Daldal, Fevzi

Dong-Woo, Lee

El Khoury, Youssef

Hellwig, Petra

Elsevier - Publisher Connector 

The Zn2+ inhibitive effects on the cytochrome bc1 complex from Rhodobacter capsulatus as revealed by the FTIR difference spectroscopy 

to narrow down the sites conferring the functional specificity of
ACPM1 and ACPM2.
doi:10.1016/j.bbabio.2010.04.055
1P.8 Electron transfer in Eschericha coli respiratory complex I
Katerina Dörner, Klaudia Morina, Marius T. Schulte, Annemarie Horn,
Thorsten Friedrich
Institut für Organische Chemie und Biochemie,
Albert-Ludwigs-Universität Freiburg, Germany
E-mail: katerina.doerner@ocbc.uni-freiburg.de
The respiratory complex I couples the transfer of electrons from
NADH to ubiquinone with the translocation of protons across the
membrane. One flavin mononucleotide and seven iron–sulfur (Fe/S)-
clusters build an electron transfer path from the NADH binding site to
the ubiquinone binding site. The role of the protein in electron transfer
was recently questioned. It is generally accepted that electron transfer
takes place via tunnelling, that means through-space electron transfer
with the Fe/S-clusters as the only centers of excess electrons. Recently it
was proposed that electron transfer is performed via a hopping
mechanism involving conserved aromatic amino acids between the
Fe/S-clusters as stepping stones. The calculated transfer rates of the
individual intramolecular electron transfer steps are in agreement with
the experimental determined total transfer rate of 170±10 s−1. To
examine this hypothesis several aromatic amino acids in complex I from
E. coli were mutated by λ-Red recombineering. The mutations led to a
dramatic decrease in the NADH:oxidase activity of the mutant
membranes, when the aromatic amino acid was replaced by a non-
aromatic amino acid. Conservative substitution with another aromatic
amino acid had a mild effect on the activity. The mutations showed the
same effect on the kcat of the NADH:ubiquinone oxidoreductase activity
of the isolated complex I variants.
References
[1] L.A. Sazanov, P. Hinchliffe, Science 311 (2006) 1430–1436.
[2] C.C. Moser, et al., Biochim. Biophys. Acta 1757 (2006) 1096–1109.
[3] C. Wittekindt, et al., J. Am. Chem. Soc. 131 (2009) 8134–8140.
doi:10.1016/j.bbabio.2010.04.056
1P.9 On complex I of Neurospora crassa — Role of the chaperone
B17.2L
Margarida Duarte1, Bruno Pereira1, Arnaldo Videira1,2
1Instituto de Biologia Molecular e Celular (IBMC), Portugal
2Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Porto, Portugal
E-mail:mduarte@ibmc.up.pt
Complex I or the NADH:ubiquinone oxidoreductase deficiency is a
common cause of mitochondrial oxidative phosphorylation diseases,
including Leigh syndrome. Mutations in nuclear genes encoding
structural subunits or assembly factors of complex I have been
increasingly identified as the cause of the diseases. One such assembly
factor is a paralogue (B17.2L) of the B17.2 structural subunit of the
enzyme, but the mechanism by which it exerts its function is still
unclear. To better understand the requirement of B17.2L for complex I
assembly we analyzed the mitochondrial respiratory chain of Neuro-
spora crassa strains using one and two-dimensional blue-native PAGE.
The results obtained indicate that disruption of both Neurospora genes,
the one encoding the structural subunit (nuo-13.4) and its paralogue
(nuo-13.4L), has no effect on the assembly or activity of complex I.
Moreover, an anti-13.4L antibody does not recognize the holoenzyme
but specifically associates with subassemblies present in several
complex I mutant strains. The analyses performed revealed that the
13.4L protein associates with different subassemblies in different
complex I mutants. Our results indicate that the 13.4L protein is a
molecular chaperone involved in the assembly of complex I in N. crassa,
probably stabilizing a subcomplex of assembly containing the mem-
brane arm and the connecting part. Furthermore, dissociation of the
13.4L protein seems to occur only upon full complex I assembly,
suggesting that the assembly factor functions as a sensor of integrity.
doi:10.1016/j.bbabio.2010.04.057
1P.10 Crystallisation of the membrane domain of complex I from
Escherichia coli
Rouslan G. Efremov, Leonid A. Sazanov
Medical Research Council, Mitochondrial Biology Unit, Wellcome Trust/
MRC Building, Hills Road, Cambridge, CB2 0XY, UK
E-mail: rge@mrc-mbu.cam.ac.uk
Complex I is the first enzyme of the respiratory chain and plays a
central role in cellular energy production, coupling electron transfer
between NADH and quinone to proton translocation. The enzyme is
L-shaped, consisting of peripheral and membrane arms. The structure
of peripheral arm was previously determined, however the complete
structure and the coupling mechanism of this large molecular
machine are currently unknown. To determine the structure of the
membrane domain of complex I form E. coli, we have developed an
effective procedure for its separation from the hydrophilic domain.
Isolated membrane domain was crystallised. Initially poor X-ray
diffraction properties were improved after extensive optimisation of
crystallisation conditions. After a broad screening of conditions for
heavy atom derivatisation, MIRAS data were collected. The prelimin-
ary findings from crystallographic data will be discussed.
doi:10.1016/j.bbabio.2010.04.058
1P.11 The Zn2+ inhibitive effects on the cytochrome bc1 complex
from Rhodobacter capsulatus as revealed by the FTIR difference
spectroscopy
Youssef El Khoury1, Lee Dong-Woo2, Fevzi Daldal2, Petra Hellwig1
1Laboratoire de spectroscopie vibrationnelle et électrochimie des
biomolecules, Institut de chimie, UMR 7177, UDS, France
2Department of Biology, Plant Science Institute,
University of Pennsylvania, Philadelphia, USA
E-mail: y.elkhoury@unistra.fr
The cytochrome bc1 complex is the third enzyme in the respiratory
chain and couples the redox reactions to the proton translocation across
themembrane. It iswidely accepted that the enzyme functions following
the modified Q-cycle [1], first proposed by Mitchell [2]. Metal dications
such as Zn2+ can bind to the proton translocating enzymes and blocks
the proton transfer, preventing thus the generation of the proton
gradient, ubiquitous for the ATP production [3, 4]. It is indeed crucial to
understand the mechanism of inhibition of Zn2+. The Zn2+ binding site
was suggested to be located at the interface between the hemebL and the
Rieske protein. The binding site consists of two histidines, one aspartic
acid, one asparagine, and one glutamic acid; all belong to the heme b
subunit [5]. The effect of the Zn2+ binding to the bc1 complex from
Rhodobacter capsulatus is studied with the help of a redox induced FTIR
difference spectroscopy combined with site directed mutagenesis. The
experiment show that redox reaction rates of the protein becomes
considerably slower in presence of Zn2+. Furthermore, the FTIR
difference spectroscopy shows that the mutation of the Glu295 to
Val295 leads to a loss of the signature of the protonated acidic residue in
Abstracts 13
the region of 1750–1700 cm−1. The Glu295 is a crucial acidic residue to
the proton translocation. In addition, the intensity of the Glu295 is
decreased by the addition of Zn2+meaning that the dication leads to the
deprotonation of theGlu295. The bc1 complex lacking theRieske protein,
shows a slower reaction rates inpresence of Zn2+without showing a loss
of the signature arising from the redox active protonated acidic residue.
References
[1] J.W. Cooley, et al., Biochemistry 48 (2009) 1888–1899.
[2] P. Mitchell, J. Theor. Biol. 62 (1976) 327–367.
[3] V.P. Skulachev, et al., Biochem. Biophys. Res. Commun. 26 (1967)
1–6.
[4] T.A. Link, et al., J. Biol. Chem. 270 (1995) 25001–25006.
[5] L. Giachini, et al., Biophys. J. 93 (2007) 2934–2951.
doi:10.1016/j.bbabio.2010.04.059
1P.12 Localization and dynamics of the OXPHOS complexes in
Escherichia coli by in-vivo fluorescence microscopy
H. Erhardt, T. Friedrich
Institut für Organische Chemie und Biochemie,
Albert-Ludwigs-Universität Freiburg, Germany
E-mail: heiko.erhardt@pluto.uni-freiburg.de
Biological membranes are known to be highly organized. Lipids and
membrane proteins are not regularly distributed within biological
membranes but are organized in distinct and dynamic clusters. It has
been proposed that protein complexes connected by a common
substrate chain form supercomplexes which are located in specific areas
within themembrane. Besides their structural role for stabilization of the
individual complexes, the functional role of supercomplexes is catalytic
enhancement.We focusedon theOXPHOS systemof aerobicallygrownE.
coli to determine whether the OXPHOS complexes are localized in
distinct lipid areas and if so, whether there is a dynamic exchange of the
complexes between the areas. To address these questions,we labeled the
membrane protein complexes with different fluorescent proteins and
visualized their distribution in the membrane by fluorescence micro-
scopy. FP-decorated variants of the NADH:ubiquinone oxidoreductase,
the succinate dehydrogenase, the cytochrome-bd complex and the FOF1-
ATP-Synthase, were created by means of λ-RED mediated mutagenesis.
Biochemical analysis revealed that the modified enzymes were
catalytically active and assembled to stable complexes in themembrane.
Fluorescence microscopy of living E. coli cells showed an uneven
distribution of the respiratory complexes in the cytoplasmatic mem-
brane. These data suggest that all complexes reside in distinctmembrane
domains, which might be of functional importance.
References
[1] T. Pohl, et al., Biochemistry 46 (2007) 10694–10702.
[2] T. Lenn, et al., Mol. Microbiol. 70 (2008) 1397–1407.
[3] E. Mileykovskaya, W. Dowhan, J. Bacteriol. 182 (2000) 1172–1175.
[4] A.S. Johnson, et al., Microbiology 150 (2004) 2815–2824.
doi:10.1016/j.bbabio.2010.04.060
1P.13 A new method of preparation of cytochrome c oxidase
vesicles from bovine heart mitochondria
Marek Stupak1, Graham Palmer, Marian Fabian
Rice University, Department of Biochemistry and Cell Biology,
Houston, USA
E-mail: fabian@rice.edu
Cytochrome c oxidase is an integralmembrane protein that catalyzes
the oxidation of ferrocytochrome c by molecular oxygen. This electron
transport reaction is coupled to the generation of transmembrane
gradient of protons. Investigations of the coupling mechanism between
the electron and proton transfer processes by spectroscopic methods
rely on the availability of small vesicles with a high concentration of
incorporated enzyme. Typically proteoliposomes are prepared by
reconstitution of the purified protein into phospholipid membranes in
the presence of detergent. However, we have found that the process of
preparation of oxidase vesicles that exhibit respiratory control can be
simplified by the fusion of isolated mitochondrial membranes with
small preformed asolectin vesicles. These proteoliposomes, formed
without detergent, can be purified further by chromatographicmethods
and used in studies of the proton translocation processes.
Support from National Institutes of Health (GM 0843348) is
gratefully acknowledged.
1 Permanent address: P. J. Safarik University, Faculty of Medicine, Department of
Medical Chemistry, Biochemistry and Clinical Biochemistry, Kosice, Slovak Republic.
doi:10.1016/j.bbabio.2010.04.061
1P.14 Mitochondrial respiratory supercomplexes: Structural
organization and functional role
Marco Faccioli1, Giovanna Barbero1, Anna Ida Falasca2,
Maria Luisa Genova1, Giorgio Lenaz1
1Dipartimento di Biochimica “G. Moruzzi”, Alma Mater Studiorum –
Università di Bologna, Via Irnerio 48, 40126 Bologna, Italy
2Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche
Applicate, Università di Parma, 43100 Parma, Italy
E-mail: marco.faccioli4@unibo.it
The model of the respiratory chain depicting the enzyme complexes
as independent units embedded in the lipid bilayer of the inner
mitochondrial membrane and connected by randomly diffusing
coenzyme Q and cytochrome c is mostly favored [1], although the
presence of stable supramolecular aggregates (respiratory supercom-
plexes) has been also demonstrated in mitochondria [2]. Besides the
structural evidence reported in the literature, the functional analysis of
the supercomplexes is still poor. In the present study, we have applied
the flux control analysis method [3] to intact liver mitochondria from
aged rats in order to measure the extent of metabolic control that each
respiratory complex exerts over respiration under phosphorylating
condition (state 3). Our results indicate that both complex I and complex
III are rate limiting, thus supporting the idea that they physiologically
behave as a supercomplex [4, 5]. The presence of the I+III assemblywas
also confirmed by 2D BN/SDS PAGE in mitochondria after digitonin
solubilization. Further experiments are available to investigate the role
of complex IV in frozen–thawed rat livermitochondria. In this condition,
we can observe that complex IV kinetically behaves as an independent
enzyme, not comprised in a functional supramolecular assembly,
although I+III+IV supercomplexes can be detected by 2D BN/SDS
PAGE. We suggest that the role of cytochrome c can be crucial for the
functionality of such supercomplexes.
References
[1] C.R. Hackenbrock, et al., J. Bioenerg. Biomembr. 18 (1986) 331–368.
[2] H. Schägger, et al., J. Biol. Chem. 276 (2001) 37861–37867.
[3] H. Kacser, et al., Symp. Soc. Exp. Biol. 27 (1973) 65–104.
[4] M.L. Genova, et al., Biochim. Biophys. Acta 1777 (7–8) (2008)740–746.
[5] G. Lenaz, et al., Biochim. Biophys. Acta (2010). doi:10.1016/j.
bbabio.2010.01.025.
doi:10.1016/j.bbabio.2010.04.062
Abstracts14


file:///data/remote/core/dit/data/elsevier/pdf/b79/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS9zMDAwNTI3MjgxMDAwMTkyMQ==.pdf

The Zn2+ inhibitive effects on the cytochrome bc1 complex from Rhodobacter capsulatus as revealed by the FTIR difference spectroscopy

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector

Elsevier - Publisher Connector