Quaternary structure of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa and its reoxidation with a novel cytochrome c from this organism

Quaternary

structure

of

quinoprotein ethanol dehydrogenase

from

Pseudomonas

aeruginosa

and its reoxidation with

a

novel cytochrome

c

from this

organism

Johanna M. J.

SCHROVER,* Johannes FRANK,* John E. VAN WIELINKt

and Johannis A.

DUINE*t

*Department ofMicrobiology and Enzymology, Delft University ofTechnology, Julianalaan 67, 2628 BC Delft,The Netherlands,

and tDepatmnent of Microbiology, Biological Laboratory,Vrije Universiteit, deBoelelaan 1087, 1081 HV Amsterdam, The Netherlands

Quinoprotein

{2,7,9-tricarboxy-IH-pyrrolo-[2,3-fJquinoline-4,5-dionequinoneform (PQQ)-containing} ethanol dehydrogenase

(EDH)from Pseudomonas aeruginosa ATCC 17933 was purified

to homogeneity. EDH has an

a2/.2

configuration and subunits comparable in size to those of methanol dehydrogenase (MDH).

Comparedwith otherPQQ-containingdehydrogenases, Ca2+ is rather loosely bound and it seems necessary for PQQ binding and stabilityofEDH. Two solublecytochromes c were detected inextractsfrom ethanol-growncells and both were purified.One

of these has an a-band at 551 nm for its reduced form, the

oxidized form being an excellent electron acceptor for the

semiquinone form of EDH. Since this cytochrome is quite different from the already known cytochromec551(operating in

INTRODUCTION

Gram-negative, methane- ormethanol-utilizing bacteriaoxidize methanolviaquinoprotein (PQQ-containing) methanol

dehydro-genase (EC 1.1.99.8, MDH). Theenzymehasanal24 structure

with subunits of 60 and 10 kDa[1,2].Electrontransferfromthe fully- and half-reduced forms of MDH occurs to a specific,

soluble, haemc-containingcytochrome, commonlyindicatedas

cytochrome cL. Reduced cytochrome CL becomes subsequently oxidized by acytochrome namedcytochrome CH [3].

Ethanol-grown Pseudomonas aeruginosa contains an

NAD-dependentas wellasaquinoprotein alcoholdehydrogenase [4].

Thelatterenzyme,which has been named ethanoldehydrogenase (EDH),hasbeenpurifiedfromP.aeruginosa [5,6]and P.putida ([7]; B. W. Groen and J. A. Duine, unpublished work) and its

quinoproteinnatureestablished [5]. EDH and MDHappear to

haveproperties incommon: therequirementforahigh pHand thepresence ofammoniaorprimaryaminesasactivators in the in vitroassaywithartificial electronacceptors[8];theshapeand maxima of theirabsorption spectra and theeffect of the suicide inhibitorcyclopropanone, suggestingasimilarcatalyticrole for

PQQin bothenzymes[5,9]andthepresenceof Ca2+[10,11].To

enable further comparison this paper describes studies which

attempt toelucidate the quaternary structure andto detect the electronacceptorfor EDH.

MATERIALS AND METHODS

Organism

and growth conditions

P.aeruginosaLMD 89.1(ATCC 17933)wasculturedaerobically

ina20 1 fermentorat37°Con amineral medium[12]with-0.5%

nitrate respiration) of this organism, it is indicated here as cytochrome CEDH. Comparison of the N-terminal amino acid sequence of cytochrome CEDH with the complete sequence of

cytochrome CL (the electron acceptor of MDH), cytochrome CH

(the electron acceptor ofcytochrome CL) and cytochrome

c551

revealed some similarityonly tointernalstretchesof amino acids

ofthelasttwo.Theother solublecytochromeappearedtobe the already-known cytochrome c556. Since it was not an electron acceptorforcytochromeCEDH (neitherforEDH),cytochromeCH is lacking in thequinoprotein-EDH-ethanoloxidation system of P.aeruginosa. It seems, therefore, that the respiratory chainsfor MDHand EDH aredifferent.

ethanolas acarbon and energysource.The pHwasmaintained

at 7.0 with 3 MKOH, the airflow was 3

1/min

and the vessel

wasstirredat300rev./min.Cellswereharvestedattheendof the

exponential growth phase (A400-A600 1.0). Theyieldwas 1.7 g

cells (wet weight)/l.Cell pastewas stored frozen.

Isolation

of

ethanol

dehydrogenase and soluble cytochromes

c

Cell paste (30 g) was suspendedin anequal volumeof 10 mM

Mops buffer, pH7.0

(buffer

A). After

adding

DNAase to the

suspension, the cells were disruptedtwice in a French pressure cellat110MPaand thesuspensionwascentrifuged for 10 minat

16300g. Protein in the supernatant precipitating at

400%

and 40-80%

(NH4)2SO4

saturation was collected by centrifugation

at48 200 gfor 20 min. The 40-80% precipitatewasdissolved in

a minimal amount of buffer A and dialysed against the same

buffer.

The dialysed preparationwasloaded on a DEAE-Sepharose

column (Pharmacia)

(1O

cmx5cm)

equilibrated

with 10 mM Mops buffer, pH7.0, containing 10 mM

CaCI2

(buffer B).

Ethanol dehydrogenase and

cytochrome

c556 are not retained

under these conditions andwerecollected in a total volume of 100ml. The bound cytochrome

c1EDH

was eluted with a linear NaCl

(1

M) gradient in buffer B in 60 min at a flow rate of

2ml/min at a concentration of 0.4 M NaCl in a fraction of

70ml. This was brought to 3M in ammonium acetate and

subjected to

hydrophobic-interaction chromatography

on a

Fractogel TSK Butyl-650 (S) column (Merck) (15cmx3

cm)

equilibrated with buffer B and

containing

3 M ammonium

acetate.The columnwaselutedwithalinear

gradient

from 3.0 M

to zeroammoniumacetatein buffer B in 60 minat aflowrateof

Abbreviations used:PQQ, thequinoneform of2,7,9-tricarboxy-1H-pyrrolo-[2,3-f]quinoline-4,5-dione; MDH,methanoldehydrogenase;EDH,ethanol dehydrogenase;

EDH,,,

EDHin thesemiquinonestate; EDHOX,fullyoxidized formof EDH.

1ml/min. Cytochrome

cEDH

was eluted at a concentration of

0.8 M ammoniumacetate.Afterconcentrating the fractionwith a Centriprep-lO concentrator (Amicon) to 2ml, cytochrome CEDH wasapplied to a Superdex 75 gel-filtration column (Phar-macia)equilibrated with 0.1 M sodium phosphate buffer, pH 6.5.

Chromatographywascarried outby elutingwith the samebuffer ataflow rate of 0.5 ml/min. The cytochrome c

EDH-containing

fractions were pooled and dialysed against buffer A.

After adding ammonium acetate (3 M final concentration) to the solutioncontaining EDH and cytochrome

c556,

hydrophobic-interaction chromatography was carried out as described for

cytochrome cEDH. EDH and cytochrome c556 were eluted at concentrations of 1.0 and 0.2 M ammonium acetate, respectively. After concentrating, each protein was subjected to gel-filtration

chromatography by injecting on a Superose 12 gel-filtration

column (Pharmacia), equilibrated with 10 mM Mops buffer, pH7.0, containing 10 mMCaCl2 and 0.1 M NaCl. The

EDH-containing fractions were dialysed against buffer B and the cytochrome c556 fractionsagainstbuffer A.

In all cases, the purification was carried out at room tem-peratureand thefinal preparations were stored at -80 'C.

Enzyme assays

EDH activitywas measuredbyfollowing the reduction rate of Wurster's Blue (NNN'N'-tetramethyl-p-phenylenediamine free radical [13]),at612nm

(e612

12.7mM-1 cm-1).Forthat purpose, 33

,1

0.3 mM Wurster's Blue, 900,1 of 0.1 M sodium borate

buffer,pH 9.0,containing50 mMethylamineand 1 mM ethanol and 5,ul of 0.2 M KCN were mixed. The reaction was started

by adding 100,1 of enzyme solution. One enzyme unit is definedas theamount reducing 1,umolWurster's Blue per min

at25 'C.

Protein determinations

Protein concentrations during thepurification were determined by themethodofBradford[14]withbovineserumalbuminas a

standard. Absorption coefficients of thepurified proteins were

determinedaccordingto vanlerseletal.[15]usingthe equation:

AO-'% =34.14A

280/A205-002

Isoelectric

point and molecular

mass

determinations

The molecular mass of native EDH was determined by gel

filtration on a Superose 12 column equilibrated with 0.1 M

sodium phosphate buffer, pH6.5, using ferritin, y-globulin (150 kDa),yeastalcoholdehydrogenase (140kDa), bovineserum

albumin(67kDa), cytochromecfromhorse heart(13kDa)and

potassium

ferricyanide

asmarkers. The subunit

composition

and

contentofEDH weredetermined with thesamecolumn,except that0.1 M sodiumphosphate buffer, pH6.5, containing0.1% (w/v) sodiumdodecylsulphate (SDS)and 0.1M NaClwasused forequilibrationandelution. For that purpose, 100

,d}

ofEDH

solution (7.2mg/ml) was mixed with 400 1l of 0.1 M sodium

phosphate buffer, pH 6.5, containing 1.0% (w/v) SDS and

0.1 M NaCl after which the mixture was heated for 2min at

100'C. After cooling and centrifugation, 100,ul of the

super-natant was injected on the column. The eluatewas monitored

byaphotodiodearraydetector. The molar ratio of theaand ,

their peaks in the chromatogram (taken at 205nm) by the respectivemolecularmasses ofthe subunits.

The molecular mass of the native cytochromes c was

de-termined byelectrophoresisongradientpolyacrylamide (8-25%)

gels with the Phast system equipment (Pharmacia). The gels

werecalibrated with the highmolecularmassmarkerkit (Phar-macia). The molecularmass ofthe subunits was determinedin

the same way except that 1% (w/v) SDS was included in the

buffer. The proteinsweredenatured for2minat 100°C and the low molecularmassmarker kit wasused for calibration.

The isoelectric points of the proteins were determined by

isoelectric focusing in the pHrange3-9. Theisoelectric focusing

marker kit (Pharmacia)wasused for calibration.

Protein stainingwasperformed with Coomassie Brilliant Blue

R250 and detection of cytochromescalso by haem stainingwith

3,3',5,5'-tetramethylbenzidine (Janssen Chimica) [16].

Midpoint

potential

of

the cytochromes

Potentiometric titrations were performed as described by van

Wielink et al. [17]. Cytochrome CEDH (5.0,M) was titrated in 0.1 M Hepes buffer, pH 7.0, in the presence of 0.1 mM

quinhydrone (E0'+ 280 mV) (Merck), 0.4 mM diaminodurene (E0'+275mV) (Fluka Chemie), 0.2mM 2,5-dimethyl-p-benzo-quinone

(Eo

+180mV) (ICN Pharmaceuticals) and 0.1 mM

trimethylhydroquinone

(Eo

+115mV) (ICNPharmaceuticals).

N-terminal amino

acid sequence

determination

Theamino acidsequenceof the N-terminalpartofcytochrome

CEDH was determined by Edman degradation with agas-phase amino acid sequenator [18] at the Laboratory for Medical Biochemistry, Sylvius Laboratories, Leiden, The Netherlands. Comparison of the sequence with that of other proteins was carriedoutbyusingthe data intheSwissprot-, the GenPept-, the

Gen-bank andthe EMBL-data libraries[19].

Stopped-flow

kinetics

Kineticexperimentswere performedat20.0 °Cwitha HI-Tech SF 50 stopped-flow spectrophotometer. Data acquisition was

performedwitha100 kHzDASH 16FA/Dconvertercontrolled byanOlivetti M24 SPcomputer. Reduction offerricytochrome CEDE bythesemiquinone form ofEDH(EDHsem)was monitored

at418nm(givingthehighestdifference inabsorbancesbetween ferri- andferro-cytochromecEDH).

Pseudo-first-orderrateconstantswerecalculatedby non-linear

regressionwith theuseofaGauss-Newtonalgorithm,available withASYST (Keithley). The data used were the averageofat

least three measurements. Experiments were carried out by diluting the EDHsem preparation with 10mM Mops buffer, pH7.0, and mixing this with an equal volume ofcytochrome

cEDH diluted in the same buffer. Concentrations of EDHsem

mentioned in thetextarethe finalconcentrations of subunit after mixing.

RESULTS

AND DISCUSSION

Purfflcation and

properties

of ethanol

dehydrogenase

Apurificationschemewaselaborated forEDH(Table 1)and its

potential electron acceptors,the solublecytochromesc.Thefinal

EDHpreparation appearedtobehomogeneous,asjudgedfrom

electrophoresis and gel filtration (the identity of thepeak was checked by comparingthe absorption spectra). Comparisonof

Table 1 Purfflcatlon of quinoprotein ethanol dehydrogenase

Total Total Specific

protein activity activity Recovery Purification Step (mg) (units) (units/mg) (%) (fold) Cell-freeextract* 3651 2357 0.65 100 1 (NH4)2SO4 2411 1849 0.77 78 1.2 fractionation DEAE-Sepharose 1109 1224 1.1 52 1.7 TSK Butyl-650(S) 313 945 3.0 40 4.6 Superose12 24 943 40 40 62

* Originating from30g cell paste.

1.0 0.8 0.6 0.4 0.2 0.0 20 Time (min)

Figure 1 Gel filtration of denatured EDH

Asampleof EDH(7.2mg/ml)in 0.1 M sodiumphosphate buffer, pH 6.5, containing1%SDS and 0.1 MNaCI,washeatedat 100OCfor 2minandappliedtoaSuperose12 column in 0.1 M

sodium phosphate buffer, pH 6.5, containing0.1%SDSand 0.1 M NaCI. Elutionoccurred at aflow rate of 0.2ml/minand the eluatewasmonitored at 205nm.

redox forms ofMDH [20] indicated that the final EDH

prep-aration consistedofenzyme in itssemiquinoneform (EDHsem).

ThiswasconfirmedbysimilarA280/A340ratios(6.3)and the fact

that EDHsem could be oxidizedby ferricytochrome CEDH. Initially, purification was carried out using buffers without

CaC12.The finalpreparation inthatcase hada specific activity of 2.4unit/mg proteinandwasverylabileuponstorageat4'C. Upon addition of CaCl2 (10 mM) to such a preparation, an increase in specific activity to 26 occurred and an additional

increase occurred upon adding PQQ (1O,uM). Since

incorpor-ation ofCaC12 in the buffers prevented the lability and gave

preparations

with a

high specific activity (40 unit/mg)

which

showed no increase upon

CaCl2

and

PQQ addition, CaCl2

was

routinely

addedtothebuffers.The results show that incontrast to other

PQQ-containing

enzymes,

Ca2+

in EDH is

loosely

bound,

its detachment

leading

toloss of

PQQ

and

lability

of the apo-enzyme.

Gel filtration indicateda molecular massof 136+12

(n

=

3)

kDa for native EDH.

Polyacrylamide gel

electrophoresis

of

denatured EDH

(approximately

4

ug

per

lane)

in the presence ofSDSrevealedtwo

bands,

onewithamolecularmassof 60 kDa and theother of9kDa. This

finding

contradicts

previous

reports

[5,6]

where

only

oneband was observed.

However,

asthe two

bandswere

consistently

found in

electrophoresis

of active prepa-rations and

peaks

corresponding

to the same size were also observedin the

gel-filtration chromatogram

of denatured EDH

(Figure 1),

weconcludethatEDHcontainsaand,subunitswith

similar sizes to those

reported

for MDH

[1,2].

To

explain

the

contradictory

findings,

the / subunit is much smaller in size than the asubunit

and,

as aconsequence, the

intensity

ofthe bands

from denaturedEDHobservedafter

electrophoresis

and

protein

staining

will differ

(7-fold

ifboth have thesame

dye-affinity)

such

that the 3 subunit will be

easily

lost or not visualized under

inappropriate

conditions. This may be

why

the , subunit of MDH has been overlooked for more than 20 years

by

many

research groups

[1]

andit may also

apply

toEDH for the two cases

reported

[5,6].

On

integrating

thesurfaceareasofthe

peaks

inthe chromato-gramofdenaturedEDH

(Figure 1),

avalue of2.2wasfoundfor

the first

(corresponding

to the a

subunit)

and of 0.3 for the secondone

(corresponding

tothe /3

subunit).

Sincethe chromato-gramwastakenat205nmandabsorbanceatthis

wavelength

is

directly

related to the

protein

concentration

[15],

dividing

the surface areas

by

the

corresponding

subunit molecular masses

(60

and9

kDa)

gives

figures

relatedtothesubunitconcentration.

Fromthese

experiments,

aratioof 1.0+0.2

(n

=

6)

was

found,

indicating

that EDH hasan

a2/32

composition, just

ashas been

recently

found for anumber of MDHs

[1,2,21].

The

soluble

cytochromes

c

Soluble

cytochromes

c were

only

detected in the

40-80%

(NH4)2SO4

fraction,

not in the

40%

precipitate

(only

minor

amounts ofmembrane-bound

cytochromes

were

present)

norin

the 80

%

supernatant

(devoid

ofany

spectral

peaks

reminiscent

of

cytochromes). Upon

purification

of the 40 80%

precipitate,

it

appeared

tocontaintwosoluble

cytochromes

c,asdemonstrated

by

the

typical

spectra ofthe final

preparations

and the

positive

responsewith

tetramethylbenzidine

staining

after

electrophoresis

ofthe denatured

samples.

Moreover,

probably

acopper

protein

is present also sinceblue-colouredfractionswereobtained from

the

hydrophobic-interaction

chromatography

column after

cyto-chrome c556 had eluted.

The

preparation

showing

an

absorption

maximumat551nm

on reduction is named here

cytochrome

cEDH in view of its

functioning

as anelectronacceptortoEDH

(see below)

and the differences fromthe

previously-described

cytochrome

c5,1

from

P.

aeruginosa

(strain

PAO1

161)

(Table 2).

The final

preparation

appeared

to be

homogeneous

and to consist ofa monomeric

protein

asthesamemolecularmasses werefound fornative and

denatured

samples (14.5

kDa).

Thus,

cytochrome

CEDH

is

signifi-cantly larger

than

cytochrome

c..,

(8.7

kDa).

Since

cytochrome

CEDH has the same functional role as

cytochrome

CL, it was

interesting

toseewhetheroverall

similarity

existed.As shown in Table 2, the

properties

of

cytochrome

CEDH fall in the ranges

Table 2

ComparIson

of cytochromesc

Properties Cytochrome CEDH Cytochrome C551* CytochromecLt Cytochrome cHt Molecular mass (kDa)

Absorption maxima (nm) Oxidizedform Reduced form Absorption coefficient of

the reduced forms (mM * cm-1)

At 551nm At 416nm Absorbanceratios of the

reduced forms

A551 /A522

A551/A280 Isoelectric point Midpoint potential (mV) 14.5 410 416, 522, 551 19 104 8.7 409 416, 521,551 29 165 1.5 0.5 4.8 +258 1.7 1.1 4.7 +286

* Data forcytochromeC551of P.aeruginosaPAO1 161 arefrom [22,23].

t Data forcytochrome cL andcytochrome

LH

ofmethylotrophicbacteriaarefrom [3].

Cytochrome cEDH Al G D V T P Q AV D T KG1 IHE P LG K19

Cytochromec551 KIG ICL A C H A] D T K M V GP A Y K CytochromecH

cJLJ

G A C H SIl

JA

LV[LGP

AYK

CytochromecL DLVLjS Q G K EG G R D T P A V K K

Figure 2 Comparison of amino acidsequences of cytochromesc

Data(positions 139-157) for cytochrome C551of P.aeruginosa PA01161 arefrom [22,23], for cytochromecL(positions 20-38) and cytochrome H(positions13-31)ofM. extorquensAM1

arefrom[3]. The typical haem c-binding amino acid residues [31] (CXXCH)aremarked*for

cytochrome C551 and cytochrome cH.

of the N-terminal amino acid sequence (19 amino acids) of

cytochrome CEDH with that of cytochrome CL from Methyl-bacteriumextorquensAM1 showednosimilarityatall.Although

an internal stretch was found having some similarity (21 %), internal stretches exist in cytochrome c551 of P. aeruginosa PAO1161 [22,23] andcytochromeCH ofMethylbacterium

extor-quens AM 1 [3] having evenhigher similarity (420% and 320%,

respectively) (Figure 2). Since the lattertwo contain thetypical

haemc-bindingsites but thesequence ofcytochromeCEDHdoes

not, the similarity is questionable. In conclusion, cytochrome

CEDHis

structurallydifferent fromcytochromec551 andthe amino acid sequence of the N-terminal part also suggests structural

dissimilarityto cytochrome CL.

The other coloured fraction, which showed an absorption

maximum at 556nm upon reduction, was also purified to

homogeneity, asjudgedfrom the results ofelectrophoresis and

gel filtration of the final preparation. Determination of the

molecular mass ofthe native protein gave a value of 80kDa,

whilst that for the denaturedproteinwas40kDa, indicatingthat itconsists oftwosubunits ofequalsize.These characteristicsare verysimilar tothose ofacytochromeC556isolatedfrom

anaero-bically grownP. aeruginosa PAO1 161, usingnitrate aselectron acceptor[24]. Sincenoothersolublecytochromecwasdetected

and soluble cytochrome CH is the electron acceptor for

cyto-chrome CL in methylotrophic bacteria [25,26], we investigated whether cytochrome c556 could function as a cytochrome CH. However, it appeared that ferricytochrome C556 was unable to

oxidizeferrocytochromeC

EDH.

Since itwasalso unable to actas

electron acceptor for

EDHsem'

a role of cytochrome c556 in ethanoloxidation viaquinoproteinEDH ishighly unlikely. The

latter conclusion is in accordance with the observation that

growth with substrate limitation (batch-fed on ethanol) gave

cells which did contain EDH and cytochrome CEDH but no

cytochrome c556.The results indicate thatacytochrome cR-like

redox protein does not occur inethanol-grown P. aeruginosa.

The blue-coloured protein fraction was able to oxidize ferro-cytochrome CEDH (results not shown). In view ofthis and the

tentativeconclusionsonthestructural differences between

cyto-chromec1EDH andcytochrome CLofM. extorquensAMI, EDH seemsto formpartofadifferenttypeofrespiratory chain from

that inwhich MDH isoperating.

Electron transfer between

EDHIPIL

and cytochrome CEDH

Since EDHsem appeared to react very rapidly with

ferricyto-chrome CEDH in spectrophotometric experiments, stopped-flow experiments were carried out to measure the rate constant of

electron transfer and the dissociation constant of complex

formation. For thatpurpose the

kobs

(observed rate constant)

was measuredat418nm forvarying EDHSem subunit concen-trations at a constant ferricytochrome CEDH concentration

(0.75 ,uM). Figure3 shows that saturation kinetics exists for this reaction. The simplest reaction mechanism explainingthis be-haviourconsists oftworeactionsteps:

EDHsem +ferricytochrome CEDH k±1 [EDHsem* ferricytochrome

(complex)

>[EDHOX * ferrocytochromeCEDHI

In thisscheme,it is assumedthatk.2isnegligible,whichseems

justified since EDH will probably have a redox potential of +100 mV(suchavalue has beenfound forquinoprotein glucose dehydrogenase [27] and quinoprotein methylamine

dehydro-genase [28] and the value ofcytochrome CEDH is significantly

larger [+258 mV (Table 2)]. Since the ferricytochrome CEDH 14-22 414-417,549-552 22-27 65-139 3.5-4.9 +190-324 8.5-15 414-418, 550-554 21-33 128-162 4.5-9.4 +224-373

80 - 60-.40 20 0 0 2 4 6 8 10 12 [EDHI (#M)

Figure 3 Reaction of

EDH:,,m

with ferricytochrome

CEDH

The reaction between terricytochrome

cEDH

(tinal concentration 0.75

/1M)

and varying concentrations ofEDHsem(1.5, 3.0, 6.0 and 11.0 uM tinal subunit concentrations) was studied withstopped-flow kinetics, as described in the Materials and methods section.

concentration was kept constant, pseudo-first-order-rate con-ditions are present sothat the steady-state approximation [29]

canbeapplied toyield thefollowing equation:

kobs =k+2

[EDHsemI]/([EDHsem

]+

K,)

inwhich

K.

=

(k-1

+

k+2)/k+l

Using the experimental data, k+2 and

K.

were calculated by

non-linear regression analysis. The value calculated for

k+2,

(99.6s-1)ishigherthan that for

MDHsem

andferricytochrome CL

(0.3-27s-1) [30].BindingofferricytochromecEDH to

EDHsem

is rather tight since the dissociation constant

(K.)

is 5.7x 106M, comparable to that for

MDHsem

and ferricytochrome CLfrom

Methylophaga marina

(K,

=5.0x10-6 M,

unpublished results).

Inconclusion,theratesandaffinityobserved and the fact thatno

otherredoxproteinwasfound

having

this

activity

make it very

likely that cytochrome CEDH is the natural electron acceptor of

EDH inP. aeruginosa.

This work wassupported by the Netherlands Organization for Scientific Research (NWO). The amino acid sequence determinations were carried out by Dr Amons at theNetherlands Organization for Chemical Research (SON) protein sequence facility,

Sylvius Laboratory, Leiden, The Netherlands. We thank Joyce Ras for comparing the N-terminal amino acid sequence of cytochromeCEDHwith those in the data libraries.

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