Biochimica et Biophysica Acta 1857 (2016) 863–871

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Review

Oxidation of NADH and ROS production by respiratory complex I☆

Andrei D. Vinogradov ⁎, Vera G. Grivennikova
Department of Biochemistry, School of Biology, Moscow State University, Moscow 119991

a r t i c l e i n f o a b s t r a c t

Article history: Kinetic characteristics of the proton-pumping NADH:quinone reductases (respiratory complexes I) are reviewed.
Received 29 September 2015 Unsolved problems of the redox-linked proton translocation activities are outlined. The parameters of complex
Received in revised form 2 November 2015 I-mediated superoxide/hydrogen peroxide generation are summarized, and the physiological significance
Accepted 7 November 2015
of mitochondrial ROS production is discussed. This article is part of a Special Issue entitled Respiratory complex
Available online 10 November 2015
I, edited by Volker Zickermann and Ulrich Brandt.
Keywords:
© 2015 Elsevier B.V. All rights reserved.
NADH:quinone oxidoreductase
Proton pumping
Hydrogen peroxide
Superoxide
Bacterial plasma membranes
Mitochondria

1. Introduction bound to a membrane freely permeable for protons catalyze reaction

Respiratory complex I (proton-translocating NADH:quinone oxido-
reductases, mitochondrial complex I, bacterial NDH-1)1 catalyzes a
reaction of great metabolic significance:
(1) irreversibly due to a significant thermodynamic gap between the
standard redox potentials of NADH/NAD+ (− 320 mV) and QH2/Q
(+60 mV).
The membrane-bound and all solubilized or detergent-dispersed
preparations of the enzyme catalyze the irreversible reaction:

ð1Þ NADH þ 2Aox →NADþ þ 2Ared þ 2Hþ ð2Þ

where Q stands for the natural membrane-bound quinone (ubiquinone
with various number of isoprenoid units at position 6 of benzoquinone
ring) or menaquinone, H+ +
in and Hout stand for intramitochondrial
(cytoplasmic) and extruded protons, respectively, and p.m.f. (proton-
motive force) is the transmembrane gradient of electrochemical activity
of H+ (Δμ~ Hþ ).
Reaction (1) is only reversible when the oxidoreduction is tightly
coupled to proton pumping activity. Thus, this reversible reaction only
proceeds when the enzyme is bound to a membrane that is poorly
permeable to protons. The enzyme detached from the membrane or
+
Abbreviations: APAD , 3-acetylpyridine adenine dinucleotide; Ferri, ferricyanide;
FMN, flavin mononucleotide; HAR, hexaammineruthenium(III); Qn, 2,3-dimethoxy,5-
methyl,6-(n)isoprenyl,1,4-benzoquinone; ROS, reactive oxygen species; SMP, submito-
chondrial particles; O•2 , superoxide anion; SOD, superoxide dismutase; p.m.f., proton-
motive force (transmembrane gradient of electrochemical activity of H+, Δμ~ Ηþ ).
☆ This article is part of a Special Issue entitled Respiratory complex I, edited by Volker
Zickermann and Ulrich Brandt.
⁎ Corresponding author.
E-mail address: adv@biochem.bio.msu.su (A.D. Vinogradov).
1
Bacterial plasma membrane proton pumping NADH:quinone oxidoreductases (NDH-1)
and corresponding mitochondrial enzymes are designated as complex I throughout the text
for the sake of simplicity.
where Aox and Ared stand for artificial oxidized and reduced forms
of the A red /A ox couple. Ferricyanide 3 − anion (Ferri) and/or
hexaammineruthenium3 + cation (HAR) are routinely used in assays
of the enzyme. Reaction (2) is not coupled to proton translocating activ-
ity. Membrane-bound and various solubilized preparations of complex I
as well as their FMN-containing fragments catalyze the reversible
NADH:APAD+ transhydrogenase reaction. Mitochondrial and bacterial
complex I also catalyzes NADH oxidation by oxygen:
NADH þ О2 þ Нþ →NADþ þ Н2 О2 ð3Þ
and/or
NADH þ 2О2 →NADþ þ 2O•2 þ Hþ ð4Þ
the formation of so-called ROS (reactive oxygen species – hydrogen
peroxide and superoxide) at the highest rate of about 0.2–0.3% of
those depicted by Eqs. (1) and (2).
Spectacular progress in visualization of atomic structures of the frag-
ments [1,2] and the entire complex I [3–5] isolated from various sources
have been achieved during the last decade. However, as important as
the information provided by the molecular architecture of the enzyme

http://dx.doi.org/10.1016/j.bbabio.2015.11.004
0005-2728/© 2015 Elsevier B.V. All rights reserved.
864 A.D. Vinogradov, V.G. Grivennikova / Biochimica et Biophysica Acta 1857 (2016) 863–871
is, it serves only as excellent groundwork for hypotheses on possible
catalytic mechanisms. We believe that studies on the catalytic activities
still are and will be the powerful tool for understanding the mechanistic
and, perhaps more physiologically important, regulatory properties of
complex I.
This short review discusses available information on some features
of eukaryotic and bacterial complex I as they appear from studies of
the reactions (1 and 2). We will also discuss reactions (3 and 4) and
their possible physiological significance. Because little information is
available on the catalytic properties of plant complex I, they are not
discussed here; an interested reader is addressed to Refs. [6,7].
2. Intramolecular electron transfer
Complex I contains up to ten active redox components (FMN, 7–8
iron-sulfur clusters, and bound quinone(s)). The structures of complex
I and fragments derived therefrom [1–4] unambiguously suggest the
following sequence of intramolecular electron transfer on the way
from NADH to the final quinone acceptor:
NADH→FMN→N3ðN1aÞ→N1b→N4→N5→N6a→N6b→N2→Q ð5Þ
During steady-state coupled or uncoupled NADH oxidation by bo-
vine heart submitochondrial particles (SMP), all EPR-detectable iron-
sulfur centers of complex I are almost completely reduced [8,9]. Thus,
the rate-limiting step of the overall NADH oxidase is reoxidation of
the terminal electron-transferring component (N2, or specifically and
tightly bound quinone) by bulk quinone. Only a very short description
of the intramolecular electron transfer is needed for further discussion
of the steady-state catalytic activity. Recently, the kinetics of intramo-
lecular electron transfer in purified Escherichia coli complex I using
ultrafast freeze-quenching techniques followed by EPR and optical
spectroscopic analysis have been reported [10–12]. The results obtained
by these groups and their interpretation significantly differ. Biphasic
reduction of iron-sulfur clusters N1a and N2 (~ 50 μsec) followed
by much slower reduction of N1b and N6b was interpreted by
Verkhovskaya et al. so as to suggest that NAD+ dissociation from the
nucleotide-binding site is the rate-limiting step in the overall reaction
(rapid oxidation of the first NADH molecule results in reduction of
N1a and N2, and further reduction of the enzyme proceeds significantly
more slowly because the next NADH molecule can bind to the empty
active site only after dissociation of NAD+) [10,11]. De Vries et al.
interpreted their result as to suggest that rapid reduction of N2
results in six-fold decrease in electron tunneling rate between other
iron-sulfur centers, thus synchronizing electron transfer with the
proton pumping activity [12]. Whatever interpretation is correct, the
enzyme turnover when it operates as a member of the respiratory
chain (~hundreds per sec) is substantially slower than any intramolec-
ular redox reaction.
3. Steady-state activity
The accumulated data on NADH-dehydrogenating activities of
membrane-bound complex I scattered in the literature are given in
Table 1. Averaged NADH oxidase activity of fully activated2 uncoupled
bovine heart SMP is about 1.5 μmol of NADH oxidized per min per mg
of protein (Table 1), a value that is close to that seen at “saturating”
concentrations of artificial quinone acceptors and other analogs or
homologs of ubiquinone (Q1). Assuming a molar content of complex I
in heart mitochondrial membranes of about 0.1 nmol per mg of protein
[37], these numbers correspond to maximal steady-state enzyme
turnover of ~ 250 s−1. Does this turnover reflect the maximal capacity
2
The mitochondrial enzyme exhibits complex kinetics in the quinone reductase activity
due to slow transition between catalytically inert D- and active A-forms (operationally
called the A/D-transition). This phenomenon reviewed in Refs [35,36] is beyond the scope
of the present discussion.
of the enzyme, and how it can be regulated in situ? Natural ubiquinones
are practically insoluble in aqueous media, and their analogs or homo-
logs at “saturating” concentrations of about 100 μM are routinely used
for assays of the membrane-bound or detergent-solubilized enzyme.
The content of ubiquinone in heart inner mitochondrial membrane is
about 5 nmol per mg of protein [37]. The bulk quinone is located in
the lipid phase of the membrane. The content of phospholipids in the
inner mitochondrial membrane (bovine heart) is about 0.4 mg per mg
of protein [37]. Simple approximate calculations show that the concen-
tration of ubiquinone in the lipid phase is as high as about 10 mM. Thus,
in situ quinone reduction by complex I proceeds at concentrations of the
substrate of about two orders of magnitude higher than that used under
standard assay systems with added artificial quinone acceptors. The
true concentration of “water-soluble” quinones in the membranes and
their respective reactivity certainly depend on the lipid/water partition
coefficient [38]. The sequence of events during the steady-state
NADH:externally added quinone-acceptor reductase reaction catalyzed
by particulate complex I is not clear. External quinone might either
directly bind to the ubiquinone-specific site or oxidize tightly bound
Q, or oxidize bulk natural reduced quinone swimming in the lipid phase.
In studies on Q-pool behavior in the respiratory chain [39] recently
confirmed by others [40], linear dependence of NADH oxidase activity
of bovine heart SMP on the content (concentration) of oxidized ubiqui-
none have been demonstrated. If extrapolated to the state where
ubiquinone is fully oxidized, the data would correspond to turnover
number of complex I of about 500 s−1 (pH 7.4, 25 °C), a value that is
substantially higher than those (averaged) measured at saturating con-
centrations of water-soluble quinones or in steady-state NADH oxidase
assays (~250 s−1, see above). This is not surprising because during the
steady state, fully uncoupled NADH oxidase operates at Qred/Qox ratio
of about 1. Linear, not hyperbolic, dependence of the NADH oxidase
rate on molar fraction of oxidized ubiquinone (Qox/(Qox + Qred)) has
been demonstrated [39]. Such dependence is expected either if the
total concentration of quinone is much lower than the apparent Km for
Qox or if Qred competes with Qox for binding at the reactive site with
similar affinity. The first possibility seems to be unlikely because of
high concentration of ubiquinone in the membrane (see above). Kinetic
competition between oxidized and reduced forms of ubiquinone (with
similar affinity) seems the more plausible explanation and corroborates
with well-established reversibility of reaction (1). Weak (competitive
with oxidized Q1) inhibition of NADH:Q1 reductase activity catalyzed
by highly purified bovine heart complex I by the product (Q1H2) have
been reported [41]. Also, a competitive relation between oxidized
and reduced quinone acceptor (Q2) was documented for respiratory
complex II [42].
The linear dependence of NADH oxidase activity on the molar frac-
tion of oxidized ubiquinone might shed some light on the mechanism
of the respiratory control phenomenon. NADH oxidation in controlled
state 4 can equally be slow either because p.m.f. inhibits proton
translocating activity (see Eq. (1)) or because an increase in reduced
ubiquinone concentration due to a decrease in Q-pool oxidation
by further components of the respiratory chain (complex III and
cytochrome c reductase), which are under the control of p.m.f. The rela-
tive contributions of “thermodynamic” (p.m.f.) and “kinetic” (Qred/Qox
ratio) factors to the respiratory control phenomenon at the level of
complex I remain to be established.
4. Artificial electron acceptors
Ferricyanide (Ferri) [43,44] and hexaammineruthenium III (HAR)
[21] are the most frequently used artificial electron acceptors for quan-
titation of complex I catalytic activity of preparations of different degree
of resolution. The substrate (NADH) concentration–activity profile
for Ferri reductase catalyzed by the membrane-bound or soluble, so-
called high molecular mass preparations of complex I appears as a
bell-shaped curve with a relatively sharp maximum. For any
 A.D. Vinogradov, V.G. Grivennikova / Biochimica et Biophysica Acta 1857 (2016) 863–871
Table 1
Specific NADH-oxidizing activities of mitochondrial or plasma membrane respiratory complex I (representative values) a
Activity (μmol/min per mg)
NADH oxidase NADH:quinone reductase
Eukaryotes
Bos taurus 0.7–2.0 [13–17] 0.3–1.6 [13,18,19]
Y. lipolytica 0.2 [25] 0.4 [25]
N. crassa 0.2–0.3 [27] 0.1–0.4 [27,28]
Prokaryotes
E. coli 0.4 [29] 0.4–3.1 [29,30]
P. denitrificans 0.8 [14,15,33,34] 0.8 [34]
R. capsulatus 0.5 [34] 0.5 [34]
a
Specific activities reported in the literature even for the same type of preparations are inherently variable due to somewhat different assay conditions. This is particularly true for data
where artificial electron acceptors were used. To the best of our knowledge, no study was reported where oxidase and acceptor reductase activities extrapolated to infinity concentrations
of the latter have been compared.
b
Extrapolated to infinity HAR concentration.
c
1 mM Ferri in the reaction mixture.
d
0.35 mM HAR in the reaction mixture.
oxidoreductase reaction, the enzyme turnover number or Vmax is mean-
ingful only if the activity determined at various concentration of an elec-
tron acceptor is extrapolated to its infinite concentration. Because of the
evidently non-hyperbolic NADH concentration–activity profile, such
extrapolation is somewhat ambiguous, and great precautions should
be taken if complex I catalytic activity is to be quantitated using Ferri
as the electron acceptor.
The simplest mechanistic explanation for the bell-shaped depen-
dence is that the reaction proceeds by a ping-pong mechanism, and
NADH (at high concentrations) binds to the active site of the reduced
enzyme, thus sterically protecting accessibility of the Ferri anion to
FMN buried in a deep cleft. This interpretation hardly agrees with the ki-
netics of NADH-HAR reductase: it proceeds according to a ternary com-
plex mechanism and shows no inhibition at high NADH concentration
[20,21,45]. Thus, the kinetic behavior makes HAR a convenient acceptor
for studies directed to quantitation of the enzyme turnover or its con-
tent (that is assumed to be proportional to Vmax) in various membrane
or solubilized preparations. The specific NADH dehydrogenating activity
of membrane-bound complex I in bovine heart SMP as extrapolated to
Vmax is at least by an order of magnitude higher than their NADH
oxidase corresponding to turnover of about 2500 s−1. This number de-
termines the lower limit for the rate of product release from the enzyme
active site: NAD+ dissociation is an inevitable step independent of the
particular site where any electron acceptor interacts with the enzyme.
Thus, at least for the mammalian enzyme, the half time of NAD+ release
should be less than 0.3 msec. This value is substantially lower than that
for the slow phase of N1b and N6b reduction (about 2 msec) in purified
E. coli complex I interpreted as a characteristic time for NAD+ dissocia-
tion [10,11].
The NADH:HAR reductase activity of SMP and purified bovine
heart complex I at low concentrations of the acceptor is strongly
(up to 10-fold) stimulated by ATP and other purine tri- and
diphosphonucleotides [45,46]. This effect was interpreted as evidence
for an allosteric nucleotide-binding site in the mammalian enzyme
[46] or, alternatively, as a change in the reaction pathways of the reac-
tion [45] in the presence of ATP, ADP, and ADP-ribose, competitive
inhibitors of the NADH/NAD+-binding site [47]. Kinetically, ATP acts
as a competitive (with NADH) activator, thus decreasing the apparent
Km for NADH [48]. Grivennikova et al. suggest that ATP bound at a puta-
tive allosteric site increases the midpoint potential of an enzyme com-
ponent that donates electrons to HAR [46]. Remarkably, no activating
effect of ATP was observed for prokaryotic Paracoccus denitrificans
membranes, whereas it was seen for eukaryotic Yarrowia lipolytica
SMP. This species-dependent activation by ATP hardly corroborates
with reaction pathway change induced by nucleotides, the mechanism
865
NADH:ferricyanide reductase NADH:HAR reductase NADH:APAD+ reductase
(Ferri 0.5 mM) (HAR 2 mM)
2.0–8.4 [20,21] 18.0b [22] 0.8 [24]
2.7–8.0 [20,21,23]
0.9–1.2 [25,26]
2.5 [28]
1.4–4.6 [29–31] 2.2d [32]
0.7 [20] 2.0 [20]
1.6–2.1c [33]
proposed by Birrell et al. [45]. Whether an allosteric ATP-binding site
presumably located at some accessory subunit [49] of eukaryotic com-
plexes has any physiological significance is an open question. Only the
HAR reductase activity of eukaryotic complex I is affected by ATP.
Recently, a striking observation relevant to HAR reductase activity
was reported by Varghese et al. [50]. A point mutation (A341V) in the
Y. lipolytica FMN-containing subunit eliminated NADH:HAR reductase
activity, leaving all other activities including paraquat reductase unaf-
fected [50]. This observation suggests a different reaction mechanism
for paraquat and HAR reduction and demonstrates that our current
knowledge on the reduction of the artificial electron acceptors is far
from complete.
5. Proton pumping activity
At present, the molecular mechanism of the redox-linked proton
translocation at coupling Site 1 remains a black box. Numerous earlier
Mitchellian direct transmembrane redox loop models should be
discarded in light of the structural arrangement of multiple redox com-
ponents within the enzyme structure. The ubiquinone reactive site
(iron-sulfur cluster N2) is located approximately 30 Å from the coupling
membrane plane [51,52]. The hydrophobic domain is composed of a
number of subunits including three transmembrane homologs of bacte-
rial Na+/H+ antiporters [53] (subunits L, M, and N according to E. coli
nomenclature) [2]. A simple and attractive possibility is that these
subunits operate as the proton-conductive channels required for the
pumping mechanism. For any model of coupling, the stoichiometric
coefficient, n in Eq. (1), should be defined. A consensus has been
reached that 4 protons are translocated per molecule of NADH oxidized
and ubiquinone reduced (2ē) [54,55,and references cited therein]. Here
we will briefly discuss several points that might cast a new perspective
on studies of the coupling mechanism.
(i) The basic structure of Thermus thermophilus complex I is remark-
ably similar to that of the E. coli and eukaryotic enzymes.
Menaquinone (E7m ~ −70 mV) serves as the electron acceptor in
T. thermophilus membranes [56], whereas ubiquinones
(E7m ~ +60 mV) are the acceptors in eukaryotic membranes. The
thermodynamic restrictions hardly permit the T. thermophilus
enzyme and ubiquinone-specific complexes I to operate by the
same proton pumping mechanism. To the best of our knowledge,
the value of n in Eq. (1) as applied to T. thermophilus is not known.
In fact, among prokaryotes the n value was experimentally evalu-
ated only for E. coli [57] and P. denitrificans [58,59]. Presumably,
the same number of proton-conducting subunits is present in
866 A.D. Vinogradov, V.G. Grivennikova / Biochimica et Biophysica Acta 1857 (2016) 863–871
prokaryotic and eukaryotic enzymes. The possibility cannot be
excluded that variable stoichiometry exists in the reaction
(Eq. 1), being controlled by some special regulatory mechanism
that turns “on” one, two, or three proton-translocating subunits
depending on particular physiological conditions (steady-state
p.m.f.). It does not seem unlikely that if a channel-forming subunit
is specialized for redox-linked proton translocation its functional
state (conformation) would be voltage-dependent, as it is well
established for a number of cation-specific transporters [60–62].
The structures of putative H+-translocating subunits N, L, and M
are different, thus their “voltage sensitivity” (if it exists) might
also be different, resulting in variable stoichiometry of the overall
reaction (1) at different p.m.f. values. It should be emphasized that
an attractive possibility of the direct participation of N, L, and M
subunits in redox-linked energy coupling remains just a proposal
having no strong experimental evidence, although “partial”
uncoupling has been observed for Yarrowia complex I: variants
in Yarrowia complex I deleted by two of the three subunits that
are assumed to be candidates for proton pumping translocate
protons with half of the stoichiometry observed for the intact
parental enzyme [63].
(ii) Counter cation translocation is required to measure proton
movement across the coupling membrane as a pH-change be-
cause the electrical component of the p.m.f. should be dissipated.
Unexpectedly, valinomycin did not affect the initial rate or the
maximal pH change if the proton translocation was initiated by
NADH-external quinone oxidoreduction in coupled SMP [54].
Strong stimulation of proton translocation by valinomycin, as ex-
pected, was seen under the same experimental conditions if the
overall NADH oxidase (all three coupling sites) was activated.
Formally, these data can be interpreted as to suggest that, in con-
trast to complex III and cytochrome oxidase, complex I operates
solely as the ΔpH generator. Work aimed to confirm or discard
such a possibility is in progress in our laboratory.
(iii) The NADH oxidase activity of bovine heart SMP is completely
(more than 90%) inhibited by specific N2-ubiquinone junction
site-directed inhibitors – rotenone and piericidin. When com-
plex I activity is assayed with externally added ubiquinone
homologs, a substantial rotenone-insensitive reaction is observed.
The trivial explanation of this phenomenon is that water-soluble
quinones accept electrons from some other than the natural
ubiquinone reactive site, just as other artificial acceptors do
(Ferri, HAR). However, studies on the proton pumping activity of
bovine heart SMP do not agree with this simple interpretation
[54]. Rotenone-inhibited particles catalyze the NADH:Q1 reductase
reaction coupled with proton translocation with the same stoi-
chiometry of 4 H+/2ē [54]. Moreover, the irreversibly stabilized
D-form of complex I, which is phenomenologically equivalent
to the rotenone-inhibited enzyme, also shows proton
translocating activity with the same stoichiometry [64]. These
unexpected observations are certainly relevant to the mecha-
nism of redox coupled proton translocation. The particular step
of the electron transfer from N2 to bulk ubiquinone blocked by
Table 2
Oxidase activities and superoxide generation by bovine heart coupled SMP (pH 8.0, 30 °C)a
Substrate-donor Oxidase activity, μmol/min per mg
– uncoupler (state 4)
1. Succinate (10 mM) 0.3
+ rotenone 0.3
2. Succinate (10 mM) + NADH (1 mM) 0.7
3. NADH (1 mM) 0.6
4. NADH (50 μM) 0.6
+ rotenone b0.002
a
Adapted from Ref. [17,74].
rotenone (and other multiple rotenone-like inhibitors) or by
transformation of the enzyme to its inactive D-form is not
known. It could well be the initial one-electron reduction of the
specifically tightly bound Q, or accepting the second electron to
form reduced bound quinol, or dismutation of two semiquinones
to form bound QH2, or electron exchange between bound QH2
and Q arriving from the bulk quinone pool. The key question of
what the particular step(s) is(are) coupled with proton pumping
remains to be answered.
6. Purified complex I
Only limited data on catalytic activities of purified complex I are
available, although several properties of the enzymes isolated from
mammalian [65–67] and yeast [26,68] mitochondria, or plasma mem-
branes of E. coli [30–32], P. denitrificans [33], and thermophilic bacteria
(T. thermophilus [69], Aquifex aeolicus [70], Rhodothermus marinus [71])
have been described. A requirement for added phospholipids for the
rotenone-sensitive activity of purified bovine heart complex I was dem-
onstrated many years ago [72]. More recently, the same phenomenon
was demonstrated for the detergent solubilized purified enzymes
from Bos taurus [66], Y. lipolytica [73], and E. coli [32]. The turnover
numbers of purified complex I are significantly lower than for complex
I-catalyzed NADH oxidase activity of the parent membranes. This is
explained either by an inadequate assay using externally added
“water-soluble” quinones (see above) or by the presence of some, yet
unidentified specific factor(s) required for full catalytic activity in the
natural membranes.
7. Complex I-mediated ROS production
Membrane-bound or purified complex I from all species studied so
far catalyze reactions (3) and/or (4). Quantitative and even qualitative
appraisals of the contribution of complex I to overall ROS production
by intact mitochondria are extremely difficult due to the presence of
multiple intramitochondrial enzymes involved in the formation (for
example, dihydrolipoyl dehydrogenase) and utilization of superoxide
(superoxide dismutases) and hydrogen peroxide (thioredoxins, gluta-
thione peroxidase, catalase). Here we discuss only the data obtained
for systems where ROS production can be unambiguously attributed
to the membrane-bound or purified complex I. NADH or succinate
oxidation by tightly coupled bovine heart SMP is accompanied by super-
oxide production at the rate of about 1 nmol/min per mg of protein
corresponding to about 0.2–0.3% of the total oxygen consumption. The
contribution of particular respiratory complexes to the total superoxide
production can be quantitatively evaluated as shown in Table 2. The
highest production, which is inhibited by rotenone and uncouplers, is
seen during coupled succinate oxidation, thus suggesting that about
70% of one-electron oxygen reduction proceeds via energy-dependent
reverse electron transfer (reversal of reaction (1)). The residual
rotenone-insensitive generation corresponds to the combined activities
of complexes II and III. It was somehow surprising that the rate of gen-
eration during more rapid coupled NADH oxidation is about three-fold
Generation of O•2 , nmol/min per mg
+ uncoupler (state 3) – uncoupler (state 4) + uncoupler (state 3)
0.9 1.0 0.1
– 0.3 –
– 0.3 –
1.9 0.4 0.1
1.8 1.0 0.9
b0.002 1.4 –
 A.D. Vinogradov, V.G. Grivennikova / Biochimica et Biophysica Acta 1857 (2016) 863–871
lower than the succinate-supported reaction. Moreover, the addition of
NADH (1 mM) to a sample where complex I produces superoxide via
succinate-supported reverse electron transfer inhibits the reaction.
The superoxide production increases and reaches the level of the
succinate-supported rate at low concentration of NADH. The depen-
dences of superoxide production and ferricyanide reductase activity of
complex I on NADH concentration are almost the same: both appear
as bell-shaped curves with a maximum at about 50 μM [17,75]. The
simplest interpretation of bell-shaped dependence is that oxygen
reduction proceeds via a ping-pong mechanism, and the accessibility
of a one-electron donating site for oxygen is restricted in the reduced
enzyme–NADH complexes. Other possible interpretations, such as the
presence of two NADH/NAD+-binding sites in mammalian complex I,
have been discussed [17]. Superoxide production at low (optimal)
concentration of NADH is activated by rotenone. At very low NADH
concentrations, purified bovine heart complex I produces ROS mostly
(90%) as superoxide [18], whereas purified E. coli enzyme generates
ROS as hydrogen peroxide [76]. Recently, membrane-bound complex I
(SMP) as well as the purified enzyme were shown to generate both
hydrogen peroxide and superoxide [75,77]. The partitioning between
the products depends on NADH concentration as shown in Fig. 1. The
superoxide production reaches a maximum at 10–50 μM NADH and
gradually decreases in the millimolar range of the substrate [17,75].
The apparent KNADH
m as determined from the linear double-reciprocal
plot for the ascending part of the superoxide production titration
curve is as low as about 0.5 μM [75]. As noted above (see 4. Artificial
electron acceptors section), the apparent Km value for the substrate-
donor in the reaction catalyzed by any oxidoreductase depends on the
redox potential gap between the primary electron acceptor (FMN for
complex I) and the component that reacts with an acceptor. The very
low KNADH
m for superoxide production [18,75] indicates that a compo-
nent immediately reacting with oxygen has a substantially higher
redox potential than FMN. The iron-sulfur cluster N2 might serve as a
one-electron donor for oxygen reduction as it has been proposed by
Genova et al. [78]. This cluster is located close to the ubiquinone-
binding site in a funnel-like area at the distance of 25–30 Å from the
membrane plane [51,52]. All other iron-sulfur centers are well insulated
by the protein environment. A direct approach to exclude cluster N2 as
the site of one-electron oxygen reduction was used by Galkin and
Brandt [25]. They found that a variant form of Y. lipolytica isolated
complex I (R141M) that showed no EPR-detectable center N2 produced
superoxide with the same rate (96%) as did the enzyme from the wild
strain. They normalized their data to NADH:HAR reductase activities
Fig. 1. Partitioning between superoxide and hydrogen peroxide as the products of complex I-mediated ROS production. Red and blue bars are superoxide and hydrogen peroxide,
respectively. The lines on left diagram are drawn to emphasize complex kinetics of the NADH concentration dependence. Adapted from Ref. [75].
867
of the wild and mutated strains. Normalization of their data to the
“natural” activities shows 60% quinone reductase. This agrees with
data reported previously by the same group [79] and originally
interpreted as to suggest that the mutants lacking N2 were still capable
of ubiquinone reduction at near normal rates, a possibility that seems
hardly probable. Rather, the R141M mutation changes the spectral
and redox properties of N2, as later demonstrated by Zwicker et al. for
the H226M mutant, showing 80 mV negative shift of N2 midpoint
potential, absence of its pH-dependence, and still being capable of
redox-linked proton translocation with unaltered stoichiometry [80].
We believe that exclusion of iron-sulfur cluster N2 as a possible site of
superoxide production should wait for more experimental verification.
Hydrogen peroxide formation depends on NADH concentration quite
differently. At low substrate concentrations (up to 3 μM), no production
is seen (Fig. 1), and the rate reaches a constant value at higher (up to
millimolar) range of NADH concentration, where its relative contribu-
tion to the overall ROS production is about 60% [75].
Complex I-catalyzed ROS production is inhibited by μmolar NAD+
concentrations [17]. Because of the very low activity of complex I in
ROS generation as compared with major oxidase or NADH:artificial
acceptor reductase reactions, it is safe to assume that all redox compo-
nents of the enzyme are in equilibrium with the NAD+/NADH couple
during the steady-state reaction (the contribution of the kinetic term
to the apparent Km is negligible). Thus, the dependence of ROS produc-
tion on NAD+/NADH ratio is indicative of the midpoint redox potential
of the component(s) reacting with oxygen. The NAD+/NADH ratios
reported in the literature for half-maximal ROS production by complex
I are greatly variable (from 0.01 up to 7.0) [8,18,81–83]. Titration of
complex I by the NAD+/NADH couple is not a true redox titration
because the relative binding affinities of the reduced and oxidized
enzyme to NAD+ and NADH [84] can significantly contribute to the
apparent (NAD+/NADH)0,5 ratio required for ROS production. This
ratio is dependent on the total pool nucleotide concentration. For exam-
ple, it decreases from 0.2 to 0.05 for total ROS production by SMP when
total NAD+ plus NADH concentration increases from 50 to 500 μM [83].
At optimal nucleotide concentration (50 μM), the rate of Н2О2
production fits the Nernst equation for a two-electron reaction with
midpoint redox potential of − 350 mV ((NAD+/NADH)0.5 = 0.13)
[75], a value close to the midpoint potential of FMN in complex I
(− 370 mV at рН 8.0) [85]. The same dependence for superoxide
production does not fit the Nernst equation, and half-maximal activity
is observed at a significantly higher NAD+/NADH ratio (0.33) [75].
Superoxide production does proceed even when the nucleotide pool is
868 A.D. Vinogradov, V.G. Grivennikova / Biochimica et Biophysica Acta 1857 (2016) 863–871
90% oxidized, i.e. at much more positive potential than that of the
FMNH−/FMN couple. The dependence of superoxide production by
purified complex I on the NAD+/NADH ratio measured by Kussmaul
and Hirst fits the theoretical curve of the two-electron titration of the
FMNH−/FMN couple with midpoint potential of −360 mV [18].
A single amino acid replacement (E95, a glutamate residue located
near FMN) by glutamine in E. coli complex I results in a 15-fold increase
in the rate of NADH-dependent hydrogen peroxide production [86]. The
authors proposed the existence of closed and open states of the
nucleotide-binding site in the FMN-containing subunit with different
reactivity (accessibility) of flavin to oxygen, a model where the E95
residue keeps the site in the closed state. This observation seems to be
relevant to the well-known stimulatory effects of guanidine on the
ferricyanide reductase [87] and superoxide producing activities [75] of
mammalian complex I. The electrostatic interaction between the large
guanidinium cation and the glutamate anion might stabilize the open
state. Interestingly, this negatively charged/neutral residue replacement
was accompanied by about 5-fold decrease in HAR reductase activity
[86].
The well-known “burst” of ROS upon anaerobic–aerobic state transi-
tion, such as organ reperfusion after ischemia, is a phenomenon that
might be relevant to the unusual hysteretic kinetics of complex I. If no
oxidized ubiquinone is available, the enzyme is transformed to the so-
called deactivated state where electron transfer from N2 to ubiquinone
is blocked (see Refs. [35,88] for reviews). The active (A) state-to-
deactivated state (D) transition has been detected for isolated complex
I [89], SMP [13], intact heart mitochondria [90], and in ex vivo studies of
perfused hearts [91]. In terms of catalytic activities, deactivation of
complex I is equivalent to its inhibition by rotenone, which is known
to increase ROS production. The back transformation of the D- to A-
form is a slow process that is inhibited by free fatty acids and divalent
metal cations [92–94]. Taken together, these data suggest the following
scenario of the normal state → ischemia → reperfusion transition.
Negligible complex I-mediated ROS production under the initial normal
state occurs. It stops when no oxygen is available, and complex I become
deactivated. A sudden increase in oxygen to the normal level would
result in a burst of ROS because the deactivated enzyme will be directly
oxidized by oxygen, not by ubiquinone. Increased ROS production is
expected for the time needed for the slow D-to-A transformation and
restoration of the normal ubiquinone reductase activity.
When discussing forward and reverse electron transfer in complex
I-mediated ROS production, a note should be made concerning the use
of succinate as the respiratory substrate. The respiratory chain of
coupled mitochondria or SMP oxidizing externally added succinate
cannot be considered as a model of any physiologically conceivable
situation. Succinate, an intermediate of the Krebs cycle, is produced
and utilized in the mitochondrial matrix, providing one fifth of the
reducing equivalents during complete oxidation of pyruvate. No other
quantitatively significant cytoplasmic sources of succinate exists in
aerobic metabolic pathways (α-oxoglutarate-dependent proline
hydroxylation [95] and succinic semialdehyde transformation [96] are
minor contributors to the total respiratory activity of mammalian
tissues). This by no means excludes reverse electron transfer as a
pathway for ROS production under some pathophysiological condi-
tions, such as anoxia, where a significant amount of intramitochondrial
succinate is accumulated [97]. Also, ubiquinol, the actual substrate for
the reversal, is produced in several mitochondrial metabolic pathways
such as fatty acid β-oxidation or oxidation of α-glycerophosphate.
As discussed above, ROS-producing activity of complex I depends on
the matrix redox potential, i.e. NAD+/NADH ratio [83]. The total amount
of pyridine nucleotides in heart mitochondria can be approximated as
4–7 nmol per mg of protein [98–100], these values corresponding
to 4–7 millimolar concentration. It can thus be assumed that the
nucleotide-binding sites of complex I are always saturated. However,
the concentrations of free NAD+/NADH nucleotides are not known.
The physiologically relevant NAD+/NADH ratio in liver mitochondria
was approximated as about 8 [101]. In isolated cardiomyocytes respir-
ing on 10 mM glucose [102] or in heart perfused by 10 mM glucose,
this ratio is closed to 1 [103]. Thus, the apparent redox potential in the
matrix is significantly higher than that conventionally used in the
model experiments on ROS production. It appears that in vivo complex
I-mediated ROS production is many-fold lower than measured under
experimentally “optimal” conditions.
In recent years, the term “p.m.f.(or energy)-dependent ROS-
production” is frequently used in the literature, thus implicitly assuming
that the membrane energization and deenergization increases and de-
creases ROS production, respectively. We believe that this terminology
can be misinterpreted by a reader who is only superficially familiar
with mitochondrial bioenergetics. The production of ROS depends on
the amount (concentration) of reactive sites accessible to oxygen. The
steady-state NADH/NAD+ ratio, indeed, is decreased when respiration
is activated by ADP (small drop of р.m.f.) or by uncoupler (complete
dissipation of р.m.f.). However, it seems important to emphasize
that р.m.f. itself does not affect the generation rate; it influences redox
state of the oxygen reactive sites. No correlation between membrane
energization and ROS production exists; for example, rotenone stimu-
lates mitochondrial ROS production, whereas it completely deenergizes
membranes. It might appear that this note is a matter of semantics;
however, the expression “energy-dependent ROS generation” (by
complex I) is misleading.
8. Notes on physiological and pathophysiological significance of
complex I and other mitochondrial enzyme-mediated
ROS production
Mitochondria contain a number of enzymes capable of ROS genera-
tion. Their relative contributions along with complex I to the level of
intracellular hydrogen peroxide are expected to be species- and
tissue-dependent. To the best of our knowledge, only one study
published more than 40 years ago was designed for very approximate
quantitation of the relative contribution of various enzyme systems to
H2O2 production in rat liver [104]. According to that report, about 15%
of intracellular hydrogen peroxide is produced by mitochondria. The
widespread opinion that mitochondria are the major source of ROS,
apparently because mitochondria are indeed the major consumers of
oxygen, is exemplified by citing D. Harman who wrote, “Mitochondria
would be expected to be particularly subject to free radical induced
change as over 90% of oxygen utilized by mammals takes place in
them” [105]. This and other similar statements circulating in the litera-
ture are somehow misleading. Mitochondrial respiration inevitably
decreases the local concentration of oxygen in the vicinity of the centers
potentially capable of ROS production, which results in a decrease in the
generation rate.
The increase in atmospheric oxygen in the history of the Earth has
led to great improvement in the energetics of life. On the other hand,
it also necessitates protection against undesired deteriorating oxidative
reactions. One strategy to solve the problem is to protect potentially
reactive centers (flavins and/or iron-sulfur clusters) by the specific
arrangement of the protein structure, including attachment of addition-
al subunits that have no other function than to avoid their oxygen
reactivity. Numerous experimental data show that almost all flavo-
and iron-sulfur proteins including those knowingly not operating
functionally as oxidases do react with oxygen, thus producing either su-
peroxide or hydrogen peroxide. In other words, their specific protection
is not perfect. This is compensated by the widespread presence of SODs,
peroxidases, and catalase. Does mitochondrial ROS production have any
physiological function? Numerous reports have recently appeared in
the literature where signaling cascade reactions with the participation
of hydrogen peroxide are suggested and discussed (see for example re-
view [106]). Note should be made that signaling molecules are defined,
in contrast to metabolites, as those that bind to a receptor and induce its
structural change without their chemical transformation. The structural
 A.D. Vinogradov, V.G. Grivennikova / Biochimica et Biophysica Acta 1857 (2016) 863–871
change of a receptor then leads to activation (or inhibition) of some
metabolic pathways. Many hormones, second messengers (c-AMP,
Са2 +), and protein factors are true signaling molecules. If expanded
(unjustified in our opinion), the meaning of “signaling molecule”
could be applied to any metabolite. In most schemes, the mechanism
of hydrogen peroxide “signaling” is postulated as a trivial nonenzymatic
oxidation of deprotonated sulfhydryl groups of the “target” that results
in their catalytic (or further signaling) activities. It appears that if a reg-
ulatory signaling cascade does exist, all its steps must be enzymatically
catalyzed, as is evident for the classic cascade regulation of glycogen
metabolism.
Another aspect worth brief discussion relevant to the physiological
significance of mitochondrial ROS production is its dependence on
oxygen concentration. In contrast to cytochrome oxidase, a reaction
which has an apparent Km for oxygen significantly lower than the
physiologically conceivable concentration (about 5-fold less than that
in air-saturated aqueous solutions at normal pressure [107]), the specif-
ic activity of “major” respiratory chain-linked ROS generator, complex I
[74,108,109], is simply proportional to oxygen concentration. Hyperbol-
ic or other complex kinetics of complex I-mediated ROS production
might be expected if it does have physiological function. On the other
hand, simple proportionality of ROS formation to oxygen concentration
might serve as an ideal oxygen sensing mechanism.
The enzymes utilizing superoxide and hydrogen peroxide are
frequently considered as an “antioxidant defense system”, whereas
generation of ROS is considered as an evolutionarily unfavorable
leakage reaction (except for NAD(P)H oxidases, which perform a clearly
defined function). An alternative view is that hydrogen peroxide is a
normal metabolic intermediate that fulfills some important not yet
clear function(s). If a “leakage” hypothesis and “antioxidant defense
system” are to be accepted, many more or less experimentally justified
efforts to improve the defense (antioxidant drugs, particularly those
mitochondrially targeted [110,111]) are certainly warranted. On the
other hand, if the alternative view is correct, the use of so-called antiox-
idant drugs could result in unfavorable consequences. When discussing
physiological and pathophysiological “oxidative stress”, one should
keep in mind metabolic effects of the “reductive stress” that can be
induced by the antioxidants [112]. Obvious expected consequences of
“overreduction” are: 1) abnormal anabolic activity including malignant
growth; 2) an increase in fatty acid and fat biosynthesis; 3) disorder
of amino acid transport systems (participation of glutathione in the
γ-glutamate cycle [113]); 4) a decrease of normal insulin level due
to an increase in the insulin:glutathione transhydrogenase reaction
[114,115]. Last, not the least consequence of reductive stress is a possi-
ble paradoxical activation of ROS production.
Conflict of Interest
We declare no conflict of interest.
Acknowledgements
We are indebted to Dr. J. Hirst for providing us data (Ref. [50]) before
publication. We are grateful to anonymous Reviewer for his (her) kind
linguistic corrections of our manuscript. The experimental works done
in our laboratory were supported by Russian Foundation for Fundamen-
tal Research Grant 14-04-00279 to A.D.V. and Grant 15-04-02957 to
V.G.G.
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