Biochemical, Spectroscopic, And Thermodynamic Properties

7/31/2019 Biochemical, Spectroscopic, And Thermodynamic Properties

1/25

C H A P T E R T W O

Biochemical, Spectroscopic, and

Thermodynamic Properties ofFlavodiiron Proteins

Joao B. Vicente, Marta C. Justino, Vera L. Goncalves,Lgia M. Saraiva, and Miguel Teixeira

Contents1 . Introduction 222 . Cloning of Genes Encoding Flavodiiron Proteins and

Their Truncated Domains 243 . Production and Purification of Recombinant Flavodiiron Proteins 254 . Biochemical Characterization of Flavodiiron Proteins 265 . Spectroscopic Properties 296 . Redox Properties 327 . Conclusions 37

7 .1 . Functional properties 39Acknowledgments 42References 42

AbstractThe flavodiiron proteins (FDPs), present in Archaea, Bacteria, and some proto-zoan pathogens (mostly anaerobes or microaerophiles), have been proposedto afford protection to microbes against nitric oxide and/or oxygen (toxic foranaerobes). The structural prototype of this protein family is a homodimerassembled in a head-to-tail configuration, with each monomer being com-posed of two domains: an N-terminal metallo- b-lactamase module harboring anonheme diiron center (active site of NO/O 2 reduction) and a C-terminal flavo-doxin module, where a flavin mononucleotide moiety is embedded. SeveralFDPs bear C-terminal extra domains, which influence the composition of therespective electron transfer chains that couple NAD(P)H oxidation to NO/O 2reduction. Herein are described methodologies employed to successfully pro-duce, isolate, and characterize fully operative recombinant flavodiiron proteins.Spectroscopic techniques, namely absorption (visible and near-ultraviolet) and

Methods in Enzymology, Volume 437 # 2008 Elsevier Inc.ISSN 0076-6879, DOI: 10.1016/S0076-6879(07)37002-X All rights reserved.

Instituto de Tecnologia Qu mica e Biolo gica, Universidade Nova de Lisboa, Oeiras, Portugal

21


2/25

electron paramagnetic resonance spectroscopies, allowed redox-sensitive spec-tral fingerprints to be obtained, used further in the functional characterization of isolated flavodiiron proteins.Altogether, these studies on pureproteins contributeto understanding the molecular determinants that govern the in vivo function of the FDPs.

1. Introduction

The first report on a flavodiiron protein (FDP) focused on Desulfovibrio gigasrubredoxin:oxygen oxidoreductase (Dg_ROO), the terminal compo-nent of a soluble electron transfer chain proposed to be involved in oxygen

detoxification, affording this (then considered) strict anaerobe protectionfrom an otherwise toxic dioxygen ( Chen et al ., 1993b ). Dg_ROO, a flavin-binding homodimer of 43-kDa monomers, was proposed to fully reduceoxygen to water, using electron equivalents from NADH, shuttled byrubredoxin and a NADH:rubredoxin oxidoreductase ( Chen et al ., 1993a;Gomes et al ., 1997 ).

The flurry of complete genome sequences led to the discovery of severalDg_ROO homologues widespread in Bacteria and Archaea and to estab-lishment of the family of A-type flavoproteins (the former designation of

flavodiiron proteins) ( Wasserfallen et al ., 1998 ). It was proposed that there isa common sequence core of about 400 amino acids, where a putativeflavodoxin-like domain could be identified at the C terminus, and it wasnoted that some members of the protein family had extra C-terminalextensions. These extensions were identified as possible redox activedomains, namely a rubredoxin domain in the Escherichia coli protein and aNAD(P)H:flavin oxidoreductase domain in the Synechocystisone. It was notuntil the crystallographic structure of Dg_ROO was solved that further insights into its functional properties were attained ( Frazao et al ., 2000 ). Thisstructure elucidated that the previously proposed core of this protein familyis indeed composed of an N-terminal b -lactamase-like domain fused to theflavin mononucleotide (FMN)-binding flavodoxin domain and revealedthe active site of oxygen reduction: a nonheme diiron center in the lacta-mase fold, with carboxylate and histidine residues in its coordination sphere.It is worth noting that the structure revealed that a head-to-tail homo-dimeric quaternary arrangement is required to place the FMN cofactor of one monomer in close contact with the diiron center from the other monomer, allowing otherwise impaired electron transfer (the two cofactorsare % 25 A apart in each monomer)( Vicente et al ., 2007a ).

A survey of available FDP sequences suggested four structural classes for this protein family [adding one class to a previous classification ( Saraiva et al .,2004 )], accounting for the C-terminal extensions ( Fig. 2.1 ), whose naturereflects itself in the composition of the electron transfer chains that couple

22 Joao B. Vicente et al.


3/25

NAD(P)H or F 420 H 2 oxidation to nitric oxide (NO) or O 2 reduction. Class AFDPs are the simplest, consisting solely of the bidomain structural core ( % 400residues), and represent the majority of the found sequences. Class B FDPs(% 480 residues) bear a C-terminal rubredoxin domain and are restricted, sofar, to enterobacteria. Class C FDPs ( % 600 residues), also found so far only in

cyanobacteria, have a NAD(P)H:flavin oxidoreductase C-terminal domain,and often there are multiple genes encoding these FDPs within the sameorganism. The newly proposed class D ( % 900 residues) comprises FDPswhere two extra C-terminal domains are fused: a rubredoxin domain and aNADH:rubredoxin oxidoreductase domain (homologous to the cognatereductase of class B FDPs). Class D FDPs were found in the genome sequencesof some Clostridiales and of the protozoan pathogen Trichomonas vaginalis.Phylogenetic analyses revealed two interesting observations: (i) FDPs bearingC-terminal extensions cluster together according to their class ( Saraiva et al .,2004 ) and (ii) genes encoding FDPs are prone to be transferred via lateral genetransfer among coexisting organisms ( Andersson et al ., 2003 ). This observationaccounts for the finding of FDP-encoding genes in pathogenic protozoa, so far the only known eukaryotic FDPs.

It is envisaged that the complexity of the modular arrangement of FDPscontrasts with the number of components of the corresponding electrontransfer chains, i.e., class C and D FDPs should accomplish coupling of NAD(P)H oxidation to substrate reduction within the same polypeptidechain. Class B FDPs require one extra redox protein, and class A mayrequire as many as two more redox partners to accomplish the same. Thisidea has been challenged only recently by a class A FDP (from a methano-genic source) that oxidizes F 420 H 2 directly ( Seedorf et al ., 2004 ), an abun-dant redox cofactor in methanogenic organisms, and thus dispenses theinvolvement of other redox proteins.

FMN Fe-Fe

FMN Fe-FeFlv

FMN Fe-FeFe-S

FMN Fe-FeFe-SFAD

Class A

Class B

Class C

Class D

Figure 2 .1 Modulararrangements in the flavodiironprotein familyand correspondingstructural classes. FMN, flavodoxin-like module, binding FMN; Fe-Fe, metallo- b-lacta-

mase module, harboring the nonheme diiron active site; Fe-S, rubredoxin-like module,harboring a Fe-Cys 4 center; Flv, NAD(P)H:flavin oxidoreductase module, bindingFAD or FMN; FAD, predicted NAD(P)H:rubredoxin oxidoreductase module.

Properties of Flavodiiron Proteins 23


4/25

The idea of flavodiiron proteins as oxygen reductases came to a halt withmolecular genetics studies on the E. coli flavodiiron protein (a class B FDPnamed flavorubredoxin because of its C-terminal rubredoxin domain) that

proposed a role for this protein in NO detoxification. It was demonstratedthat expression of the norV geneencoding flavorubredoxinwas inducedby NO and that an E. coli norV mutant strain was more sensitive to NO thanthe wild-type strain ( Gardner et al ., 2002; Justino et al ., 2005b ), withdeleterious effects to NO-sensitive metabolic enzymes and affecting cellsurvival (Gardner et al ., 2002 ). A role in anaerobic NO detoxification wasthus proposed for flavorubredoxin, acting as an NO reductase, an activitythat was confirmed further in vitro(Gomes et al ., 2002 ). Flavodiiron proteinsare presently considered a prominent family of NO-detoxifying enzymes,

in the line of flavohemoglobin, although some members of the proteinfamily retain a preference for oxygen as their substrate ( Rodrigues et al .,2006; Seedorf et al ., 2007 ).

Research efforts have been employed to clarify the ambiguity of thepossible roles for flavodiiron proteins. A thorough biochemical characteri-zation of each studied member (and the corresponding electron transfer chains) is essential to understanding the molecular basis for the substratepreference (NO vs O 2). In parallel, molecular genetics studies provide cluesto understanding the function and relative role of each FDP in (i) NOdetoxification (e.g., as a subversive mechanism of pathogens to counteractthe host immune response) and/or (ii) O 2 detoxification in anaerobicorganisms (to allow survival of transient exposure to toxic environments).

2. Cloning of Genes Encoding FlavodiironProteins and Their Truncated Domains

The production of recombinant flavodiiron proteins from variousmicrobial sources has been successfully achieved by overexpression inE. coli , with T7 promoter-based expression vectors ( Gomes et al ., 2000,2002; Rodrigues et al ., 2006; Seedorf et al ., 2004; Silaghi-Dumitrescu et al .,2003, 2005; Vicente et al ., 2002; Wasserfallen et al ., 1998 ).

In our laboratory, flavodiiron proteins from classes B and C have beenisolated as recombinant proteins overexpressed in E. coli : the E. coli flavo-rubredoxin(FlRd) and its truncated rubredoxin and flavodiiron domains, theSynechocystissp. PCC6803 SsATF573 (the original designation for the 573amino acid FDP from this organism, encoded by gene sll 0550), and itsC-terminal domain ( Gomes et al ., 2000, 2002; Vicente et al ., 2002 ).The coding regions were amplified by polymerase chain reaction fromgenomic DNA, using primers containing restriction sites that allow cloninginto the T7 expression vectors pET24a or pT77 (for the rubredoxin



5/25

domain, Rd domain). Cloning of DNA fragments encoding the C-terminaldomains required the introduction of a Nde I site in the sense primers thatchanged to initiation codon (ATG) the codons of residue 422 of E. coli FlRd

(Gomes et al ., 2002 ) and residue 402 of SynechocystisSsATF573. Sequencingof the recombinant plasmids confirmed the correct nucleotide sequences(Vicente et al ., 2002 ).

3. Production and Purification of RecombinantFlavodiiron Proteins

Overexpression of E. coli flavorubredoxin (and its truncated domains)(Gomes et al ., 2000 ) and SynechocystisSsATF573 (and the C-terminaldomain) ( Vicente et al ., 2002 ) is performed in BL21-Gold(DE3) cells(Stratagene) under conditions that have been progressively optimized. Initi-ally, Luria-Bertani (LB) broth (supplemented with 10 mM ferrous sulfate)was used to attempt the overexpression of FDPs. However, an improve-ment of iron and flavin incorporation was achieved by decreasing the rate of protein synthesis. This was attained by changing the growth medium tominimal medium M9 ( Gomes et al ., 2002; Silaghi-Dumitrescu et al ., 2003 ),

reducing the air chamber, and decreasing the growth temperatures from 37to 28 C. Under optimized conditions, freshly transformed cells are grownin M9 minimal medium with 10 m M glucose ( Ausubel et al ., 1995 ) supple-mented with 10 mM FeSO 47H 2O, in flasks filled to 70% of the volume, at28 C and 130 rpm. Induction of expression is made with 100 mM isopro-pyl-1-thio- b-D-galactopyranoside (IPTG) when the cultures reach OD 600 0.30.4, and the cells are harvested after 7 h by centrifugation (11,000 g ,10 min, 4 C). Cells resuspended in 10 m M Tris-HCl, pH 7.6, are dis-rupted in a French press cell at 130 MPa, followed by a 2-h ultracentrifuga-tion (100,000 g , 4 C) to remove cell debris. The soluble extracts aredialyzed against 10 m M Tris-HCl, pH 7.6, containing 18% (v/v) glycerol(buffer A). Complementing the buffers with glycerol increases the stability of the enzymes, preventing the loss of flavin moieties throughout the purifica-tion. In the purifications of intact flavorubredoxin, 500 mM of the proteaseinhibitor phenylmethanesulfonyl fluoride is added to all buffers to preventpeptidic breakage between the structural modules of FlRd ( Vicente andTeixeira, 2005 ). All purification steps are done at 4 C. Dialyzed solubleextracts are applied onto a Q-Sepharose column (Amersham) equilibratedpreviously with buffer A and, by applying a gradient up to 1 M NaCl, proteinsare eluted at 400450 m M NaCl ( Gomes et al ., 2000, 2002; Vicente et al .,2002 ). After desalting, fractions are introduced into a Fractogel EMD TMAEcolumn (Merck), eluted at 250 m M NaCl, concentrated, and further appliedonto a gel filtration column (Superdex S-75 or S-200, both from Amersham)



6/25

equilibrated with buffer A containing 150 m M NaCl. Regarding purificationof the C-terminal domain of SynechocystisSsATF573, the protein is purifiedfrom the soluble extract, in two steps: a Q-Sepharose fast flow column

(Amersham) equilibrated in 20 m M KP buffer at pH 6 (buffer B) and aSP-Sepharose column (Amersham). The truncated form of SsATF573 iseluted with 200 m M KCl (Vicente et al ., 2002 ). Protein purity is evaluatedthroughout the purification steps by SDS-PAGE ( Garfin, 1990 ).

The purifications of recombinant FDPs from Moorella thermoacetica(Silaghi-Dumitrescu et al ., 2003 ), Desulfovibrio vulgaris(Silaghi-Dumitrescuet al ., 2005 ), and D. gigas (Rodrigues et al ., 2006 ) are conducted in similar ways, although with minor differences on the expression conditions.The production and purification of Methanobrevibacter marburgensisFprA

are significantly different from other FDPs, namely the fact that the proteinis only successfully isolated under anaerobic conditions. M. marburgensisFprA is overexpressed in E. coli Rosetta(DE3)pRare cells are induced atOD 600 % 0.8 by the addition of 1 m M IPTG. Harvested cells are disruptedby ultrasonication and heated at 60 for 20 min. FprA is isolated from thesoluble extract (obtained after a 150,000 g ultracentrifugation) in one purifi-cation step, using a DEAE-Sepharose fast flow column equilibrated with50 mM Tris-HCl, pH 7.6. The protein is recovered in the 400 m M NaClfraction ( Seedorf et al ., 2004 ).

4. Biochemical Characterization ofFlavodiiron Proteins

Flavodiiron proteins from three (of the aforementioned four) classeshave been studied, the majority of which belong to class A (propertiessummarized in Table 2.1). The monomeric molecular masses are deter-

mined by SDS-PAGE ( Garfin, 1990 ), and the measured molecular massesare in accordance with the expected values inferred from the peptidesequences, namely 4348 kDa per monomer for class A FDPs ( % 400residues), % 54 kDa for class B FDPs ( % 480 residues), and % 70 kDa for class C FDPs (% 600 residues). The quaternary structure of isolated FDPs ismeasured by analytical gel permeation chromatography using the appropri-ate molecular mass standards. FDPs alternate between homodimers andhomotetramers, satisfying the prerequisite of a dimer as the minimal func-tional unit, to allow proximity between the FMN from one monomer and

the diiron center from the other monomer ( Frazao et al ., 2000; Seedorf et al ., 2007; Silaghi-Dumitrescu et al ., 2005 ).The purified proteins have been assayed for their cofactor content,

namely in terms of flavin and iron incorporation. For the FDPs (and



7/25

Table 2 .1 Physical-chemical properties and cofactor content of avodiiron proteins

Protein Microorganism a.a length

Monomermolecular

mass *

Quaternary

structure

Class A

Rubredoxin:oxygenoxidoreductase(ROO)

Desulfovibrio gigas

402 43 kDa(44.8)

Homodimer

Flavodiiron protein Moorellathermoacetica

399 45 kDa(44.3)

Homodimer

Flavoprotein(Tm0755)

Thermotogamaritima

410 n. d.(47.1)

Homodimer

Flavodiiron protein Desulfovibriovulgaris

402 45 kDa(45.1)

Homodimer

FlavoproteinA (FprA)

Methanobrevibacter arboriphilus

45 kDa(46.1)

n. d.


Methanothermobacter marburgensis

404 43 kDa(45.3)

Homotetram er

FlavoproteinA (FprA) Rhodobacter capsulatus 420 48 kDa(46.2) Homodimer


8/25

Table 2 .1 ( continued )

Protein Microorganism a.a length

Monomermolecularmass *

Quaternarystructure

FlavoproteinA (FprA) Methanobacteriumthermoautotrophicumstrain D H

409 45 kDa(46.0) Homodimer 1.


MethanobacteriumthermoautotrophicumMarburg

404 43 kDa(45.7)

Homotetramer 0.

Class B

Flavorubredoxin(FlRd)

Escherichia coli 479 54 kDa(54.2)

Homotetramer 2.

Class C

SsATF573 Synechocystis 573 70 kDa(63.5)

Homodimer 1.

* between brackets, molecular mass estimated from the aminoacid (a.a.) sequence.{ between brackets, experimental methodology by which the cofactor was identified and quantified: XRC, X-ray crystallography; P

AE-UVS, acid extraction plus visible spectroscopy; AE-HPLC, acid extraction followed by HPLC analysis; N/C colorimetric mn.d. not determined.


9/25

truncated domains) of E. coli and Synechocystis, the protein concentrationsare measured by the 2-bicinchoninic acid protein assay (Pierce) ( Walker,1994 ). The iron content is determined by the 2,4,6-tripyridyl-1,3,5-triazine

method ( Fischer and Price, 1964 ). Flavin quantification (adapted from Susinet al ., 1993 ) is performed by acid extraction with TCA (10%) followed bycentrifugation and supernatant neutralization with 1 M NH 4CH 3COO,pH 7. The nature of the flavin cofactors (FMN or FAD) is determined,when needed ( Wasserfallen et al ., 1998 ), by reversed-phase chromatographyusing a Nucleosil 1005 C18 column equilibrated with 10 m M ammoniumformate, pH 6.4 (containing 12% methanol), and performing a three-stepgradient of increasing methanol concentration. The appropriate commer-cial flavin standards (FAD and FMN from Fluka) are treated and measured

identically to flavins extracted from the protein samples. The extractedflavins are quantified spectrophotometrically using the following molar absorption coefficients: EFMN (l 445 ) 12,200 M 1 cm 1; EFAD (l 450 ) 11,300M 1 cm 1; and EFMN FAD (l 447 ) 11,750 M 1 cm 1 (Sober and Harte, 1968 ).The cofactor content of studied FDPs (Table 2.1) yields % 12 Fe and % 0.71FMN per monomer. E. coli flavorubredoxin binds instead three iron ions per monomer, one in the rubredoxin domain and two in the diiron site. Byreplacing Fe 2 with Zn 2 in the growth medium of E. coli cells overexpressingM. thermoaceticaFDP ( Silaghi-Dumitrescu etal ., 2003 ), the isolated FDP comeswith a binuclear zinc site in place of the diiron site. This promiscuity isexplained by equivalently high affinities of the center for Fe and Zn(Schilling et al ., 2005 ).

5. Spectroscopic Properties

To probe the functional properties of isolated flavodiiron proteins,spectroscopic methods proved to be essential, namely in characterization of the redox-active cofactors. Whereas visible spectroscopy was used mainly tocharacterize the flavins, electron paramagnetic resonance (EPR) spectros-copy allowed characterization of the diiron center.

Visible and near-ultraviolet absorption spectra of as-isolated flavodiironproteins are mostly dominated by the contribution of their flavin moieties.Nonheme diiron centers ( Solomon et al ., 2000 ) have much lower extinc-tion coefficients than flavins (free or protein bound) ( Ghisla and Edmonson,2001 ), and therefore spectra of class A ( Silaghi-Dumitrescu et al ., 2003 ) andclass C (Vicente et al ., 2002 ) FDPs (which have only flavin and diironcofactors) have features almost solely attributable to the flavin moieties(Fig. 2.2A and B ). It is noteworthy that visible spectra of FDPs are slightlyheterogeneous among different members of the protein family, in the sensethat the band centered at % 450 nm is broad and smooth in some cases



10/25

( Jouanneau et al ., 2000; Silaghi-Dumitrescu et al ., 2003; Wasserfallen et al .,1998 ) and has two shoulders in others ( Nolling et al ., 1995; Vicente et al .,2002; Wasserfallen et al ., 1995 ). This band is commonly assigned to chargetransfer transitions within the isoalloxazine core, from the xylene ring to thepyrimidine ring. To understand this spectral heterogeneity, an inspection of the flavin pocket in the Dg_ROO structure was undertaken and comparedwith structural models generated with that structure as the template. In thestructure of Dg_ROO, a tryptophan residue is coplanar with the FMNisoalloxazine ring (Trp347 in Dg_ROO). This Trp residue is conserved in

FDPs where the same broad spectrum ( Fig. 2.2A ) is observed and appears inthe same position in the modeled structures of other FDPs ( Saraiva et al .,2004 ). However, in FDPs where this Trp is lacking, the spectral bandcentered at 450 nm has two shoulders ( Fig. 2.2B ). Therefore, it has been

Wavelength (nm)

A

B

C

300 400 500 600 700

FMN Fe-Fe

FMN Fe-FeFlv

FMN Fe-FeFe-S

Class A

Classes

A and C

Class B

Figure 2 .2 Visible spectra of flavodiiron proteins. (A) Flavodiiron domain of Escheri-chia coli flavorubredoxin (i.e., with the rubredoxin domain truncated); (B) flavodiironprotein from Synechocystis sp. PCC6803, named SsATF573; and (C) E . coli flavorubre-doxin. All spectra in 20 m M Tris-HCl,18% glycerol, pH 7.6, at 25 .



11/25

proposed that this Trp residue may account for the spectral heterogeneity(Saraiva et al ., 2004 ) by interacting with the FMN moiety ( Vicente et al .,2008a ).

On top of the flavin absorption spectrum, the E. coli flavorubredoxin(class B FDP) has the contribution of the [Fe-Cys 4] center from therubredoxin domain ( Fig. 2.2C ) (Gomes et al ., 2000; Vicente and Teixeira,2005 ). Although the spectrum of FlRd overlaps in almost the entire visibleregion, above % 560 nm, the observed broad band is almost exclusivelybecause of the rubredoxin domain. This observation is of great value,namely in deconvoluting the functional behavior of the different cofactors.

Electron paramagnetic resonance spectroscopy has proved to be a valu-able tool in characterizing the cofactors of flavodiiron proteins, providing in

fact the first direct spectroscopic evidence for the diiron center for a member of this protein family. Initially, EPR was used to characterizethe flavin cofactor in D. gigas ROO ( Gomes et al ., 1997 ), where a signal atg % 2.0 obtained under reductive conditions was attributed to the oneelectron-reduced semiquinone state of the flavin, proposed to correspondto the red anionic radical, based on the 1.6-mT line width (whichconcurred with visible spectroscopy data) ( Gomes et al ., 1997 ).

Electron paramagnetic resonance spectroscopy is essential in studyingthe diiron site, which has very low absorptivity in the visible region. Inoxidized states, only FDPs containing a rubredoxin core are EPR active,with the characteristic g % 4.3 resonance typical of high-spin (S 5/2) ferriciron ( Gomes et al ., 2000; Vicente and Teixeira, 2005 ). Upon reduction,because of the spin change to S 2, the rubredoxin resonance vanishes.The diiron center is EPR silent in the oxidized state, as the two ferric ionsare coupled antiferromagnetically [as confirmed by Mo ssbauer spectroscopyfor the M. thermoaceticaFDP ( Silaghi-Dumitrescu et al ., 2003 )]. For thisreason, the diiron center is only clearly detected by EPR spectroscopy in itsone electron-reduced, mixed-valence (Fe III -Fe II ) state, displaying a rhom-bic signal with g values at g < 2.0 (Fig. 2.3 ), which has its maximal intensityat 7K (Vicente and Teixeira, 2005 ). An interesting observation is thatobtained spectra differed in their shape and g values according to the wayby which the mixed-valence state was obtained, i.e., in the presence (line 1in Fig. 2.3 ) or absence (line 2 in Fig. 2.3 ) of redox mediators (in both cases,reduction was achieved by the addition of sodium dithionite). Nevertheless,the relaxation properties do not appear to be affected by the different shape,as their corresponding temperature dependences are practically identical(not shown). Full reduction of the diiron center to the Fe II -Fe II state resultsin the disappearance of this signal, leaving as sole EPR evidence for thediferrous state a g % 11 signal in parallel-mode EPR, indicating an S 4 spinstate. Hence, the achievement of a spectroscopic signature for the diironcenter in E. coli FlRd allowed characterization of its thermodynamic



12/25

properties (see later), and it is envisaged that the same methodology can beapplied to study the diiron centers in other proteins of this family.

6. Redox Properties

The thermodynamic properties of FDPs have been determined essen-tially for the FMN moiety and only for a few cases. The redox titration of D. gigas ROO, followed by visible spectroscopy, yielded reduction potentialsof 0 15 mV for the FMN ox /FMN sq stepand 130 15 mV for the FMN sq/FMN red step (Gomes et al ., 1997 ). For M. thermoaceticaFprA (Mt_FDP), thedetermined reduction potentials were 117 mV for the FMN ox /FMN sq stepand 220 mV for the FMN sq/FMN red step (Silaghi-Dumitrescu et al ., 2003 ).In both cases, the observed semiquinone was of the red anionic type, as judgedby the corresponding spectral features ( Gomes et al ., 1997; Silaghi-Dumitrescuet al ., 2003 ).

Concerning E. coli flavorubredoxin, the visible absorption spectrum(Fig. 2.2C ) comprises features of both FMN and rubredoxin cofactors(Gomes et al ., 2000 ), with overlapping features, which hamper the decon-volution of their individual redox properties. Herein is described a meth-odology that allows identification of the reduction potentials of each

1.95

1.80

1.74

1.93

1.88

1.82

300 350 400 450

Magnetic field (mT)

I n t e n s i t y

( A . U

. )

Fe-Fe

1

2

Figure 2 .3 EPR spectra of the mixed-valence nonheme diiron center in flavorubre-doxin. EPR spectra of the flavodiiron structural core (FDP-domain) of Escherichia coliflavorubredoxin, obtained in thecourse of a redox titration (line1) andby mild chemicalreduction (line 2) with sodium dithionite. Spectra focus on the g < 2 region, wheremixed-valence nonheme diiron centers have known EPR signatures.The FDP-domain(250 mM ) was titrated at 25 C, in 50 m M Tris-HCl,18% glycerol, pH 7.5. Arrows indicate g values assigned to each signal. Spectra collected at 7K; microwave power: 2.4 mW;microwave frequency: 9.64 GHz; modulation amplitude:1 mT.



13/25

cofactor in E. coli FlRd. The experimental approach combined potentio-metric methods with visible and EPR spectroscopies and relied significantlyon the use of truncated proteins for the separate characterization of each

domain, thus allowing the deconvolution of superimposing spectral fea-tures. This methodology was further employed to analyze modulation of the redox properties of FlRd that occurs upon interaction with its cognatereductase partner (FlRd-reductase) ( Vicente et al ., 2007b,c ).

Redox titrations are performed anaerobically at 25 C, with the isolatedE. coli FlRd whole protein or truncated domains (Rd domain and FDPdomain) and with a stoichiometric (1:1) mixture of FlRd (or its Rd domain)with the FlRd-reductase partner. Reduction is attained by the stepwiseaddition of sodium dithionite (250 m M Tris-HCl, pH 8) or NADH, the

physiological electron donor of FlRd-reductase. Anaerobic conditions weremaintained by continuously degassing on surface the titration buffer (50 m M Tris-HCl pH 7.6, 18% glycerol) with argon and by the addition of oxygenscavengers (glucose, glucose oxidase and catalase). The redox mediators usedare methylene blue (E o

0 11 mV), indigo tetrasulfonate (E o0 30 mV),

indigo trisulfonate (E o0 70 mV), indigo disulfate (E o

0 82 mV), indigodisulfate anthraquinone 2,7-disulfonate (E o

0 182 mV), safranine (E o0

280 mV), neutral red (E o0 325 mV), benzyl viologen (E o

0 359 mV),and methyl viologen (E o

0 446 mV). The concentration of redox media-tors ranges between 30 and 80 mM in the EPR-monitored titrations andbetween 0.25 and 0.5 mM in the visible monitored ones. A silver/silver chloride electrode is used, calibrated with a saturated quinhydrone solutionat pH 7, and the reduction potentials are quoted against the standardhydrogen electrode ( Gomes et al ., 1997; Vicente and Teixeira, 2005 ).Spectral deconvolution and experimental data analysis is performed usingMATLAB (Mathworks, South Natick, MA) for Windows.

Analysis of titration of intact flavorubredoxin (followed by visible spec-troscopy) is initiated, taking into account that the absorbance changes at560 nm are almost exclusively attributable to the Fe-Cys

4center in the

rubredoxin module ( Vicente and Teixeira, 2005 ). Plotting the absorbancechanges at 560 nm as a function of the reduction potential and fitting datawith a Nernst equation for a one-electron transition yield a reductionpotential of 123 15 mV. Since the redox titration of the truncated Rd-domain (following by visible spectroscopy the bleaching at 484 nm) revealedan identical reduction potential to the Fe-Cys 4 centre in the intact protein, itwas possible to subtract the spectra of the Rd-domain titration(Fig. 2.4C) tothe ones of the intact FlRd titration, where the corresponding experimentalredox values match. The subtraction procedure yields a matrix comprisingsolely spectral features of the flavodiiron core of FlRd, which is clearlydominated by the FMN moiety (see Fig. 2.4D ). Spectra in Fig. 2.4D showformation of the red semiquinone upon one-electron reduction of the FMN,characterized by the decrease in the absorbance at % 450 nm accompanied by



14/25

an increase at % 390 nm, and its further disappearance resulting from fullreduction to hydroquinone, i.e., the two-electron-reduced flavin. Thisobservation is confirmed by the bell-shaped curve of data corresponding tothe difference in absorption at 390 and 458 nm as a function of the redoxpotential, which was fitted with reduction potentials of 40 15 mV for theFMN

ox/FMN

sqstep and 130 15 mV for the FMN

sq/FMN

redstep. These

values are similar to the aforementioned reduction potentials measured for D. gigasROO( Gomes etal ., 1997 ) and are each approximately 80 mV higher than those determined for M. thermoaceticaFDP ( Silaghi-Dumitrescu et al .,2003 ). As stated earlier, EPR spectroscopy proved to be essential to probe the

FAD

FMN Fe-FeFe-S

A

C

B

D 0.3

700400

FMN Fe-FeFe-S

FAD

600500

700400 600500 600300 500400

700400 600500

l (nm) l (nm)

l (nm) l (nm)

0.0

0.2

0.1

0.3

0.0

0.2

0.1

0.5

0.4

0.6

0.0

0.4

0.2

1.0

0.8

0.3

0.0

0.2

0.1 A b s o r b a n c e

A b s o r

b a n c e

A b s o r b a n c e

A b s o r

b a n c e

Figure 2 .4 Redox properties of Escherichia coli flavorubredoxin and its partner NADH:flavorubredoxin oxidoreductase. Flavorubredoxin and its cognate reductase titrated ina stoichiometric mixture to probe a possible modulation of the redox properties uponinteraction of the two partner proteins. An elaborate spectral deconvolution (describedin the text) allowed us to isolate the redox behavior of each cofactor (in both FlRd andthe reductase). (A) Absolute absorption changes in visible spectra of a stoichiometricmixture of FlRd and FlRd-Red (both at 20 mM , in 50 m M Tris-HCl, 18% glycerol,pH 7.5) titrated with NADH, at 25 C; (B) matrix comprising the optical contribution of the FlRd-reductase to the titration of the mixture (A); (C) matrixof the spectral contri-bution of the Fe-Cys 4 center in the rubredoxin domain; (D) spectral progression of theflavodiiron domain in FlRd, obtained by subtraction of the optical contributions of theother cofactors (B and C) to the overall changes (A); block arrows depict progression of the absorbance changes, i.e., upon reduction absorbance at % 450 nm decreases, whereasabsorbance at % 390 nm initially increases (with the formation of the one electron-reducedflavin) and then decreases upon full reduction of FMN.



15/25

redox properties of the diiron center: data obtained by monitoring this EPR-active species (the mixed valence Fe III -Fe II form) yielded bell-shaped curves,corresponding to its appearance (one electron reduction of fully oxidized

FeIII

-FeIII

to FeIII

-FeII

) and subsequent disappearance (one electron reduc-tion of Fe III -Fe II to the fully reduced Fe II -Fe II state). Data were fitted asintermediate species of two consecutive one-electron step processes withpotentials for isolated FlRd of 20 20 mV for the Fe III -Fe III /Fe III -Fe II stepand 90 20 mV for the Fe III -Fe II /Fe II -Fe II step. Equivalent data for thetruncated form of FlRd consisting of its flavodiiron core (FDP-domain) yielded slightly higher redox potentials (0 20 and 50 20 mV)(Vicente and Teixeira, 2005 ).

With the knowledge of the reduction potentials for all cofactors in

isolated FlRd, studies were undertaken to evaluate the possible changes inits redox properties upon formation of an electron transfer (eT) complexwith its partner, FlRd-reductase. This was accomplished by titrating bothproteins, combined in stoichiometric amounts, and using NADH as thereducing agent. FlRd (or the truncated Rd domain) does not acceptelectrons directly from NADH, so its reduction is exclusively accomplishedby reduced FlRd-reductase. In the titration followed by visible spectros-copy, data comprise absorption features from three almost overlappingredox centers (see Fig. 2.4A ): the FAD from FlRd-reductase, the FMNfrom the flavodoxin module, and the [Fe-Cys

4] center from the Rd module.

Because the reduction potentials of FAD in FlRd-reductase are significantlylower than those of the FlRd centers (depicted in Fig. 2.5 ) (Vicente andTeixeira, 2005 ), deconvolution of FlRd-reductase data is immediate. Thereduction potentials used to fit the data with two consecutive one-electronNernst curves (FAD ox /FAD sq: 250 15 mV and FAD sq/FAD red : 220 15 mV) reflect that reduction of FlRd-reductase proceeds macroscop-ically as a two-electron process. Using the reduction potentials for FlRd-reductase and spectra of its oxidized and reduced forms, it was possible tocreate a matrix of the FlRd-reductase spectral contribution (see Fig. 2.4B )to be subtracted from the titration of the mixture in the following manner:for each experimental potential, a fraction of oxidized and reduced FlRd-Red was assigned and concomitantly a spectrum with the contribution of FlRd-reductase to the overall spectrum of the mixture titration. After subtracting the FlRd-reductase matrix, a matrix was obtained consistingsolely of FlRd spectra in the course of the titration, which were treated inthe same manner as the data obtained for the titration of isolated FlRd(described earlier). Data for the Rd domain were fitted with a one-electronNernst curve with a reduction potential of 65 15 mV (see Fig. 2.5 ),which is upshifted with respect to the isolated FlRd titration. Because thisreduction potential was identical to that measured in another titration of thetruncated Rd domain in a stoichiometric mixture with FlRd-reductase,the analysis proceeded to deconvolution of the reduction potentials for the



16/25

FMN in FlRd-reductase/FlRd titration. This was achieved by subtracting amatrix with the contribution of the Rd domain (see Fig. 2.5C ) from the onecontaining FlRd data (i.e., obtained after subtraction of the FlRd-reductasecontribution). The isolated Rd-domain matrix was in turn obtained bysubtracting the FlRd-reductase contribution from data obtained for titrationof the truncated Rd domain in the presence of FlRd-reductase, as describedearlier for the FlRd-reductase/FlRd titration. By subtracting the Rd-domain matrix from the FlRd matrix (both obtained after subtracting theFlRd-reductase component out of their mixed titrations), a matrix wasobtained comprising solely the flavodiiron core spectral features dominatedby the FMN moiety (see Fig. 2.4D ), identical to the one observed for theisolated FlRd titration. It was then possible to fit potentials for the FMNone-electron reduction to the semiquinone state (40 15 mV) and fullreduction to flavin hydroquinone (130 15 mV) that are identical tothose obtained earlier for the isolated FlRd. To probe the influence of thepresence of FlRd-reductase on the reduction potentials of the nonhemediiron site of FlRd, the 1:1 FlRd/FlRd-reductase titration was repeatedand followed by EPR spectroscopy. Resulting data were treated in thesame manner as the titration of the isolated FlRd and revealed an upshift of 40 mV for each transition. The resulting modulation of the reduction

0.0

0.2

0.4

0.6

0.8

1.0

N o r m a l

i z e d

A b

s

E0 (mV)

300 2000

100 100

FMN

Fe-FeFe-S

FAD

Figure 2 .5 Titration curves for the redox cofactors of Escherichia coli flavorubredoxinand its reductase partner. The best fits to experimental data (described throughout thetext) are represented for eachof the redoxcofactors in the twointeracting partners. FAD,flavin cofactor in FlRd-reductase, ^220 mV for the FAD ox /FAD sq step and ^260 mV forthe FAD sq /FAD red ; FMN, flavin mononucleotide bound to the flavodoxin module, ^ 40 mV for the FMN ox /FMN sq step and ^260 mV for the FMN sq /FMN red ; Fe-S, Fe-Cys 4center in the rubredoxin module, ^65 mV; Fe-Fe, nonheme diiron center in the metallo-b -lactamase domain (measured by combining potentiometry with EPR spectroscopy), 20 mVfor the Fe III -Fe III /Fe III -Fe II stepand^50 mVfor theFe III -Fe II/Fe II-Fe II step.



17/25

potentials of FlRd by its cognate reductase led to the proposal of an electrontransfer mechanism, which is discussed in the next section.

7. Conclusions

The work summarized herein described several experimental meth-odologies that altogether contributed to a successful characterization of flavodiiron proteins from different source organisms. Studies undertaken ata molecular level are essential to complement the functional in vivostudies,with each approach reciprocally contributing to a deeper knowledge on the

structurefunction relationship of a novel family of proteins.Most of the studies on FDPs were performed with recombinant proteinsoverexpressed heterologously in E. coli . The quality of recombinant pro-teins in terms of cofactor incorporation and sample homogeneity is arecurrent challenge. Through a permanent search for improving expressionconditions of flavodiiron proteins, it has been observed that flavin and ironincorporations are favored by lower temperature and decreased aeration of growing cultures, with the latter being achieved by reducing the gasheadspace and stirring speed. Flavodiiron proteins from different sources

were purified successfully in a small number of chromatographic steps,which include anion-exchange and gel filtration columns. A developmentin the quality of purified FDPs was the observation that cofactor integrity(namely FMN) throughout the purification steps benefited from the inclusionof glycerol ( % 18%) in all the buffers.

The redox-active cofactors in FDPs were readily extractable by standardprocedures (typically acid extraction) and easily quantified by spectropho-tometric (flavin) and colorimetric (iron) methodologies. As summarized inTable 2.1, the cofactor content of isolated FDPs (0.71 FMN and 12 Feper monomer of flavodiiron core) corresponds to what can be inferred fromthe peptide sequences, where each monomer comprises one FMN-bindingflavodoxin module and a b-lactamase module where a nonheme diironcenter is embedded. Consistently, FDPs with extra C-terminal structuralmodules have a higher cofactor content. For instance, the E. coli flavoru-bredoxin contains three Fe ions per monomer, one from the [Fe-Cys 4]center in the rubredoxin domain and the diiron center in the lactamasemodule.

The characterization of flavodiiron proteins by spectroscopic techniquesallowed redox-sensitive spectral fingerprints assigned to each cofactor to beestablished. Visible spectroscopy has been used to characterize essentially theflavin moieties (and also the rubredoxin domain in flavorubredoxin) andEPR and Mo ssbauer spectroscopies were employed to study the nonhemediiron center.



18/25

The thermodynamic properties of flavodiiron proteins, in terms of reduction potentials of their cofactors, have only been determined for afew cases. Whereas known potentials for M. thermoaceticaFDP ( Silaghi-

Dumitrescu et al ., 2003 ) and D. gigas ROO ( Gomes et al ., 1997 ) regardsolely their flavin moieties, a thorough redox characterization has beenundertaken with E. coli flavorubredoxin ( Vicente and Teixeira, 2005 ).The determined reduction potentials of FDP-bound FMN moieties rangebetween 0 ! 117 mV for the FMN ox /FMN sq pair and 130 ! 220 mVfor the FMN sq/FMN red pair. These potentials differ from the establishedredox behavior of canonical flavodoxins. Flavodoxins greatly stabilize thesemiquinone state because of the large difference of reduction potentialsbetween the FMN ox /FMN sq pair ( 121 ! 229 mV) and the FMN sq/

FMN red pair (372!

522 mV), an effect attributed to conformationalrearrangements and a series of hydrogen bonds ( Hoover et al ., 1999; Kasimand Swenson, 2000; OFarrell et al ., 1998; Paulsen et al ., 1990 ). Moreover,flavodoxins stabilize the blue neutral semiquinone radical, contrasting withthe red anionic semiquinone observed in FDP-bound flavodoxin modules.Formation of a red anionic semiquinone in FDPs may be related to aprevalence of basic over acidic residues in the FMN pocket ( Frazao et al .,2000 ), which could contribute to lower the p K a of 8.3 for the equilibriumbetween the red and the blue semiquinone forms of free FMN ( Ghisla andEdmonson, 2001 ). The extensive redox characterization focusing on E. coli flavorubredoxin and its cognate reductase combined potentiometric andspectroscopic methods, as described in detail earlier. It should beemphasized that complete understanding of the thermodynamic propertiesof this system was significantly supported by studying the truncated modulesof flavorubredoxin in parallel with the whole enzyme. Results are summar-ized in Fig. 2.6 , depicting the reduction potentials of each cofactor and theproposed electron transfer mechanism inferred on a strictly thermodynamicbasis. By titrating flavorubredoxin in a stoichiometric mixture with thereductase, it was observed that upon interaction of the two redox partners,the reduction potentials of the iron cofactors are upshifted with respect tothe values obtained for the isolated FlRd, whereas those of the flavins inFlRd (FMN) and FlRd-reductase (FAD) remain essentially unaltered.Modulation of the redox properties of FlRd by its reductase poses thepossibility of intramolecular eT steps to be inferred differently, with respectto the reduction potentials of the isolated FlRd. In the isolated protein themore favorable intramolecular eT mechanism involves full reduction of FMN, to further allow two-electron reduction of the diiron site. However,the observed redox shifts resulting from the interaction with FlRd-reduc-tase change the situation regarding possible eT mechanisms. On the onehand, the upshift in the Rd potential creates a thermodynamic barrier for full reduction (two electrons) of FMN. On the other hand, the upshiftobserved in the reduction potentials of the diiron center allows the flavin



19/25

semiquinone to act as a one-electron shuttle to the diiron center, withoutthe need to reach the hydroquinone state.

Assuming this eT mechanism on a pure thermodynamic basis, thefully reduced FlRd under normal operative conditions would have atotal of four electrons available for reductive chemistry, sufficient to catalyzethe reduction of four NO to two N 2O or full reduction of oxygen to water.This mechanism was supported by a thorough kinetic study ( Vicente et al .,2007b,c ).The complexity of the redox behavior of the eT chain composedby flavorubredoxin and its reductase suggests that a redox characterization of other flavodiiron proteins, taking into account all the eT components, mayprovide clues for the efficiency and functionality of the corresponding eT

chains, whereby substrate (NO and/or oxygen) reduction is coupled toNAD(P)H oxidation.

7 .1 . Functional propertiesSince the establishment of the family of flavodiiron proteins, their functionalproperties have been assessed through a combination of parallel in vitroandin vivo studies. Whereas studies on the isolated proteins focused on thestructurefunction relationship and the corresponding in vitro NO and/or oxygen reductase activities, in vivostudies attempted to provide clues for therelative role of each FDP in its source organism.

The first function assigned to a flavodiiron protein concerned theoxygen reductase activity of D. gigas rubredoxin:oxygen oxidoreductase,

FAD

Fe-S

FMN ox

FMN sq

FMN red

Fe III Fe III

Fe III Fe II

Fe II Fe II

+ 20

50 40 65

130

238

Stoichiometric mixture

R e d u c t

i o n

P o t e n t i a l ( m

V )

Figure 2 .6 Electron transfer mechanism of Escherichia coli flavorubredoxin and itsreductase on a strictly thermodynamic base. The scheme depicts reduction potentialsupon interaction of the two redox partners. Curved full arrows indicate probable elec-tron transfer steps inferred on a pure thermodynamic basis. Dotted arrow depicts the(unlikely) formation of the flavin two-electron-reduced hydroquinone.



20/25

proposed to provide a protective mechanism against oxygen toxicity for thisanaerobic organism ( Chen et al ., 1993b; Gomes et al ., 1997 ). Later on, it wasdemonstrated that the E. coli orthologous enzyme flavorubredoxin had

considerable NO reductase activity ( Gomes et al ., 2002 ), in the order of respiratory heme b3:nonheme iron NO reductases. More reports on other flavodiiron proteins revealed that their substrate selectivity is different,despite the structural similarity of the studied FDPs. Whereas E. coli flavo-rubredoxin has a clear preference for NO, FDPs from M. thermoacetica,D. gigas, and D. vulgaris have comparable NO and oxygen reductase activ-ities (refer to Vicente et al ., 2007b ). At the other extreme is the FDP fromM. marburgensis, which reduces oxygen to water exclusively, displaying noactivity toward NO ( Seedorf et al ., 2004, 2007 ).

The role of E. coli flavorubredoxin in in vivoNO detoxification was firstproposed by Gardner and colleagues (2002) based on observations that adeletion of norV , encoding flavorubredoxin, results in a strain with higher sensitivity to nitric oxide releasing compounds ( Gardner et al ., 2002;Hutchings et al ., 2002; Justino et al ., 2005b ). More recently, it has beendemonstrated that a mutant strain of D. gigas where the Dg_ROO encod-ing gene has been silenced is also more sensitive to nitrosative stress (bothNO and GSNO) than the wild-type strain ( Rodrigues et al ., 2006 ). More-over, it has been shown by complementation studies in an E. coli norV mutant that D. gigas ROO ( Rodrigues et al ., 2006 ), as well as FDPs fromM. thermoacetica(Silaghi-Dumitrescu et al ., 2003 ) and D. vulgaris (Silaghi-Dumitrescu et al ., 2005 ), can protect in vivo E. coli from NO toxicity.

In E. coli , the norV gene is cotranscribed in a di-cistronic unit with norW ,which encodes its redox partner, the NADPH:flavorubredoxin oxidore-ductase (da Costa et al ., 2003 ). The norW mutant does not show suchpronounced phenotypes as the norV mutant, and indeed this strain stillretains some NO reductase activity, which is completely abolished in thenorV strain, suggesting an ancillary role for NorW that may be accomplishedby other reductases ( Gardner et al ., 2002 ).

Transcriptional regulation of the norVW operon of E. coli has beenstudied extensively. The norV promoter is activated by reactive nitrogenspecies, both aerobically and anaerobically ( da Costa et al ., 2003; Gardner et al ., 2002; Hutchings et al ., 2002 ), and also during nitrate/nitrite respirationwhen traces of NO may be formed ( da Costa et al ., 2003; Hutchings et al .,2002 ). The regulation of norVW is controlled by the oxygen-sensitivetranscription factor FNR and the nitrate/nitrite responsive regulatorsNarL/NarP ( Constantinidou et al ., 2006; da Costa et al ., 2003 ). Studiesshowing that deletion of the divergently transcribed gene norR causedsimilar phenotype as the deletion of norV (Gardner et al ., 2002; Hutchingset al ., 2002; Justino et al ., 2005b ) and completely abolished the nitrosativeinduction of norVW (Gardner et al ., 2003; Hutchings et al ., 2002;



21/25


22/25

These two systems differ also in their protein expression rates, with FlRdshowing the faster response. Under anaerobic conditions, NO induced max-imal expression of the FlRd protein within 515 min, whereas Hmp

maximal level requires longer times, being reached only after 45 min,suggesting that the faster response is achieved by the enzyme that in vitrohas the higher NO reductase activity, i.e., flavorubredoxin ( Justino et al .,2005b ). Interestingly, the aerobic transcriptional response of hmp and norV to a constant nitrosative stress showed oscillatory behaviors with distinctfeatures. The genes showed two peaks of induction, one after 5 min and theother after 90 min, but while the norV gene showed the highest induction inthe first peak, the induction of hmp was more pronounced in its second peak(Mukhopadhyay et al ., 2004 ). These results further show that flavorubre-

doxin appears to have a more efficient initial response. Thus, it has beendemonstrated that in E. coli , which possesses two inducible NO-detoxifyingsystems, Hmp is an active participant in a broad range of O 2 concentrations,whereas FlRd seems to contribute essentially when oxygen is limited. Itshould be noted that this does not entirely diminish the role of FlRd inprotection from NO toxicity in in vivosituations, as pathogen colonizationoccurs close to anaerobic environments.

So far, only flavohemoglobin appears to have a role in protectingS . enterica(Stevanin et al ., 2002 ) or E. coli from macrophage NO-dependentkilling, as an E. coli norV mutant strain showed similar survival ability as theparent strain ( Pullan et al ., 2007 ). Furthermore, Hmp, but not FlRd, isrequired for Salmonellavirulence in mice ( Bang et al ., 2006 ) under the testedconditions. However, the functional characterization of E. coli flavorubre-doxin (both in vivo and in vitro) clearly shows its involvement in NO-derived stress response, revealing that more studies need to be performedon flavodiiron proteins to fully elucidate their physiological role.

ACKNOWLEDGMENTS

This work was partially supported by FCT Projects POCTI/1999/BME/36558, POCTI/2002/BME/44597, and POCI/SAU-IMI/56088/2004. JBV, MCJ, and VLG benefitedfrom FCT Ph.D. grants, respectively, SFRH/BD/9136/2002, SFRH/BD/13756/2003,and SFRH/BD/29428/2006.

REFERENCES

Andersson, J. O., Sjogren, A. M., Davis, L. A., Embley, T. M., and Roger, A. J. (2003).Phylogenetic analyses of diplomonad genes reveal frequent lateral gene transfers affectingeukaryotes. Curr. Biol. 13, 94104.

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., andStruhl, K. (1995). Current Protocols in Molecular Biology. Greene PublishingAssociates and Wiley Interscience, New York.



23/25

Bang, I. S., Liu, L., Vazquez-Torres, A., Crouch, M. L., Stamler, J. S., and Fang, F. C.(2006). Maintenance of nitric oxide and redox homeostasis by the Salmonella flavohe-moglobin hmp. J. Biol. Chem. 281, 2803928047.

Chen, L., Liu, M. Y., Legall, J., Fareleira, P., Santos, H., and Xavier, A. V. (1993a).

Purification and characterization of an NADH-rubredoxin oxidoreductase involved inthe utilization of oxygen by Desulfovibrio gigas. Eur. J. Biochem. 216, 443448.Chen, L., Liu, M. Y., LeGall, J., Fareleira, P., Santos, H., and Xavier, A. V. (1993b).

Rubredoxin oxidase, a new flavo-hemo-protein, is the site of oxygen reduction to water by the strict anaerobe Desulfovibrio gigas. Biochem. Biophys. Res. Commun.193, 100105.

Constantinidou, C., Hobman, J. L., Griffiths, L., Patel, M. D., Penn, C. W., Cole, J. A., andW., O. (2006). A reassessment of the FNR regulon and transcriptomic analysis of theeffects of nitrate, nitrite, NarXL, and NarQP as Escherichia coli K12 adapts from aerobic toanaerobic growth. J. Biol. Chem. 281, 48024815.

da Costa, P. N., Teixeira, M., and Saraiva, L. M. (2003). Regulation of the flavorubredoxinnitric oxide reductase gene in Escherichia coli : Nitrate repression, nitrite induction, and

possible post-transcription control. FEMS Microbiol. Lett. 218, 385393.Eriksson, S., Lucchini, S., Thompson, A., Rhen, M., and Hinton, J. C. D. (2003). Unravel-

ling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica. Mol. Microbiol.47, 103118.

Fischer, D. S., and Price, D. C. (1964). A simple serum iron method using the new sensitivechromogen tripyridyl-s-triazine. Clin. Chem. 10, 2131.

Flatley, J., Barrett, J., Pullan, S. T., Hughes, M. N., Green, J., and Poole, R. K. (2005).Transcriptional responses of Escherichia coli to S-nitrosoglutathione under defined chemo-stat conditions reveal major changes in methionine biosynthesis. J. Biol. Chem. 280,1006510072.

Frazao, C., Silva, G., Gomes, C. M., Matias, P., Coelho, R., Sieker, L., Macedo, S.,Liu, M. Y., Oliveira, S., Teixeira, M., Xavier, A. V., Rodrigues-Pousada, C., et al .(2000). Structure of a dioxygen reduction enzyme from Desulfovibrio gigas. Nat. Struct.Biol. 7, 10411045.

Gardner, A. M., and Gardner, P. R. (2002). Flavohemoglobin detoxifies nitric oxide inaerobic, but not anaerobic, Escherichia coli : Evidence for a novel inducible anaerobic nitricoxide-scavenging activity. J. Biol. Chem. 277, 81668171.

Gardner, A. M., Gessner, C. R., and Gardner, P. R. (2003). Regulation of the nitric oxidereduction operon ( norRVW ) in Escherichia coli : Role of NorR and sigma54 in the nitricoxide stress response. J. Biol. Chem. 278, 1008110086.

Gardner, A. M., Helmick, R. A., and Gardner, P. R. (2002). Flavorubredoxin, an induciblecatalyst for nitric oxide reduction and detoxification in Escherichia coli . J. Biol. Chem. 277,81728177.

Garfin, D. E. (1990). One-dimensional gel electrophoresis. Methods Enzymol. 182, 425441.Ghisla, S., and Edmonson, D. (2001). Flavin coenzymes. In Encyclopedia of Life Sciences.Gomes, C. M., Giuffre, A., Forte, E., Vicente, J. B., Saraiva, L. M., Brunori, M., and

Teixeira, M. (2002). A novel type of nitric-oxide reductase: Escherichia coli flavorubre-doxin. J. Biol. Chem. 277, 2527325276.

Gomes, C. M., Silva, G., Oliveira, S., LeGall, J., Liu, M. Y., Xavier, A. V., Rodrigues-Pousada, C., and Teixeira, M. (1997). Studies on the redox centers of the terminaloxidase from Desulfovibrio gigasand evidence for its interaction with rubredoxin. J. Biol.Chem. 272, 2250222508.

Gomes, C. M., Vicente, J. B., Wasserfallen, A., and Teixeira, M. (2000). Spectroscopicstudies and characterization of a novel electron-transfer chain from Escherichia coli involv-ing a flavorubredoxin and its flavoprotein reductase partner. Biochem.39, 1623016237.

Goncalves, V. L., Nobre, L. S., Vicente, J. B., Teixeira, M., and M., S. L. (2006).Flavohemoglobin requires microaerophylic conditions for nitrosative protection of Staphylococcus aureus. FEBS Lett. 580, 18171821.



24/25

Hoover, D. M., Drennan, C. L., Metzger, A. L., Osborne, C., Weber, C. H.,Pattridge, K. A., and Ludwig, M. L. (1999). Comparisons of wild-type and mutantflavodoxins from Anacystis nidulans: Structural determinants of the redox potentials. J. Mol. Biol. 294, 725743.

Hutchings, M. I., Mandhana, N., and Spiro, S. (2002). The NorR protein of Escherichia coli activates expression of the flavorubredoxin gene norV in response to reactive nitrogenspecies. J. Bacteriol.184, 46404643.

Jouanneau, Y., Meyer, C., Asso, M., Guigliarelli, B., and Willison, J. C. (2000). Characteri-zation of a nif-regulated flavoprotein (FprA) from Rhodobacter capsulatus:Redox proper-ties and molecular interaction with a [2Fe-2S] ferredoxin. Eur. J. Biochem.267, 780787.

Justino, M. C., Almeida, C. C., Goncalves, V. L., Teixeira, M., and Saraiva, L. M. (2006).Escherichia coli YtfE is a di-iron protein with an important function in assembly of iron-sulphur clusters. FEMS Microbiol. Lett. 257, 278284.

Justino, M. C., Almeida, C. C., Teixeira, M., and Saraiva, L. M. (2007). Escherichia coli di-iron YtfE protein is necessary for the repair of stress damaged iron-sulfur clusters.

J. Biol. Chem. 282, 1035210359. Justino, M. C., Goncalves, V. M., and Saraiva, L. M. (2005a). Binding of NorR to three

DNA sites is essential for promoter activation of the flavorubredoxin gene, the nitricoxide reductase of Escherichia coli . Biochem. Biophys. Res. Commun.328, 540544.

Justino, M. C., Vicente, J. B., Teixeira, M., and Saraiva, L. M. (2005b). New genesimplicated in the protection of anaerobically grown Escherichia coli against nitric oxide. J. Biol. Chem. 280, 26362643.

Kasim, M., and Swenson, R. P. (2000). Conformational energetics of a reverse turn in theClostridium beijerinckii flavodoxin is directly coupled to the modulation of its oxidation-reduction potentials. Biochemistry39, 1532215332.

Mukhopadhyay, P., Zheng, M., Bedzyk, L. A., LaRossa, R. A., and Storz, G. (2004).Prominent roles of the NorR and Fur regulators in the Escherichia coli transcriptionalresponse to reactive nitrogen species. Proc. Natl. Acad. Sci. USA 101, 745750.

Nolling, J., Ishii, M., Koch, J., Pihl, T. D., Reeve, J. N., Thauer, R. K., and Hedderich, R.(1995). Characterization of a 45-kDa flavoprotein and evidence for a rubredoxin, twoproteins that could participate in electron transport from H 2 to CO 2 in methanogenesis inMethanobacterium thermoautotrophicum. Eur. J. Biochem. 231, 628638.

OFarrell, P. A., Walsh, M. A., McCarthy, A. A., Higgins, T. M., Voordouw, G., andMayhew, S. G. (1998). Modulation of the redox potentials of FMN in Desulfovibriovulgaris flavodoxin: Thermodynamic properties and crystal structures of glycine-61mutants. Biochemistry37, 84058416.

Paulsen, K. E., Stankovich, M. T., Stockman, B. J., and Markley, J. L. (1990). Redox andspectral properties of flavodoxin from Anabaena 7120. Arch. Biochem. Biophys.280, 6873.

Poole, R. K. (2005). Nitric oxide and nitrosative stress tolerance in bacteria. Biochem. Soc.Trans. 33, 176180.

Poole, R. K., Anjum, M. F., Membrillo-Hernandez, J., Kim, S., Hughes, M. N., andStewart, V. (1996). Nitric oxide, nitrite, and Fnr regulation of hmp (flavohemoglobin)gene expression in Escherichia coli K-12. J. Bacteriol.178, 54875492.

Pullan, S. T., Gidley, M. D., Jones, R. A., Barrett, J., Stevanin, T. M., Read, R. C.,Green, J., and Poole, R. K. (2007). Nitric oxide in chemostat-cultured Escherichia coli issensed by Fnr and other global regulators: Unaltered methionine biosynthesis indicateslack of S nitrosation. J. Bacteriol.189, 18451855.

Rodrigues, R., Vicente, J. B., Fe lix, R., Oliveira, S., Teixeira, M., and Rodrigues-Pousada, C. (2006). Desulfovibrio gigasflavodiiron protein affords protection againstnitrosative stress in vivo. J. Bacteriol.188, 27452751.

Saraiva, L. M., Vicente, J. B., and Teixeira, M. (2004). The role of the flavodiiron proteins inmicrobial nitric oxide detoxification. Adv. Microb. Physiol.49, 77129.



25/25

Schilling, O., Vogel, A., Kostelecky, B., Natal da Luz, H., Spemann, D., Spath, B.,Marchfelder, A., Troger, W., and Meyer-Klaucke, W. (2005). Zinc- and iron-dependentcytosolic metallo-beta-lactamase domain proteins exhibit similar zinc-binding affinities,independent of an atypical glutamate at the metal-binding site. Biochem. J.385, 145153.

Seedorf, H., Dreisbach, A., Hedderich, R., Shima, S., and Thauer, R. K. (2004). F 420 H 2oxidase (FprA) from Methanobrevibacter arboriphilus, a coenzyme F 420 -dependent enzymeinvolved in O 2 detoxification. Arch. Microbiol.182, 126137.

Seedorf, H., Hagemeier, C. H., Shima, S., Thauer, R. K., Warkentin, E., and Ermler, U.(2007). Structure of coenzyme F420H2 oxidase (FprA), a di-iron flavoprotein frommethanogenic Archaea catalyzing the reduction of O 2 to H 2O. FEBS J. 274, 15881599.

Silaghi-Dumitrescu, R., Coulter, E. D., Das, A., Ljungdahl, L. G., Jameson, G. N.,Huynh, B. H., and Kurtz, D. M., Jr. (2003). A flavodiiron protein and high molecular weight rubredoxin from Moorella thermoaceticawith nitric oxide reductase activity.Biochem.42, 28062815.

Silaghi-Dumitrescu, R., Kurtz, D. M., Jr., Ljungdahl, L. G., and Lanzilotta, W. N. (2005).

X-ray crystal structures of Moorella thermoaceticaFprA: Novel diiron site structure andmechanistic insights into a scavenging nitric oxide reductase. Biochem.44, 64926501.

Sober, H. A., and Harte, R. A. (1968). Handbook of Biochemistry: Selected Data for Molecular Biology. CRC Press, Boca Raton, FL.

Solomon, E. I., Brunold, T. C., Davis, M. I., Kemsley, J. N., Lee, S. K., Lehnert, N.,Neese, F., Skulan, A. J., Yang, Y. S., and Zhou, J. (2000). Geometric and electronicstructure/function correlations in non-heme iron enzymes. Chem. Rev. 100, 235350.

Stevanin, T. M., Poole, R. K., Demoncheaux, E. A. G., and Read, R. C. (2002).Flavohemoglobin Hmp protects Salmonella entericaserovar typhimurium from nitricoxide-related killing by human macrophages. Infect. Immun. 70, 43994405.

Susin, S., Abian, J., Sanchez-Baeza, F., Peleato, M. L., Abadia, A., Gelpi, E., and Abadia, J.(1993). Riboflavin 3 0- and 5 0-sulfate, two novel flavins accumulating in the roots of iron-deficient sugar beet ( Beta vulgaris). J. Biol. Chem. 268, 2095820965.

Vicente, J. B., Carrondo, M. A., Teixeira, M., and Fraza o, C. (2007a). Structural studies onflavodiiron proteins. Methods Enzymol. 437 [1] (this volume).

Vicente, J. B., Gomes, C. M., Wasserfallen, A., and Teixeira, M. (2002). Module fusion inan A-type flavoprotein from the cyanobacterium Synechocystiscondenses a multiple-component pathway in a single polypeptide chain. Biochem. Biophys. Res. Commun.294, 8287.

Vicente, J. B., Scandurra, F. M., Forte, E., Brunori, M., Sarti, P., Teixeira, M., andAlessandro Giuffre , A. (2007b). Kinetic characterization of the Escherichia coli nitricoxide reductase flavorubredoxin. Methods Enzymol. 437 [3] (this volume).

Vicente, J. B., Scandurra, F. M., Rodrigues, J. V., Brunori, M., Sarti, P., Teixeira, M., andGiuffre , A. (2007c). Kinetics of electron transfer from NADH to the Escherichia coli nitricoxide reductase flavorubredoxin. FEBS J. 274, 677686.

Vicente, J. B., and Teixeira, M. (2005). Redox and spetroscopic properties of the Escherichiacoli nitric oxide-detoxifying system involving flavorubredoxin and its NADH-oxidizingredox partner. J. Biol. Chem. 280, 3459934608.

Walker, J. M. (1994). The bicinchoninic acid (BCA) assay for protein quantitation. MethodsMol. Biol. 32, 58.

Wasserfallen, A., Huber, K., and Leisinger, T. (1995). Purification and structural characteri-zation of a flavoprotein induced by iron limitation in Methanobacterium thermoautotrophicumMarburg. J. Bacteriol.177, 24362441.

Wasserfallen, A., Ragettli, S., Jouanneau, Y., and Leisinger, T. (1998). A family of flavo-proteins in the domains Archaea and Bacteria. Eur. J. Biochem. 254, 325332.


Documents

Biochemical, Spectroscopic, And Thermodynamic Properties