the enzyme can function as a quinol peroxidase (Vmax = 75/s, KM=6.6 mM), but not as a catalase as suggested in [2]. The membrane-bound catalase activity separates from the cytochrome bd oxidase inthe first chromatographic step. In the oxidase reaction the cytochromebd oxidase does not produce reactive oxygen species (ROS) becausethe OO bond is split in a single four-electron reduction via a Com-pound I intermediate [3]. In the peroxidase reaction, H2O2 is reducedto water in a rapid single two-electron reaction in which heme d2+ isoxidized to d4+O2− (Compound II). This reaction occurs both in one-electron reduced enzyme and in two- or three-electron reducedenzyme, where it is followed by rapid internal electron transferyielding d3+. Together with the cellular concentration of cytochromebd oxidase (5–10 μM) and its activity under physiological conditions(~1/s), the rapid initial formation of Compound II provides the bio-chemical basis for the enzyme to act as an efficient scavenger of H2O2,which is produced at a rate of 15 μM/s in Escherichia coli [4].

References[1] S. Al-Attar, S. de Vries, Energy transduction by respiratory

metallo-enzymes: From molecular mechanism to cell physiology,Coord. Chem. Rev. 257 (2013) 64–80.

[2] V.B. Borisov, E. Forte, A. Davletshin, D. Mastronicola, P. Sarti, A.Giuffre, Cytochrome bd oxidase from Escherichia coli displays highcatalase activity: an additional defense against oxidative stress,FEBS Lett. 587 (2013) 2214–2218.

[3] A. Paulus, S.G. Rossius, M. Dijk, S. de Vries, Oxoferryl-porphyrinradical catalytic intermediate in cytochrome bd oxidases protectscells from formation of reactive oxygen species, J. Biol. Chem. 287(2012) 8830–8838.

[4] J.A. Imlay, Cellular defenses against superoxide and hydrogenperoxide, Annu. Rev. Biochem. 77 (2008) 755–776.

doi:10.1016/j.bbabio.2014.05.166

S9.P3

Fast electron transfer between hemes and binding of internalligand to heme d upon photodissociation of CO from cytochromebd oxidaseVitaliy B. Borisova, Sergey A. Siletskyb, Andrey A. Zaspaa,Robert K. PoolecaA.N. Belozersky Institute of Physico-Chemical Biology, LomonosovMoscow State University, RussiabDepartment of Molecular Energetics of Microorganisms, A.N. BelozerskyInstitute of Physico-Chemical Biology, RussiacDepartment of Molecular Biology and Biotechnology, The University ofSheffield, UKE-mail: bor@genebee.msu.su

Cytochrome bd is a tri-heme (b558, b595, d) terminal oxidase [1, 2]that couples the electron transfer from quinol to O2 with generation ofa proton-motive force [3–5]. We studied photolysis and subsequentrecombination of CO with cytochrome bd-I from Escherichia coli inone-electron reduced (MV) and fully reduced (R) states by time-resolved absorption spectroscopy at 532-nm excitation. Probing theSoret and visible band regions, it is found that CO photodissociationfrom MV enzyme causes fast (within 1.5 μs) electron transfer fromheme d to heme b595, which is not reported earlier. Unlike cyto-chrome bd in the R state, in MV enzyme the apparent contribution ofabsorbance changes associated with CO dissociation from heme d issmall, if any. Photodissociation of CO from heme d in MV enzyme issuggested to be accompanied by the immediate binding of an internalligandat the opposite side of the heme. Through the following transition,CO recombines with heme d yielding a transient hexacoordinate state.

Then the ligand slowly dissociates from heme d [6]. This work wassupported in part by the Russian Foundation for Basic Research, grants12-04-01000-a (to SAS) and 14-04-00153-a (to VBB).

References[1] V.B. Borisov, R.B. Gennis, J. Hemp, M.I. Verkhovsky, The cyto-

chrome bd respiratory oxygen reductases, Biochim. Biophys. Acta1807 (2011) 1398–1413.

[2] A. Giuffrè, V.B. Borisov, D. Mastronicola, P. Sarti, E. Forte, Cyto-chrome bd oxidase and nitric oxide: From reaction mechanismsto bacterial physiology, FEBS Lett. 586 (2012) 622–629.

[3] A. Jasaitis, V.B. Borisov, N.P. Belevich, J.E. Morgan, A.A. Konstantinov,M.I. Verkhovsky, Electrogenic reactions of cytochrome bd, Biochem-istry 39 (2000) 13800–13809.

[4] I. Belevich, V.B. Borisov, J. Zhang, K. Yang, A.A. Konstantinov, R.B.Gennis, M.I. Verkhovsky, Time-resolved electrometric and opticalstudies on cytochrome bd suggest a mechanism of electron–proton coupling in the di-heme active site, Proc. Natl. Acad. Sci.USA 102 (2005) 3657–3662.

[5] V.B. Borisov, R. Murali, M.L. Verkhovskaya, D.A. Bloch, H. Han, R.B.Gennis, M.I. Verkhovsky, Aerobic respiratory chain of Escherichiacoli is not allowed to work in fully uncoupled mode, Proc. Natl.Acad. Sci. USA 108 (2011) 17320–17324.

[6] S.A. Siletsky, A.A. Zaspa, R.K. Poole, V.B. Borisov, Microsecond time-resolved absorption spectroscopy used to study CO compounds ofcytochrome bd from Escherichia coli, PLoS ONE (2014) in press,http://dx.doi.org/10.1371/journal.pone.0095617.

doi:10.1016/j.bbabio.2014.05.167

S9.P4

Aerobic respiration in Shewanella oneidensis MR-1Myriam Brugnaa, Arlette Kpebeb, Marielle Bauzanc, Sabrina Lignond,Marc Rousseta, Sébastien Le LazaaBIP, CNRS, Marseille, FrancebBIP, CNRS/AMU, FrancecCNRS, Aix-Marseille Université, Unité de fermentation, FR3479, IMM, FrancedCNRS, Aix-Marseille Université, Plate-forme Protéomique, FR3479, IMM,MaP IBiSAE-mail:mbrugna@imm.cnrs.fr

In aerobic respiration of prokaryotic and eukaryotic organisms,the reduction of molecular oxygen to water is catalyzed by terminaloxidases, cytochrome c or quinol oxidases, which are integral mem-brane multi-subunit enzymatic complexes. Two types of terminaloxidases are known, the heme–copper oxidases and the cytochromebd-type oxidases. The heme–copper oxidases are classified intothree families: type A (mitochondrial like oxidases), type B (ba3-typeoxidases) and type C (cbb3-type oxidases). The subunit composition oftypes A and B enzymes differs from one oxidase to another but theseheme–copper oxidases always contain the catalytic subunit (subunitI) and a smaller subunit named subunit II.

Shewanella oneidensis MR-1, a gram-negative proteobacterium,inhabits a wide variety of niches in nature and has the character-istic ability to reduce, in addition to O2, a broad spectrum of electronacceptors such as metals, nitrate, thiosulfate, dimethyl sulfoxide,trimethylamine N-oxide, fumarate and azo dyes.

The genome of S. oneidensis MR-1 encodes for three terminaloxidases: a bd-type quinol oxidase and two heme–copper oxidases, aA-type cytochrome c oxidase (genes SO4606–SO4609) and a cbb3-type oxidase (genes SO2361–SO2364).

In this study [1], we used a biochemical approach and directlymeasured oxidase activities coupled to mass-spectrometry analysis to

Abstractse96

investigate the physiological role of the three terminal oxidases underaerobic andmicroaerobic conditions. Our data revealed that the cbb3-typeoxidase is the major terminal oxidase under aerobic conditions whileboth cbb3-type and bd-type oxidases are involved in respiration at low-O2

tensions. On the contrary, the low O2-affinity A-type cytochrome c oxi-dasewas not detected in our experimental conditions even under aerobicconditions and would therefore not be required for aerobic respiration inS oneidensisMR-1. In addition, the deduced amino acid sequence suggeststhat the A-type cytochrome c oxidase is a ccaa3-type oxidase since anuncommon extra-C terminal domain contains two c-type heme-bindingmotifs, an uncommon feature among A-type oxidases.

Reference[1] S. Le Laz, A. Kpebe, M. Bauzan, S. Lignon, M. Rousset, M. Brugna,

Plos One. (2014) 10.1371/journal.pone.0086343.

doi:10.1016/j.bbabio.2014.05.168

S9.P5

Diiron four-helix-bundle proteins in oxygen and hydrogenperoxide detoxificationJoana P. Carrilho, Célia V. Romão, Miguel TeixeiraInstituto de Tecnologia Quimica e Biológica-António Xavier, UniversidadeNova de Lisboa, Av. da República, 2780-157 Oeiras, PortugalE-mail: joanacarrilho@itqb.unl.pt

Oxygen and derived reactive species pose a huge challenge topresent day living forms, and specialized enzymes exist to deal withtheir toxicity. Among those, are the large family of diiron-containingenzymes, such as the so-called alternative oxidase, a quinol:oxygenoxidoreductase [1], and the rubrerythrin sub-family, putative hydro-gen peroxide reductases [2–3]. Both rubrerythrins and AOX share acommon four-helix-bundle structural fold, harboring a catalytic diiron-site bound to histidines and aspartates/glutamates and a μ-(hydr)oxobridge in thediferric state. Rubrerythrinsmay contain additional domains(one or two), namely possessing simple mononuclear rubredoxin-type[FeCys4] centers, located either before or after the four-helix bundle(e.g. [3]).

The most consensual activity for rubrerythrins is that of H2O2

reductase, linked to NADH oxidation by redox partner enzymes.Rubrerythrin-like enzymes are present in the three life domains.Biochemical and enzymatic, spectroscopic, and structural studies onseveral rubrerythrins, of the simplest one-domain enzyme (from thehyperthermoacidophilic Archaeon Acidianus ambivalens erythrin), ofa canonical Clostridium difficile (a bacterial human pathogen) two-domain rubrerythrin, and the more complex three-domain Campylo-bacter jejuni (also a human bacterial pathogen), desulforubreythrin,will be described. A detailed comparison of the 3D structures of AOXand rubrerythrins will be presented, which reinforce our previoushypothesis [4] that rubrerythrins may have been primitive ancestorsof diiron-containing oxygen reductases.

References[1] A.L. Moore, T. Shiba, L. Young, S. Harada, K. Kita, K. Ito, Unraveling

the heater: new insights into the structure of the alternativeoxidase, Annu Rev Plant Biol 64 (2013) 637–663.

[2] E.D. Coulter, N.V. Shenvi, D.M. Kurtz, Jr., NADH peroxidase activityof rubrerythrin, Biochem Biophys Res Commun 255 (1999) 317–323.

[3] A.F. Pinto, S. Todorovic, P. Hildebrandt, M. Yamazaki, F. Amano, S.Igimi, C.V. Romao, M. Teixeira, Desulforubrerythrin from Campylo-bacter jejuni, a novel multidomain protein, J Biol Inorg Chem 16(2011) 501–510.

[4] C.M. Gomes, J. Le Gall, A.V. Xavier, M. Teixeira, Could a diiron-containing four-helix-bundle protein have been a primitiveoxygen reductase? Chembiochem 2 (2001) 583–587.

doi:10.1016/j.bbabio.2014.05.169

S9.P6

Kinetic comparisons of 5A and 5B isozymes of yeast cytochromec oxidaseRaksha Dodiaa, Brigitte Meunierb, Peter Richa

aUniversity College London, UKbCentre de Génétique Moléculaire, CNRS, FranceE-mail: r.dodia@ucl.ac.uk

The nuclear-encoded subunit 5 of Saccharomyces cerevisiae cyto-chrome c oxidase (CcO) has two isoforms, 5A and 5B. Their expressionis differentially modulated by oxygen concentration. COX5A isexpressed under aerobic conditions while COX5B is expressed only atoxygen tensions below 1 μM. Since subunit 5 is essential for enzymeassembly, under aerobic growth conditions a COX5A-deleted straincontains no or very low level of CcO and so is respiratory deficient [1].Previously, respiratory growth was restored by combining mutationsof ROX1 that encodes a transcriptional repressor of COX5B expressionwith Δcox5A [1]. The level of 5B isozyme expression in these mutantswas 30–50% of wildtype (5A isozyme) and its maximum turnovernumber was up to 3 fold greater than that of the 5A isozyme [2]. Toassess the structural basis of this elevated activity, a mutant strainwasconstructed in which COX5B was inserted downstream of the COX5Apromoter. This allowed the 5B isozyme to be expressed to wildtypelevels without the complications of additional mutation in a transcrip-tion factor.When expressed in thismanner, the isozymes displayed nosignificant differences in their maximum catalytic activities or in theiraffinities for cytochrome c or oxygen. Hence, the elevated activity ofthe 5B isozyme in the rox1mutant is not caused simply by exchange ofisoforms and must arise from a secondary effect that is still to beresolved.

References[1] C. E. Trueblood, and R. O. Poyton, Identification of REO1, a gene

involved in negative regulation of COX5b and ANB1 in aerobicallygrown Saccharomyces cerevisiae, Genetics 120 (1988) 671–680.

[2] A. L. Allen, X.-J. Zhao, W. Caughey, R. O. Poyton, Isoforms of yeastcytochrome c oxidase subunit V affect the binuclear reaction centerand alter the kinetics of interaction with the isoforms of yeastcytochrome c. J. Biol. Chem. 270 (1995) 110–118.

doi:10.1016/j.bbabio.2014.05.170

S9.P7

Revisiting individual absorption spectra of reduced hemes a anda3 in bovine-heart cytochrome c oxidaseArtem V. Dyuba, Tatiana V. Vygodina, Alexander M. Arutyunyan,Alexander A. KonstantinovBelozersky Institute of Physico-Chemical Biology, Moscow State University,RussiaE-mail: dyubon@gmail.com

Cytochrome c oxidase (COX) is a terminal enzyme of the respiratorychain which catalyzes electron transport (ET) from cytochrome c to O2

coupled to transmembrane H+-pumping. The ET is mediated by two

Abstracts e97