Reductase from the Peptidolytic Sulfate-Reducing Bacterium Desulfovibrio aminophilus DSM 12254

Purification

and

Preliminary

Characterization of

Tetraheme

Cytochrome

and

Adenylylsulf te

Reductase

from the Peptidolytic Sulfate-Reducing

Bacterium

_{Desulfovibrio}

aminophilus

DSM

12254

Alejandro

L6pez-Cort6s

*,

Sergey

Bursakov

,

Angelo Figueiredo

,

Anders

E.Thapper

,

Smilja

Todorovic

,

Jos

J.G.

Moura

,

Bernard Ollivier

3,

Isabel

Moura

and

Guy

Fauque

Centro

de lnvestigaciones

Biol6gicas

del

Noroeste, (CIBNOR). Mar

Bermejo 195,

Playa Palo

Santa

Rita,

La Paz,

Baja

_California

Sur

23090, Mdxico.

2REQUIMTE/CQFB,

Departamento

de Quimica, Faculdade de

Cinciase

Tecnologia da

Universidade

Nova

de Lisboa,

2829-516 Monte

de Caparica,

Portugal.

3Laboratoire

de Microbiologie

IRD,

UR 101,

IFR-BAIM,

ESIL

UniversitOsde

Provence

etde la Mditerrane,

Campus

de

Luminy, Case 925,

13288 Marseille Cedex

09, France.

ABSTRACT

Two proteins werepurified andpreliminarilycharacterized from the solubleextract ofcells

(310

g, wet

weight) of theaminolytic and peptidolytic sulfate-reducingbacterium

_{Desulfovibrio}

(D.) aminophilus DSM

12254. The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5"-phosphosulfate,

APS)

reductase, a key enzyme in the microbial dissimilatory sulfate reduction, has been purified in three chromatographic steps (DEAE-Biogel

A,

Source 15, and Superdex 200

columns).

Itcontains two different subunits withmolecular

massesof 75 and 18 kDa.The fraction after the last purification step hadapurity index

(A278

,m/A388

nm)

of

5.34, which was used for further EPR spectroscopic studies. The D. aminophilus APS reductase is very

similar to thehomologous enzymesisolatedfromD.gigas andD.

_{desulfuricans}

ATCC27774. Atetraheme

cytochrome c3 (His-heme iron-His) has been purified in three chromatographic steps

(DEAE-

Biogel A, Source 15, and Biogel-HTP

columns)

and preliminarily characterized. It has a purity index

([A.s.s3

nm"

A570

nm]rcd/m280nm

ox)

of 2.9 and a molecular mass of around 15 kDa, and its spectroscopic characterization

(NMR

and

_EPR)

has been carried out. This hemoproteinpresentssimilarities with the tetrahemecytochrome

c3 from

Desulfomicrobium

(Des.)

norvegicurn

(NMR

spectra,and N-terminal amino acidsequence).

Corresponding author: Alejandro L6pez-Cort6s. Phone:

(612)

123-84-25Fax:

(612)

125-36-25

Vol. 3, Nos. 1-2, 2005

_{Purification.}

andPrelim&wyCharacterization

_of

Tetraheme Cylochromec3

Abbreviations: adenosine 5’-phosphosulfateor adenylylsulfate

(APS),

flavin adenine dinucleotide

(FAD),

adenosinemonophosphate

(AMP),

diethylaminoethyl

(DEAE),

hydroxyapatite

(HTP),

electronparamagnetic

resonance

(EPR),

nuclear magneticresonance

(NMR),

polyacrylamidegel electrophoresis

(PAGE),

sodium

dodecyl sulfate

(SDS),

2,2-dimethyl-2-silapentane-5-sulfonate

(DSS),

ultraviolet

(UV),

Desulfovibrio

(D.),

Desulfomicrobium

(Des.),

Deutsche Sammlung von Mikroorganismen

(DSM),

Laboratoire de Chimie Bacterienne,

(LCB),

CentreNationalde laRecherche Scientifique

(CNRS),

basiclocal alignment search tool (BLAST), D6partement Soutien et Formation des Communaut6s Scientifiques du Sud (DSF), Institut de

RecherchepourleDeveloppement,

(IRD).

INTRODUCTION

Sulfate-reducing bacteria constitute a group of anaerobic prokaryotes sharing the capacity to carry out

dissimilatory sulfate reduction to sulfide as a major component of their bioenergetics processes

[1-3],

and contain a complexand diversifiedelectroncarriersystem

[4,

5].D. aminophilus DSM 12254 is amesophilic

strainof_{sulfate-reducing}bacteriumisolated froman anaerobicsludge ofa dairywastewater treatmentplant

in Santa Fe de

Bogota,

Colombia

_[6].

This strain uses a widerrange ofenergy substratesthan reported for

most

_{Desulfovibrio}

species

[1

]. It presents, in particular, an important propertyoffermenting oroxidizing proteinaceous compounds, such as amino acids and peptides. This sulfate-reducing strain is also able to

disproportionatesulfiteandthiosulfate, suggestingthat itplaysamajorrole in regulating electronflow in the

dissimilatory sulfurcycle

[6].

We report here purification and preliminarycharacterization of two proteins

involved inthe respiratory system ofD. aminophilus: one electroncarrier, the tetraheme cytochromec3, and

oneenzyme,theadenylylsulfatereductase.

EXPERIMENTAL

Bacterial strainand

growth

conditions

D.aminophilusDSM 12254 wasgrownat37Cin a lactate/sulfatemediumunder anaerobic conditions in the Unit6 de Fermentation, Laboratoire de Chimie Bacterienne (LCB), Centre National de la Recherche Scientifique

(CNRS),

inMarseille,

France,

andcellswereharvestedaspreviouslydescribed

[7].

Preparation of the soluble fraction

A. Lopez-Cortesetal. Bioinorganic Chemistry and Applications

Proteins

purification

The soluble fraction was then loaded onto a DEAE Bio-Gel A column

(Bio-Rad,

44 x 4.5

cm)

equilibratedwith 10mM Tris-HC1 buffer,

pH

7.6.

A

gradientof 11 10mMTris-HC1

pH

7.6and 11 500mM

Tris-HC1

pH

7.6 was setup. Fivemajorproteinswereelutedfromthecolumn: tetrahemecytochromec3,two

molybdenum iron-sulfur-containing proteins

(one

withaldehyde oxidoreductaseactivity), bisulfite reductase of the desulfoviridin-type, and adenylylsulfate reductase. Two of these proteins

(tetraheme

cytochrome c3

and APS

reductase)

were completely purified in two supplementary purification steps. The tetrahemiC

cytochrome

c.

fraction, after concentration on a Diaflo apparatus using a YM-10 membrane, was then appliedto a Pharmacia Biotech ionexchangecolumnSource15

(32

x2.6

cm)

equilibratedwith10

mM

Tris-HC1

pH

7.6 and eluted with 10mMTris-HC1

pH

7.6 to 10mMTris-HC1

pH

7.6 and 500mM NaC1. After

concentration(DiafloYM-10

_membrane),

thetetraheme cytochromec3wasfinallypassedover aBiogel-HTP

column

₍₄₅

x 1.6

cm)

equilibrated with 10 mM Tris-HC1

pH

7.6 andeluted with a continuous gradientof sodium phosphate buffer

pH

7.6

(250

ml 10 mM/250 ml 250

mM).

After concentration

(Diaflo

YM-30

membrane),

theAPS reductase fractioneluted from the DEAE-Biogel Acolumnwasappliedto aSource 15

column (Pharmacia Biotech, 32 x2.6

cm)

equilibratedwith 10mMTris-HC1buffer

pH

7.6andelutedwith 10 mM Tris HC1

pH

7.6 to 10 mMTris-HC1 + 500 mM NaC1. APS reductase after concentration

(Diaflo

YM-30

_membrane)

was finallypassed over a Superdex 200column

(Amersham

Biosciences, 67 x 2.6

cm)

equilibratedwith50mMTris-HC1

pH

7.6 and 300mMNaC1.

Molecular mass and

purity

determination

Subunitcomposition, molecular mass,andpurityofproteinswere determinedbydenaturingPAGE,using

as runningbuffer Tris (0.025M)-glycine

(0.192M),

SDS

(0.1%)

pH

8.3. Lowmolecular weightkit markers forSDS electrophoresis(Pharmacia Biotech

17-0446-01)

wereusedforthe calibration ofAPS reductase and

tetraheme cytochromec3. The proteinstandards withapproximatemolecular weightswere: phosphorylaseb,

94 kDa; albumin, 67 kDa; ovoalbumin 43 kDa;carbonic anhydrase 30 kDa; trypsin inhibitor 20.1 kDa and alfa lactalbumin 14.4 kDa.Thegelswerestainedforproteinbycoomassieblue0.5%.

Ultraviolet (UV)-visible

spectroscopy

UV-visible absorption spectrawere recorded ona ShimadzuUV-2101 PC splitbeamspectrophotometer using1-cmquartz cells.

Electron

paramagnetic

resonance

(EPR) spectroscopy

Vol. 3,

Nos.

1-2,2005

_Purification

and Prelhninary Characterization

_of

Tetraheme Cytochrome

c

Nuclear

magnetic

resonance

(NMR)

spectroscopy

The NMR spectra were taken for a 0.8-1.0 mM tetraheme cytochrome c3 sample in

DeO.

The NMR

spectraofD. aminophilus tetrahemecytochromec3intheoxidized state were recorded on a400MHzBruker

ARX-400 spectrometer equipped with an inverse detection 5-mm probe and a variable temperature unit BrukerB-VT2000. The 1D-NMR spectraweremeasuredinoxidizedstate at317K, 313K, 308K, 303K, and 283.2K (pH

7.6)

with a spectral width of 40.3kHz and a transmitter power level of 1.0 dB. All experimentswere obtained with waterpre-saturationand chemical shift andpresented inppmrelative to the internalstandard2,2-dimethyl-2-silapentane-5-sulfonate

(DSS).

N-terminal amino acid

sequencing

of tetraheme

cytochrome

c3

N-terminal amino acid sequence of D. aminophilus tetraheme cytochrome c3 was determined by

automated Edmandegradation in

a

proteinsequencer (AppliedBiosystemmodel

477)

coupledtoan analyzer (Applied Biosystem model 120) following the manufacturer’s instructions, using 100 pmol oftetraheme cytochromec3.

RESULTS

AND DISCUSSION

Adenylylsulfate

(APS)

reductase

Although the firstreportsonAPS reductasewerepublishedinthe sixties,itsthree-dimensional structure was published only recently

[8].

Comparison of physicochemical and spectroscopical properties of APS reductases isolated from several

_{Desulfovibrio}

species show great similarity and high degree of homology

[9].

Only recently has APS reductase been isolated from Archaeoglobus fulgidus and found to be a heterodimer with one subunit

(75

kDa,

1FAD)

and one subunit

(18

kDa, 2

[4Fe-4S]) [10].

APSreductasehas

been purified in threechromatographic steps fromthe soluble fractionofD. aminophilus and preliminarily characterized.The UV-visiblespectrumofAPSreductaseinthenativeformshowsabroadmaximumaround 392nmwithshoulders at 445 and 475 nm andaproteinabsorption peakat278 nm(unpublished

results).

The

overallvisiblespectrumindicatesthe presence ofa flavin group andironsulfurcenters. APS reductase isa heterodimer with one subunit

(75

kDa, containing

FAD)

and one subunit

(18

kDa, containing

214Fe-4S]

centers)

(Fig.

1).

EPR spectroscopic studies were carried out at different temperatures with the D. aminophilus APS

reductase in the native state(Fig.

2-A)

inthepresence ofnaturalsubstrates

(AMP

andsulfite)(Fig.

2B)

and in th.e reduced form (Fig. 2C and

2D). Temperature

dependence studies helped separate resonances

originatingin different species. Inthe nativestate (Fig.

2A),

twoclusters ofAPSreductase are in the

[4Fe-4S]

2/oxidizedstate,withfour iron-atomsasFe

a’5+,

giving total spinofS 0. Nevertheless, theEPRspectrum showsa signal spread aroundg 2.00. The spectral shape and g valueof the signalindicate thatthe broad

resonance accountsfor only 0.1-0.25 spins/mol,which canbe attributed to the residual

[3Fe-4S]

cluster

[9].

A. Lopez-Cortesetal. _{Bioinorganic ChemistryandApplications}

94 kDa

67

kDa

43

kDa

30 kDa

2

20.1 kDa

14.4

kDa

Fig. l:"Denaturing SDS-PAGE 12.5%. Lane 1: profile of low molecular-weight markers. Lane 2: a heterodimerAPSfromD. aminophilusreductase with onesubunit, around 75 kDa, containing

FAD,

andanothersubunitvisible,around 18kDa, fromD.aminophilus.

former resonance can be detectedupto -30K andthe latterup to45K. The short reduction of the protein

with

Na2S204

(at

15 sec,

pH 9.5)

(Fig.

2C),

aswellastheadditionof thesubstratesAMPand

Na2SO3

(Fig.

2-B),

gives rise to a rhombic signal(gl 2.084,g2 1.94 andg3

1.90)

thatwasattributed to thereduced

[4Fe-4S]

Center I, S 1/2. In addition to the iron-sulfur cluster resonances, therewas ag 2.0048 signal originating from the FAD radical and the residual resonance ofthe native APS reductase. The studies of

temperature andpowerdependence ofthespectraindicatethattheFADradical ispresent upto 100K (data

not

_shown),

whilethe Center cluster can be seen upto45K.Apparently,the longreduction with

Na2S204

(Fig.

2D)

did not result in a fullyreduced APS reductase after several attemptsto fully reduce the sample.

After morethan30 min of reduction with dithionite(pH

9.5),

both

[4Fe-4S]

clustersshouldbereduced, each having total spin S 1/2, but the EPR spectrum ofAPS reductase most likely originates from only one reduced

[4Fe-4S].

It is not clear at thispointwhether this can be related to some specific characteristic of

APSreductase inD.aminophilus.

APS reductase isamajor cytoplasmic enzyme constituting2 to3%of solubleproteinsinsulfatereducers of the genus

_{Desulfovibrio}

[4,

5].

It is a nonheme iron flavoprotein which has also been found in several genera of sulfate-reducing bacteria: Desulfobacter,

Desulfotomaculum,

Desulfosarcina,

Desulfococcus,

Desulfobulbus,

Thermodesulfobacterium,andArchaeoglobus

[4,

9, 10, 11

].

APSreductase fromeightspecies

Yol. 3, Nos. 1-2, 2005

A

3000

8*K

10*g

/’’-

33"K

45"K

3200 3400 3600 3800

Magnetic Field

(Gauss)

Purification

and PreliminaryCharacterization

_of

Tetraheme

CS’tochrome

c3

B

120K

!8"K

("-22"K

28"K 35"K

____....__

450K

3000 3200 3400 3600 3800

Magnetic Field

(Gauss)

C

45"K

i

=

i

3000 3200 3400 3600 3800

Magnetic Field

D

25"K

3000 3200 3400 3600 3800

MagneticField(Gauss)

Fig. 2:

Temperature

dependece of EPR spectra of APS reductase fromD. aminophilus with:

(A)

Native

enzyme, signal from residual

_[3Fe-4S]

cluster.

(B)

nativeAPS reductaseincubated with substrates

AMP and

SO3

"2.

(C)

native APSreductasewith Na2S20, ~15sec (only Center is

reduced).

(D)

APS reductase with Na2S20, > 30 min

(both [4Fe-4S]

clusters are

reduced).

The spectrometer

conditions were: microwave power: 2

mW;

microwave frequency: 9.485 GHz; modulation

amplitude:8G;modulationfrequency:100 kHz;field center: 3400G;sweepwidth: 1000 G.

spectroscopic properties determined. They present a high degree of homology in their physicochemical

characteristicsand their visible andEPRspectra

[9].

APS reductases isolated from

_{Desulfovibrio}

speciesare

proteins containingone FAD permoleculeandeightiron atomsarranged in two

[4Fe-4S]

clusters

(Center

andCenter

II).

Theyhave a monomericmolecularmass rangingbetween 150 and 180kDaandpossesstwo different subunits with molecular masses of around 20 and 70 kDa [9]. The reaction ofsulfite with APS

A. Lopez-Corteset

aL

BioinorganicChemistryandApplications

appearance of a maximum at 320 nm, corresponding to the reaction of sulfite at the N-5 position of the isoalloxazine ringofFAD.The subsequentaddition ofAMP resultsin a decrease in absorbance at 320 nm, partial reductionof iron-sulfurcenters, andtheformation ofAPS. Acommon feature of all APS reductases

from

_{Desulfovibrio}

speciesisthe perturbationoftheEPR spectralfeatures ofCenter afterits reaction with

AMP and sulfite, as well as its high redox potential

(0

to -50

mV)

when compared with other

[4Fe-4S]

clusters. Center II is a

[4Fe-4S]

cluster with a redox potential lower than -400mV

[9].

APS reductase in

sulfate-reducingbacteria is an enzyme highlyconserved in terms of itscompositionatthe active site aswell

asitsphysiologicalproperties.

Tetrahemic cytochromec3

A tetrahemic cytochrome (,’3 (His-heme iron-His) has been purified inthree chromatographic steps from the D. aminophilus soluble extract. It has a purity index

([A553

nm-A570

nm]rc,/Aes0

ox)

equal to 2.90.

Denaturing SDS-PAGE corroborated the purity of this protein (Fig.

3).

The UV-visiblespectrum of the oxidized tetrahemiccytochromec3exhibits abroadabsorptionband around531nm

(beta

band),aSoret peak

(gamma

band)

with a maximum at410 nm, another broad bandat350 nm(delta band),andaproteinpeakat

280 nm(Fig.

4).

The tetraheme cytochrome c3 is not reduced by sodiumascorbate, but is fully reduced by

94

kDa

67

kDa

43

kDa

30

kDa

Fig. 3:

20.1 kDa

Denaturing SDS-PAGE 15%. Lane 1: profile oflowmolecular-weight markers. Lane2: tetrahcmic

cytochrome c3 (5

tl).

Lane 3: tetrahemic cytochrome c3 (2

tl)

from D. aminophilus; singlc band

ol.3, Nos. 1-2, 2005

_Purification

and PreliminaryCharacterization

_of

Tetrahetne Cytochromec3

1.6

1o4

0..0

VAVE.LENGTtl nm

Fig. 4: UV-visibleabsorption spectraofD.aminophilus tetrahemecytochromec3in the oxidized(thin line)

and reduced(thick line)forms.

5

,1.

2.

I.

.,|

...

MagneticField(Gauss)

Fig. 5: EPR spectrum ofD. aminophilus ferritetraheme cytochrome c3. Conditions ofmeasurement were:

temperature l0K, microwave power: 2 mW; microwave frequency: 9.652 GHz; modulation

amplitude" 10G;modulationfrequency: 100kHz;fieldcenter" 3400G; sweepwidth 4000G.

sodiumdithionite,showing absorptionmaxima at553nm(alpha

band),

523nm

_{(beta band),}

andaSoretpeak

at 418 nm (gamma

band)

(Fig.

4).

Figure 5 shows the EPR spectrum ofthe D. aminophilusferritetraheme

cytochromec3 recordedat 10K. The spectrum shows aprominentfeature atg 2.920 inthe gmax region

and a derivativepeakisobservedatg 2.276

(probably

g

med).

Figure6showsthe

NMR

spectrumoftheD. aminophilus ferritetraheme cytochrome

ca

recordedat313K. This NMR spectrum isvery closeto the one reported forDes. norvegicumtetrahemecytochrome c3

[12]

with twomethyl resonances around 25ppmand

A. Lopez-Cortesetal. Bioinorganic ChemistryandApplications

30 15 10

ppm

Fig. 6: NMR spectrumofD. aminophilusferritetrahemecytochromec3(concentration0.8-1.0mM,

pH 7.6)

recorded at 313K. Conditions of measurement were: spectral width 40.3 kHz and a transmitter

power level 1.0dB.

D.

aminophilus

(1-32)"

DSM12254

1 ADAPKQDIV

MKS

PGAXAQGTVTLSHAKH

GGQQ 32

ADAP

DV

MK+

PGA

Q

TV

H

Des.

norvegicum

(1-39):

1

ADAPGDDYVISAPEGMKAKPKGDKPGA-LQKTVPFPHTKH

39

P00136

Fig.7" N-terminal amino acid sequencing of tetraheme cytochrome c3 fromD. aminophilus DSM 12254.

This amino acid sequence was compared with other tetraheme cytochrome c3 sequences using

BLAST. Results showed a significant alignment with the primary structure of the homologous protein, tetraheme cytochrome c3 of 13 kDa, GenBank accession number P00136 from Des.

norvegicum. Identities 17/40

_(42%),

Positives 18/40

(44%),

Gaps 13/40

_(32%).

tetraheme cytochromec3 wasdetermined up toresidue32(Fig.

7).

Thissequencewas investigatedwiththe

protein Basic Local Alignment SearchTool software

(BLAST),

and showed a significant alignmentwith a tetraheme cytochrome c3 of 13 kDa, with 118 residues

(GenBank

accession number P00136 from Des.

norvegicum DSM1741,formerlycalledD.

_{desulfuricans}

strain

Norway 4).

Tetrahemecytochrome c3istheonlyhemoprotein presentinlargeamounts inall

Desulfovibrio

speciesso

far isolated, and it is characteristic of this genus although it has also been found in two

Thermodesulfobacterium

species, Des. norvegicum and

_{Desulfobulbus}

elongatus

[4,

5]. Tetraheme cytochrome c3 is a small

(14

kDa)

monomeric protein located in the periplasmic space that plays an

important role in the metabolism of dissimilatory sulfate reduction

[4,

5]. No unequivocal physiological

function has beenclearly establishedfor tetrahemecytochromec3 even if it can act as a sulfurreductase in

several strains of

_{Desulfovibrio}

and

_{Desulfomicrobium}

/13

]. The four hemes of tetraheme cytochromes c3

hemes have histidine-histidineligation, andasshownby EPRandNMR spectroscopies, theyare localized in

non-equivalent protein environments, where each heme has a differentredox potentialvalue ranging from-50 to-400mV

[4,

5, 14]. EPR spectraoftetrahemecytochromesc3show features of four differentlow-spin

Vol.3, Nos. 1-2, 2005

_Purification

and Preliminary Characterization

of

Tetraheme Cytochromecj

tetraheme cytochrome c3s have been determined by

X-ray

diffraction

[16-19].

The most striking

characteristics of the three-dimensional structures of the tetraheme cytochromes c3 are the compact organization of the four hemes with a relatively high degree of solvent exposure. Despite the rather low

homology among the amino-acid sequences of tetraheme cytochromes c3

(lowest

homology of

20%),

no

significantdifferences in the overallstructure and spatial arrangement ofthefour hemeshavebeen observed

[16-19].

Here,we described the purification and preliminary characterization oftwo key proteinsinvolved inthe dissimilatory sulfatereductionpathwayofD. aminophilus.Wehaveshown that, accordingto theUV-visible

and EPR spectra, D. aminophilusAPSreductase isveryclose to thehomologousenzymes isolated fromD.

gigas andD.

_{desulfuricans}

ATCC27774.D. aminophilustetrahemecytochromec3 presentsmorehomology

with the homologous protein present in Des. norvegicum (N-termimd amino acid sequence and NMR spectra).

ACKNOWLEDGEMENTS

We areindebted to R. TociandM. Bauzanforgrowing the bacteriaused in thisstudy. A. L6pez-Cort6s

received financial support from the D6partement Soutien et Formation des Communaut6s Scientifiques du

Sud

_{(DSF)-Institut}

de Recherche pour le D6veloppement

(IRD),

France, during his

postdoctoral

stay in

Marseille,France.EditorialstaffatCIBNORimproved the Englishtext.

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