Meunier, Brigitte

Rappaport, Fabrice

Trouillard, Martin

Elsevier - Publisher Connector 

The alternative oxidase is a terminal respiratory chain protein
found in plants, fungi and some parasites that still remains physically
uncharacterised. Current model of the AOX, predicts that the enzyme
is a monotopic integral membrane protein associating with one
leaﬂet of the lipid bilayer. Although it is generally accepted that AOX
is a non-haem diiron carboxylate protein in which the metal atoms
are ligated by amino acid residues that all reside within a 4-helix
bundle there is little biophysical experimental evidence in favour of
this notion. We present EPR evidence from parallel mode experi-
ments which reveal signals at approximately g=16 in both puriﬁed
plant alternative oxidase protein (g=16.9), isolated plant mitochon-
drial membranes (g=16.1), and in the trypanosomal AOX expressed
in Escherichia coli membranes (g=16.4). Of particular importance is
the ﬁnding that such signals disappear in the presence of inhibitors
of the AOX. Such signals are indicative of a dicarboxylate diiron
centre at the active site of the enzyme. To our knowledge these data
represent the ﬁrst EPR signals from AOX present in its native
environment.
This work is supported by a grant from the BBSRC.
doi:10.1016/j.bbabio.2008.05.358
S13.15 Far infrared spectroscopic studies on hydrogen bonding
features in proteins from the respiratory chain
Petra Hellwig, Youssef El Khoury, Ruth Hielscher
Institut de Chimie, UMR 7177, Université Louis Pasteur, Strasbourg,
France
E-mail: hellwig@chimie.u-strasbg.fr
In order to understand the molecular basis of energy transduction,
we have extensive interest in experiments which reveal at the mole-
cular level how protons are drawn through proteins. It is crucial to
determine the structural, dynamic and energetic requirements for the
proton transferring groups in the proton pumping enzymes and the
cofactor sites that rule them. A signiﬁcant part of the proton con-
duction is made by channels that orient speciﬁcally bound water
molecules. These water molecules can be monitored by X ray crystal-
lography. However, high resolution structures of membrane proteins
are difﬁcult to obtain. Interestingly, water molecules and their hydro-
gen bonding interactions are expected to contribute in the far infrared
spectral range (b400 cm−1). Experiments at synchrotron far infrared
beamlines have been made with large membrane proteins from the
respiratory chain and their models, clearly demonstrating the pre-
sence of this hydrogen bonding signature signal and, importantly, that
it can be manipulated.
doi:10.1016/j.bbabio.2008.05.359
S13.16 The role of the cross-linked Tyr in the catalytic cycle of
cytochrome c oxidase
Elena A. Gorbikova, Ilya Belevich, Mårten Wikström,
Michael I. Verkhovsky
Helsinki Bioenergetics Group, Institute of Biotechnology, University of
Helsinki, Finland
E-mail: elena.gorbikova@helsinki.ﬁ
Tyr-280 in cytochrome c oxidase (CcO) from Paracoccus denitriﬁ-
cans undergoes posttranslational modiﬁcation that results in a cova-
lent cross-link to histidine residue 276 that is a ligand of CuB. The Tyr-
280 has been predicted to be a proton (or a proton and electron) donor
for oxygen activation. The aim of our studies was to test the role of the
cross-linked Tyr-280 as a proton donor for oxygen reduction and to
resolve the stage in catalysis when it becomes reprotonated. The
combination of three time-resolved techniques: visible spectroscopy,
electrometry and FTIR spectroscopy were used. Electrometry showed
positive charge transfer across a distance consistent with that from
Tyr-280 to oxygen in the reaction site, and FTIR spectroscopy revealed
a band at 1308 cm−1 that was assigned to deprotonated Tyr-280. The
obtained results strongly indicate that Tyr-280 provides a proton for
oxygen activation in fully reduced enzyme. According to the FTIR
spectra it is fully deprotonated at the next step after O–O bond
splitting (ferryl) and partly reprotonated in the fully-oxidized state,
depending on pH. We showed that full reprotonation of Tyr-280 takes
place in the one-electron reduced state when CuB becomes reduced.
doi:10.1016/j.bbabio.2008.05.360
S13.17 Reverse redox loop enzymes for driving endergonic
reactions in bacterial electron transport
Gottfried Unden
Institute for Microbiology andWine Research, University of Mainz, Germany
E-mail: unden@uni-mainz.de
The study deals with enzymes that drive endergonic reactions of
electron transport by a reverse redox loop mechanism and the proton
potential. Succinate dehydrogenase (succinate: menaquinone (MK) re-
ductase) from MK-containing bacteria catalyzes an endergonic reaction
(ΔE0′=+110mV). The soluble subunits (SdhAB) of the enzyme are similar
in composition and function to succinate: ubiquinone reductase from
mitochondria and ubiquinone containing bacteria. The quinone reactive
subunit SdhC is a membrane integral diheme protein which allows
electron transfer from the cytoplasmic to the extracellular side of the
membrane. Function of succinate: MK reduction depends on the
membrane potential and is inhibited by protonophores and ionophores.
Bioenergetic studies and membrane topology of SdhC suggest that the
active site for MK reduction is close the extracellular side of the mem-
brane, resulting in the uptake of H+ from the outside, whereas succinate
oxidation and the accompanying proton release take place in the cyto-
plasm. In this way the enzyme consumes a proton potential and uses a
reverse redox loop (2H+/2e) for driving the redox reaction. The same type
of enzyme is found in anaerobic electron transport of sulphate reducing
bacteria. Database screening demonstrated that a reverse redox loop
mechanism is predicted for other bacterial respiratory enzymes catalyz-
ing endergonic reactions. Reverse redox loop enzymes therefore are of
general signiﬁcance for driving endergonic redox reactions in bacteria.
doi:10.1016/j.bbabio.2008.05.361
S13.18 Light-activating the respiratory chain: Toward the
time-resolved studies of the electron transfer chain in vivo
Martin Trouillarda, Brigitte Meunierb, Fabrice Rappaporta
aInstitut de Biologie Physico-Chimique, CNRS-Univ. Pierre et Marie Curie,
Paris, France
bCentre de Génétique Moléculaire, CNRS-Univ Pierre et Marie Curie, Gif
sur Yvette, France
E-mail: Fabrice.Rappaport@ibpc.fr
Although the structure and function of most of the individual
complexes involved in the respiratory chain are known with great
details, the accurate understanding of the function of the chain as a
whole is still missing. Yet, an increasing number of evidence suggests
it may signiﬁcantly differ from the simple combination of the func-
tions of the individual complexes. Indeed, the supramecular associa-
tion of complexes within identiﬁed super-complexes or the ultra-
structure of the inner membranewhich possibly restricts the diffusion
S92 ABSTRACTS / Biochimica et Biophysica Acta 1777 (2008) S2–S111
of soluble electron carriers may have signiﬁcant functional conse-
quences since they would introduce macroscopic heterogeneities in
the chain. The expected signature of such heterogeneities is essen-
tially kinetic calling for new methods allowing the time-resolved
analysis of the electron transfer sequence associated with the mito-
chondrial respiration in vivo. We are currently developing such a
method based on the ﬂash-induced photolysis of CO in the presence of
O2, as fruitfully conducted for the mechanistic dissection of complex
IV. The advantages and pitfalls of the approach will be described and
preliminary results will be presented and discussed.
doi:10.1016/j.bbabio.2008.05.362
S13.19 A role for sodium ions in the respiratory chain of
Rhodothermus marinus
Andreia S. Fernandesa, Ana P. Batistaa, Ricardo O. Louroa,
Miguel Teixeiraa, Julia Steuberb, Manuela M. Pereiraa
aInstituto de Tecnologia Química e Biológica, Universidade Nova de
Lisboa, Oeiras, Portugal
bDepartment of Biochemistry, University of Zurich, Switzerland
E-mail: andreiaf@itqb.unl.pt
Rhodothermus marinus is a strictly aerobic and thermohalophilic
organism isolated from submarine hot springs in Iceland and Açores.
Its respiratory complexes have been studied and include a complex I
(NADH:menaquinone oxidoreductase), a complex II (succinate:
menaquinone oxidoreductase), a novel complex III and at least
three different dioxygen reductases. Since it is a halophilic organism,
and because a proton/sodium antiporter gene was found among its
complex I genes, a possible role of sodium ions in R. marinus
bioenergetics was investigated. We prepared inside-out vesicles from
R. marinus and demonstrated that the vesicles maintained an
electrochemical K+ potential imposed by K+/valinomycin. The
membrane potential driven by the addition of substrates NADH
and succinate to R. marinus membrane vesicles was followed using
the sensitive dye oxonol V. It was observed that the NADH-driven
membrane potential was sodium ion dependent, while the build-up
of a membrane potential during succinate oxidation seems not to be
inﬂuenced by Na+. To investigate the mode of Na+ transport during
NADH respiration, 23Na in membrane vesicles was followed by NMR
spectroscopy.
doi:10.1016/j.bbabio.2008.05.363
S13.20 Structural characterization of respiratory complexes in
potato tuber
Jelle B. Bultemaa, RomanKouřila, Hans-Peter Braunb, Egbert J. Boekemaa
aDepartment of Biophysical Chemistry, University of Groningen, The
Netherlands
bInstitute for Plant Genetics, Faculty of natural Sciences, University of
Hannover, Germany
E-mail: j.b.bultema@rug.nl
The aim of this study was to determine the structures of potato
respiratory supercomplexes. Therefore, mitochondrial inner mem-
branes from potato tuber cells were isolated, mildly solubilized with
digitonin and the respiratory supercomplexes were separated by
sucrose gradient ultra centrifugation. Finally, content of sucrose gra-
dient fractions was inspected with Blue Native electrophoresis and
electron microscopy. Single particle analysis of our data revealed
several projection maps of complex I, monomeric and dimeric ATP
synthase, supercomplex III2+IV1, supercomplex I+III2 and larger
unassigned supercomplexes. In some side-view projection maps of
complex I the structure of carbonic anhydrase shows its trimeric
features. Furthermore, one projectionmap revealed an extra unknown
density at the intermembrane side of complex I. Top-view projection
maps of I+III2 supercomplex showed similar features found in other
plant species including the presence of carbonic anhydrase. Besides
the top-views, two different side-views and several angular views of
the I+III2 supercomplex were revealed which allowed a better
assignment of interaction between complex I and III2 within the
supercomplex. The side-views of the largest supercomplex most likely
do not represent the structure of the I+III2+IV1 supercomplex, also
known as the respirasome. The largest particles represent probably a
supercomplex composed of two copies of complex I and one copy of
complex III2.
doi:10.1016/j.bbabio.2008.05.364
S13.21 Production, characterization, and determination of the real
catalytic properties of the ‘succinate dehydrogenase’ from
Wolinella succinogenes
Hanno D. Juhnkea, Heiko Hiltschera, Hamid R. Nasirib, Harald Schwalbeb,
C. Roy D. Lancastera,c
aCluster of Excellence “Macromolecular Complexes”, Max Planck Institute
of Biophysics, Department of Molecular Membrane Biology, Frankfurt am
Main, Germany
bCluster of Excellence “Macromolecular Complexes”, Institut für
Organische Chemie und Chemische Biologie, Center for Biomolecular
Magnetic Resonance, Johann Wolfgang Goethe-Universität, Frankfurt am
Main, Germany
cChair of Structural Biology, Faculty of Medicine, Saarland University,
Homburg (Saar), Germany
E-mail: Roy.Lancaster@structural-biology.eu
The genomes from both of the ε-proteobacteria Wolinella
succinogenes and Campylobacter jejuni contain operons (sdhABE
operons) that encode for hitherto uncharacterized enzyme com-
plexes annotated as ‘non-classical’ succinate dehydrogenases. In the
framework of a functional genomics project, a genetic system has
been established for the homologous (over-)production and manip-
ulation of the SdhABE complex from W. succinogenes. The catalytic
properties of the puriﬁed enzyme were examined using various
possible electron donor and acceptor substrates. Strikingly, for the
SdhABE complex annotated as a ‘succinate dehydrogenase’, no
succinate oxidation activity could be detected, neither with DCPIP,
nor with methylene blue, nor with the high-potential quinone EQ-0
as electron donor. Although the complex catalyzes fumarate
reduction with the menaquinol-6 analog 2,3-dimethyl-1,4-naphtho-
quinol (DMNH2) the activities are very low. In addition to men-
aquinol-6, membranes of C. jejuni and of W. succinogenes contain a
second quinol, 8-methylmenaquinol-6 (8-MMKH2-6). Supplying an
8-MMKH2-6 analog as a substrate increased speciﬁc quinol:fumarate
reductase activity by about one order of magnitude. Furthermore,
studies on variant enzymes demonstrated that the hydrophilic
subunits of the complex are, in contrast to all other members of
the superfamily, exported into the periplasm via the tat-pathway.
Our studies reveal that the putative succinate dehydrogenase is in
fact a novel periplasmic 8-methylmenaquinol:fumarate reductase
with no detectable succinate dehydrogenase activity. These results
provide an explanation for apparently puzzling previously pub-
lished observations on the regulation of the C. jejuni sdhABE
operon.
doi:10.1016/j.bbabio.2008.05.365
S93ABSTRACTS / Biochimica et Biophysica Acta 1777 (2008) S2–S111


S13.18 Light-activating the respiratory chain: Toward the time-resolved studies of the electron transfer chain in vivo 

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S13.18 Light-activating the respiratory chain: Toward the time-resolved studies of the electron transfer chain in vivo

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