induced inhibition by BSA Rotenone is known to specifically block the electron flow within complex I at the Q-reductase region [8] The different sensitivities of SMP and SPP to rotenone indicate a difference in the organization of the Q-reductase segment of the bacterial and mitochondrial enzymes The absence of active/inactive transition of P denitrificans complex I [16], the phenomenon that is related to Q-reductase function of complex I [26], further supports the above suggestion Perhaps the most unexpected finding of this work is the inability of rotenone to inhibit the DlH+-dependent NAD+-reductase reaction The different sensitivities of NADH-oxidase and NAD+-reductase reactions catalyzed
by coupled SMP to rotenone has been shown in our earlier studies [9]; however, both reactions were completely inhibited by submicromolar concentrations of the inhi-bitor Inability of rotenone and ADP-ribose to inhibit DlH+-dependent reverse electron transfer catalyzed by the coupled SPP can be explained by assuming lower affinity of the energized complex I, compared to the affinity of the uncoupled enzyme, to both ligands It has been shown previously [3,27,28] that the affinities for NADH and NAD+ are significantly different for cou-pled and uncoucou-pled complex I, supporting the above proposal
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