Faculty of Technology and ScienceChemistry

DISSERTATIONKarlstad University Studies

2011:40

Anna Smedja Bäcklund

c Cytochromes as Electron Carrier in Microbial Chlorate

Respiration

Anna Smedja Bäcklund

c Cytochromes as Electron Carrier in Microbial Chlorate

Respiration

Karlstad University Studies2011:40

Anna Smedja Bäcklund. c Cytochromes as Electron Carrier in Microbial Chlorate Respiration

DISSERTATION

Karlstad University Studies 2011:40ISSN 1403-8099 ISBN 978-91-7063-375-1

© The author

Distribution:Karlstad UniversityFaculty of Technology and ScienceChemistryS-651 88 KarlstadSweden+46 54 700 10 00

www.kau.se

Print: Universitetstryckeriet, Karlstad 2011

Abstract

Microbial respiration of oxochlorates is important for the biotreatment of

effluents from industries where oxochlorates are produced or handled. Several

bacterial species are capable to use perchlorate and/or chlorate as an alternative

electron acceptor in absence of oxygen. The present study deals with the

electron transport from the membrane-bound components to the periplasmic

chlorate reductase, in the gram-negative bacterium Ideonella dechloratans. Both

chlorate reductase and the terminal oxidase of I. dechloratans were found to

utilize soluble c cytochromes as electron donors. For further investigation, two

major heme-containing components were purified and characterized. The most

abundant was a 9 kDa c-type cytochrome (class I), denoted cytochrome c-Id1.

This protein was shown to serve as electron donor for both chlorate reductase,

and for a terminal oxidase. The other major component was a 55 kDa

homotetrameric cytochrome c', (class II). A function for this cytochrome could

not be demonstrated but it does not appear to serve as electron donor to

chlorate reductase. A gene predicted to encode a soluble c cytochrome was

found in close proximity to the gene cluster for chlorate reduction. The

predicted sequence did not match any of the cytochromes discussed above. The

gene was cloned and expressed heterologously, and the resulting protein was

investigated as a candidate electron donor for chlorate reductase. Electron

transfer from this protein could not be demonstrated, suggesting that the gene

product does not serve as immediate electron donor for chlorate reductase.

3

Table of contents

Abstract ................................................................................................................... 2

Table of contents .................................................................................................. 3

List of papers ......................................................................................................... 4

List of abbreviations ............................................................................................. 5

1. Introduction....................................................................................................... 6

1.2 Oxyanions of chlorine ................................................................................................. 6

1.3 Perchlorate and chlorate in the environment ........................................................... 7

1.3.1 Sources .................................................................................................................... 7

1.3.2 Environmental effects .......................................................................................... 7

1.3.3 Treatment of perchlorate- and chlorate containing effluents ......................... 8

1.4 Bacterial perchlorate- and chlorate respiration ......................................................... 9

1.4.1 Mechanism and enzymes ..................................................................................... 9

1.4.2 Perchlorate- and chlorate respiring bacteria .................................................... 10

1.4.3 Electron transfer pathway in chlorate respiration .......................................... 12

1.5 c-type cytochromes ..................................................................................................... 14

2. Methods used in present study ..................................................................... 18

2.1 Analysis of periplasmic c cytochromes .................................................................... 18

2.1.1 Separation ............................................................................................................. 18

2.1.2 Detection .............................................................................................................. 19

2.2 Mass spectrometry ...................................................................................................... 19

2.2 Protein purification .................................................................................................... 21

3. Results and discussion ................................................................................... 23

3.1 Native periplasmic c cytochromes in Ideonella dechloratans ..................................... 23

3.2 Characterization of a candidate cytochrome c gene associated with the

gene cluster for chlorate respiration ....................................................................... 26

3.3 Purification and characterization of soluble cytochrome c capable of

delivering electrons to chlorate reductase in Ideonella dechloratans ....................... 29

3.4 Purification and characterization of a cytochrome c' in the chlorate

respiring bacterium Ideonella dechloratans ................................................................. 32

5. Conclusion ....................................................................................................... 37

6. Tack ................................................................................................................... 39

7. References ........................................................................................................ 41

4

List of papers

I Bäcklund A. S., Bohlin J., Gustavsson N. & Nilsson T. (2009)

Periplasmic c cytochromes and chlorate reduction in Ideonella dechloratans.

Appl Environ Microbiol 75 (8) 2439-2445

II Bohlin J., Bäcklund A. S., Gustavsson N., Wahlberg S. & Nilsson T.

(2010) Characterization of a cytochrome c gene located at the gene

cluster for chlorate respiration in Ideonella dechloratans. Microbiol Res 165 (6)

450-457

III Bäcklund A. S. & Nilsson T. (2011), Purification and characterization of

a soluble cytochrome c capable of delivering electrons to chlorate

reductase in Ideonella dechloratans. FEMS Microbiol Lett 321 (2)115-120

IV Bäcklund A. S. & Nilsson T., Purification and characterization of a

cytochrome c' from the chlorate respiring bacterium Ideonella dechloratans,

Manuscript

5

List of abbreviations

BOD Biological oxygen demand

COD Chemical oxygen demand

Cld Chlorite dismutase

Clr Chlorate reductase

Ddh Dimethyl sulfide dehydrogenase

DMS Dimethyl sulfide

DMSO Dimethyl sulfoxide

DTT Dithiothreitol

Edh Ethylbenzene dehydrogenase

GF Gel filtration

HIC Hydrophobic interaction chromatography

IEF Isoelectric focusing

IEX Ion exchange chromatography

IMAC Immobilized metal ion affinity chromatography

IPG Immobilized pH gradient

LC Liquid chromatography

LTQ Linear trap quadrupole

MALDI Matrix assisted laser desorption ionization

MES 2-(N-morpholino)ethanesulfonic acid

MS Mass spectrometry

MS/MS Tandem mass spectrometry

Nap Periplasmic respiratory nitrate reductase

Nir Nitrate reductase

ORF Open reading frame

PAGE Polyacrylamide gel electrophoresis

PCR Polymerase chain reaction

Pcr Perchlorate reductase

Q/QH2 Quinone pool

SDS Sodium dodecyl sulfate

Ser Selenate reductase

TFA Trifluoroacetic acid

TMAO Trimethylamine-N-oxide

TMBZ 3, 3’, 5, 5’- tetramethylbenzidine

TOF Time of flight

6

1. Introduction

Sustainable development of industrial processes include the minimization of the

release of waste products in the environment. In many industrial operations,

biological treatment of effluents is used to reduce their content of harmful

substances. Knowledge about the organisms involved, and their physiological

and biochemical properties, is important for the design, operation and

optimization of waste-treatment plants. Chlorate and perchlorate are examples

of toxic industrial by-products, which need to be removed from waste streams.

The present work deals with the chemistry of microbial respiration of chlorate

and perchlorate. Here, chlorate and/or perchlorate are used as alternative

respiratory electron acceptors in absence of oxygen with chloride as the end

product. The specific aim of this work was to elucidate the electron transport

pathways in chlorate respiration.

1.2 Oxyanions of chlorine

Chlorine can combine with oxygen to produce four different oxyanions, shown

in table I. The most oxidized form is perchlorate. Most salts of chlorate and

perchlorate are highly soluble in water and chemically inert under ambient

conditions (Kang et al., 2006; Urbansky & Schock, 1999). In contrast, chlorite

and hypochlorite are highly reactive in different reactions.

Table I Oxyanions of chlorine

Name

Formula

Oxidation state of chlorine

Hypochlorite

Chlorite

Chlorate

Perchlorate

ClO-

ClO2-

ClO3-

ClO4-

I

III

V

VI

7

1.3 Perchlorate and chlorate in the environment

1.3.1 Sources

For long, the only known occurrence of perchlorate in the environment was

deposits in Chile (Urbansky et al., 2001). Recently it has been shown that

perchlorate is readily formed by atmospheric processes when chloride is

exposed to high concentrations of ozone. This suggests that some natural

perchlorate background from the atmosphere should exist (Dasgupta et al.,

2005). Also, Kang et al. (2006) demonstrated that perchlorate can be generated

by photochemical transformations of aqueous chlorine anions such as

hypochlorite, chlorite, and chlorate, upon exposure to UV-radiation. In the

latter case there is a combination of anthropogenic and natural sources since

the chlorine oxyanions are introduced to the environment by human activities.

A major anthropogenic source is the manufacture of perchlorate compounds.

Perchlorate salts are used as oxidants in rockets and missiles (Urbansky &

Schock, 1999), and most of the perchlorate contamination in the ground and

surface water is a result of discharge from rocket fuel manufacturing plants,

decades ago (Urbansky, 1998).

At the present, there is no evidence for any natural sources of chlorate in the

environment, but there are several anthropogenic sources. In agriculture,

chlorate is applied as herbicide or as defoliant (Logan, 1998). When

hypochlorite is used as disinfectant in drinking water, chlorate is formed as a

by-product (van Ginkel et al., 1995). Another considerable source of chlorate is

the pulp- and paper industry. Pulp is bleached with chlorine dioxide and

chlorate is formed both by decomposition of chlorine dioxide and in the

bleaching process (Germgård et al., 1981; Rosemarin et al., 1994). Furthermore,

chlorate can be formed during the manufacture and storage of hypochlorite

solutions (Siddiqui, 1996).

1.3.2 Environmental effects

In Kalmar Strait, in the Baltic Sea, it was recorded in 1992 that the brown algae

bladder wrack had disappeared from an area of 12 km2 caused by the effluent

from Mönsterås pulp mill (Lehtinen et al., 1988; Rosemarine et al., 1994). Studies

of the marine environment have shown that groups of marine algae are

8

sensitive to high concentration of the chlorate anions, especially the benthic

brown macro algae (van Wijk and Hutchinson, 1995). The mechanism of

toxicity is not clear and appears to differ among species. It has been suggested

that chlorate can be reduced by nitrate reductase and that the resulting chlorite

ion inhibits nitrate reduction, with nitrogen starvation as the result (Åberg,

1947; Stauber, 1998). Further support for this hypothesis is the finding that the

cyanobacterium Nostoc muscorum, which is lacking the nitrate reduction system, is

chlorate resistant (Singh et al., 1977). In conflict with this hypothesis, chlorate

toxicity in plants and bacteria lacking nitrate reduction systems have also been

reported (Siddiqi et al., 1992; Prieto & Fernadez, 1993). This suggests that other

metabolic pathways are affected by chlorate. For example, in the microalga

Nitzschia closterium chlorate has no effect on the nitrate reductase activity, but

the alga is still chlorate sensitive. Stauber (1998) suggests that the toxicity can be

a result of decreasing flow of electrons in the electron transport chain, caused

by chlorate-dependent altering of membrane-bound components, or

inactivation of cytochromes.

1.3.3 Treatment of perchlorate- and chlorate containing effluents

Traditionally, waste water treatment in the pulp and paper industry has been

focused on removal of biological and chemical oxygen demand (BOD and

COD, respectively) by biological active sludge processes in aerated lagoons.

However, the introduction of chlorine-containing chemicals in the pulp-

bleaching process made the removal of the resulting chlorate necessary (Yu &

Welander, 1994). The toxicity of chlorate is suggested to be coupled to

reduction of chlorate to the highly reactive chlorite by the nitrate reduction

systems, as was described in the above section. However, non-toxic

decomposition of chlorate by microbial respiration has been known since the

beginning of the 20th century (Åslander, 1928). The difference between toxic

and non-toxic decomposition of perchlorate and chlorate depends on the

bacterial enzyme systems, as will be discussed in the next section. Several

bacterial species, able to grow with perchlorate and/or chlorate as an alternative

electron acceptor under anaerobic conditions, have been isolated (Korenkov et

al., 1976; Malmqvist et al., 1994; Rikken et al., 1996; Wallace et al., 1996; Bruce et

al., 1999; Achenbach et al., 2001; Wolterink et al., 2002). These strains can be

utilized for anaerobic biotreatment of water and waste water.

9

One method, developed by Ødegaard et al. 1994, is a process where biomass is

grown on small carrier elements that move along with waste water in reactor

tanks. The carriers are made by polyethylene and are shaped like small cylinders

(Johnson et al., 2000). This technique was introduced into Swedish paper mill

industries in the 1990th (Kindh & Johansson, 1999). Another method,

described by Kroon and van Ginkel (2004), is chlorate reduction in a gas-lift

reactor, where microorganisms are attached to pumice particles in the reactor.

1.4 Bacterial perchlorate- and chlorate respiration

1.4.1 Mechanism and enzymes

Bacterial perchlorate or chlorate reduction is coupled to cell growth (Malmqvist

et al., 1994), and therefore a part of a respiratory chain that generates an

electrochemical gradient, which can serve as driving force for ATP synthesis.

Many bacteria use several types of acceptors like nitrate, sulfate, manganese

(IV), iron (III), selenate, iodate, bromate and DMSO. Respiration of chlorate

and perchlorate is unique because it involves formation of molecular oxygen.

The complete reaction takes place in three steps: ClO4- → ClO3

- → ClO2- →

O2 + Cl- (Kengen et al., 1999) and is catalyzed by two soluble enzymes,

(per)chlorate reductase and chlorite dismutase (Bender et al., 2002; Stenklo et al.,

2001; van Ginkel et al., 1996). In bacteria able to reduce perchlorate, the first

and second step is catalyzed by the same enzyme, perchlorate reductase

(Kengen et al., 1999). However, some bacteria are not able to reduce perchlorate

and in that case the first step will be the reduction of chlorate into chlorite,

catalyzed by chlorate reductase (Kengen et al., 1999; Wolterink et al., 2003). The

last step, where chlorite is decomposed to chloride ion and oxygen, is catalyzed

by chlorite dismutase.

10

1.4.2 Perchlorate- and chlorate respiring bacteria

During the last two decades many different species and strains of bacteria,

capable of growth with chlorate or perchlorate as the sole electron acceptor,

have been isolated and further investigated. A selection of these is listed in

Table II.

The subject of the present study is chlorate respiration in the Gram-negative

bacterium Ideonella dechloratans. It belongs to the beta subgroup of proteobacteria

and was isolated at the environmental biotechnical company ANOX AB

(presently AnoxKaldnes) in 1994 (Malmqvist et al.). This bacterium can grow by

reduction of chlorate, but is not capable to reduce perchlorate. The enzyme

chlorite dismutase was isolated and purified by Stenklo et al. 2001. Also, the

gene encoding chlorite dismutase was cloned, characterized and expressed by

Danielsson Thorell et al. 2002. This enzyme, located in the periplasm, is a

homotetrameric heme b- containing protein with the molecular weight of 100

kDa.

11

Table II Examples of isolated perchlorate and/or chlorate respiring bacteria.

Bacteria strain Electron acceptor Reference

Ideonella dechloratans

ClO3-, NO3

- *, IO3-, BrO3

-, O2 Malmqvist et al. 1994

Azospira oryzae

(Strain Gr-1) ClO4-, ClO3

-, NO3-, Mn(IV) Rikken et al. 1996

Dechloromonas agitata

(Strain CKB)

ClO4-, ClO3

-, O2,

Bruce et al. 1999;

Achenbach 2001

Wolinella succinogenes ClO4-, ClO3

-, NO3-, O2 Wallace et al. 2001

Dechloromonas aromatica

(Strain RCB)

ClO4-, ClO3

-, NO3-, O2

Coates et al. 2001;

Bender et al. 2005

Pseudomonas

chloritidismutans (Strain

AW-1) ClO3-, O2 Wolterink et al. 2004

Pseudomonas

chloritidismutans (Strain

ASK-1) ClO3-, O2 Wolterink et al. 2005

Dechloromonas hortensis

(Strain MA-1) ClO4-, ClO3

-, NO3-, O2 Wolterink et al. 2005

Alicycliphilus denitrificans

(Strain BC) ClO3-, O2, NO3

-, NO2- Weelink et al. 2008

* After several subcultivations on chlorate, Ideonella dechloratans loses the ability to use nitrate as

electron acceptor (Malmqvist et al, 1994).

12

Chlorate reductase was isolated and the gene cluster for the chlorate

metabolism in I. dechloratans was investigated by Danielsson Thorell and

coworkers, 2003. Also this enzyme was found to be located in the periplasm.

The protein is heterotrimeric and contains molybdopterin, iron-sulfur cluster

and heme b bound to subunits A, B and C, respectively. The molecular weight

of the native protein was estimated by gel filtration to 160 kDa. The enzyme

belongs to the type II subgroup of the dimethyl sulfoxide (DMSO) reductase

family. For several years, the most similar known member in the DMSO

reductase family was Thauera selenatis selenate reductase (Ser) (Schröder, 1997).

The similarity between Ser and Clr is 84 %. However, selenate is a poor

substrate for chlorate reductase. More recently, the benzene-degrading

bacterium A. denitrificans strain BC was isolated and characterized by Weelink

and co-workers (2008). The similarity of the amino acid sequence of Clr from

this bacterium compared with Clr from I. dechloratans show 100 % identity for

the B- subunits, and 99 % for the A- and C-subunits. Other related enzymes,

based on sequence similarities and cofactor contents, are found in the marine

photosynthetic bacterium Rhodovulum sulfidophilum (dimethyl sulfide (DMS)

dehydrogenase, Ddh) and in Azoarcus sp. Strain EbN1 (ethylbenzene

dehydrogenase, Edh) (Danielsson Thorell et al., 2002). The A-subunit,

containing molybdopterin, is similar to subunits in several other

oxidoreductases, e.g. in DMSO reductase, TMAO reductase, and in periplasmic

nitrate reductase (NapAB) (Karlsson & Nilsson, 2005). The C-subunit of

chlorate reductase was expressed, purified, refolded, and heme reconstituted.

The results confirmed the C subunit as the cytochrome b moiety of chlorate

reductase in I. dechloratans (Karlsson & Nilsson, 2005).

1.4.3 Electron transfer pathway in chlorate respiration

Although many perchlorate- and chlorate respiring species have been isolated

and investigated, electron transport between the inner membrane components

and the periplasmic oxidoreductases is not well understood. In the case of

nitrate respiration relying on the periplasmic Nap system, electrons are

mediated from the quinone pool to the soluble periplasmic NapAB by a

membrane-anchored multi-heam c-type cytochrome, belonging to the

NapC/NirT family (Berks et al., 1995; Roldan et al., 1998) or by the NapGH

complex (Wolinella succinogenes), (Simon et al., 2003; Simon & Kern, 2008).

13

In other cases the electron transfer agent is soluble and located in the

periplasm. The oxidation of quinol is performed by the membrane-bound

cytochrome bc1 complex, followed by electron transfer to a soluble c-type

cytochrome, which further acts as electron donor to the periplasmic reductase.

In T. selenatis, electron transfer to Ser has been shown to be organized in this

way. Electrons are mediated between membrane-bound quinol:cytochrome c

oxidoreductase (bc1-complex) and periplasmic selenate reductase (Ser) by the 24

kDa di-heme cytochrome cytc-Ts4 (Lowe, et al., 2010). In the photosynthetic

bacterium R. sulfidophilum a soluble c cytochrome is utilized for transfer of

electrons, but in the reverse direction. The B subunit in Ddh donates electrons

to the membrane-bound photochemical center, mediated by the soluble

cytochrome c2 (McDevitt et al., 2002; Creevey et al., 2008). Figure 1 illustrates

different electron transfer pathways in anaerobic respiration.

Figure 1 Electron transport in anaerobic respiration, proposed for D. agitata, D. aromatica, P.

denitrificans and T. selenatis. The gray arrows show the direction of electron transfer suggested for

D. agitata, D. aromatica, P. denitrificans (napC/NirT) and W. succinogenes (Nap GH). In T. selenatis

electrons are transferred through the bc1 complex to selenate reductase via a soluble cytochrome c,

as illustrates by black arrows.

14

In the perchlorate-reducing bacteria D. agitata and D. aromatica a NapC/NirT-

type cytochrome gene was found, suggesting that a membrane-bound

cytochrome acts as the link between the quinone pool and the periplasmic

perchlorate reductase (Bender et al., 2005). In an earlier investigation we tried to

identify a gene for a NapC/NirT-like protein in I. dechloratans by touchdown

PCR, using degenerate primers based on conserved features of the NapC/NirT

proteins. Such a gene, however, was not found (unpublished, 2004). Using the

same primers we searched for a candidate in A. oryzae strain Gr-1 and obtained

a gene product which showed nucleotide sequence similarity (69 %) to a

NapC/NirT-type cytochrome gene in D. aromatica (unpublished, 2007/2008.)

I. dechloratans is classified as a facultative anaerobe, but does in fact never grow

on chlorate in strictly absence of oxygen. Decomposition of chlorite results in

formation of molecular oxygen, which further can be utilized as electron

acceptor by terminal cytochrome c oxidase. Malmqvist et al. have shown that I.

dechloratans is cytochrome c oxidase positive (1994), suggesting an electron

transport chain containing a bc1-type complex transferring electrons from the

quinone pool to terminal cytochrome c oxidase mediated by a soluble

cytochrome c. Several varieties of the latter occur in bacterial respiration. One

classic heme-copper oxidase is the aa3-type. Another oxidase is the cbb3-type,

which has an exceptional high affinity for oxygen (Preisig et al., 1996) and is

therefore more often expressed in bacteria growing in microaerophilic

environments. The cbb3 oxidase can be identified by characteristic spectral

changes, induced by CO-binding (Pitcher et al., 2002). This has been done with

I. dechloratans membrane, and the result strongly indicated presence of cbb3

oxidase (unpublished, 2004).

1.5 c-type cytochromes

Many different types of c cytochromes occur in bacteria as electron carriers.

They are characterized by covalently bound heme as a prosthetic group. Heme

consists of a Fe-atom coordinated to four nitrogen atoms of a porhyrin ring.

The Fe atom is the redox component, either in the reduced ferrous [Fe (II)] or

in the oxidized ferric [Fe (III)] state. The heme-group is attached to the protein

by two thioether linkages. These are formed by reaction of the vinyl groups of

heme with two cysteine residues within a CXXCH sequence motif (Kranz et al.,

15

Class I Class IIa

1998) of the polypeptide chain, as shown in figure 2a. The fifth ligand of the

heme iron is always a histidine residue. Several types of c cytochromes are

classified according to their binding of the sixth ligand. Class I (e.g. low-spin

soluble cytochrome c of mitochondria and bacteria) have a methionine residue

occupied as the sixth ligand. The class II subgroup, divided into class IIa and

IIb, includes the high-spin cytochrome c' (IIa), but also a number of low-spin

cytochromes (IIb) (e.g. cytc556). Class IIa (cytochrome c') is either penta- or

hexacoordinated and only small ligands (e.g. CO, NO or CN-) can be attached

to the free position of the iron ion in the former case (Kintner & Dawson

1991).

(a) (b)

Figure 2 (a) The prosthetic group of heme c consists of a central Fe ion, coordinated to four nitrogen

atoms of a porphyrin ring. (b) Hexacoordinated cytochrome c (class I) with the fifth and sixth binding

site attached to His and Met respectively. Pentacoordinated cytochrome c' (Class IIa).

16

In bacteria, cytochromes of c-type are often found in the periplasmic space

where they are connected to the respiratory components of the cytoplasmic

membrane, acting as electron carriers. They can either be parts of multisubunit

enzyme complexes or occur as mobile electron shuttles (Thöny-Meyer, 1997).

Cytochromes exhibit three characteristic absorption bands, designated alpha-

(α), beta- (β) and Soret-band. In oxidized form the alpha- and beta-bands are

broad whereas the reduced form shows three distinct peaks, as shown in figure

3. Different types of cytochromes can be identified on the basis of the position

of their alpha band in the region 550 – 610 nm (Nicholls & Ferguson, 2002).

For c-type cytochromes the maximum of the alpha band is usually found

between 550 – 557 nm, whereas the maximum for b-type is between 555 – 565

nm (Thöny-Meyer, 1997).

Figure 3 The characteristic spectra of cytochrome c, class I, (here from horse heart), in oxidized

(dashed) and reduced (solid) state. In the reduced spectrum there is a shift in the Soret-band and the

α- and β-bands appear as distinct peaks at 551 and 521 nm respectively.

17

The aim of the present work is to investigate the scenario that electron transfer

in I. dechloratans is mediated by one or several soluble c-type cytochromes. In this

case, electrons are expected to be passed from the quinone pool to a soluble c

cytochrome by a membrane-bound bc1 complex. This hypothesis was based on

the apparent absence of a NapC/NirT-gene in I. dechloratans and the similarity

between chlorate reductase and Ddh in R. sulfidophilum and Ser in T. selenatis.

Soluble c-type cytochromes were extracted from the periplasm of I. dechloratans

(paper I) and two different monoheme proteins have been purified and

investigated (papers III and VI). One of the cytochromes is a 9 kDa member of

the subgroup class I (paper III). The other cytochrome is a homotetramer

consisting of four 13 kDa subunits and is characterized as a class II or

cytochrome c' (paper IV). Further, a gene encoding a candidate c cytochrome

has been expressed heterologously (paper II). This gene is associated with the

gene cluster for chlorate reduction.

We can demonstrate that the 9 kDa soluble c-type cytochrome is able to

mediate the electron transfer between the membrane-bound components and

the soluble chlorate reductase (paper III). This ability could not be

demonstrated for neither the recombinant c cytochrome (paper II) nor the

cytochrome c' (paper IV) and the role of their participation in the chlorate

metabolism remains to be clarified. The gene product of the cytochrome c gene

associated with the gene cluster for chlorate reduction (paper II) was not found

in the periplasmic extract.

18

2. Methods used in present study

2.1 Analysis of periplasmic c cytochromes

2.1.1 Separation

A common technique for analytical separation of proteins is sodium dodecyl

sulphate polyacrylamide gel electrophoresis, SDS-PAGE using discontinuous

buffer systems. Proteins denatured by SDS are separated in a polyacrylamide gel

and the migration of the protein is a function of its size. In a discontinuous

system, introduced by Ornstein, L. and Davis, B.J. (1964) and adapted by

Laemmli (1970), the gel and running buffer consists of different ions and, most

commonly, a discontinuous pH. In the Laemmli buffer system the stacking-gel

buffer (pH 6.8) contains chloride ions and the running buffer in the upper

reservoir consists of glycine, which has a low net charge and low effective

mobility at pH 6.8. NuPAGE® Bis-Tris Discontinuous Buffer System

(Invitrogen) is a modified system where the gel buffer (pH 6.4) contains

chloride and the running buffer (pH 7.3-7.7) contains MES (2-(N-morpholino)

ethanesulfonic acid). Bis-tris is the common counter-ion present in both gel-

and running buffer. The combination of pH results in an operating pH of 7

during electrophoresis which improves the protein stability, resulting in sharper

bands and better resolution. MES buffer is optimal for resolving small

molecular weight proteins, due to its fast ion migration. When using 4-12 %

bis-tris gels and MES SDS running buffer, the separation range for proteins is

between 2.5-200 kDa compared to the Laemmli tris-glycine system which needs

18 % gels to resolve low molecular weight proteins (NuPAGE® Technical

Guide, Invitrogen). In a variation of the method, proteins can be separated

under non-denaturing conditions. Blue native gel electrophoresis is a technique

in which the proteins obtain a negative charge by using Coomassie G-250 as a

charge-shift molecule, instead of SDS (Schägger & von Jagow, 2004).

Proteins in the same molecular weight range can be distinguished by their

different isoelectric point (pI), using isoelectric focusing (IEF). The pI is the pH

at which a protein has a neutral net charge and thus, no migration in an electric

field. The combination of IEF and SDS-PAGE, referred as two-dimensional

gel electrophoresis, is widely used for analysis of complex protein mixtures. In

the first dimension IEF is performed using an immobilized pH gradient (IPG).

When proteins are loaded on to a gel strip, a voltage is applied to the system

and the proteins will migrate in the pH gradient until its net charge is zero.

19

After the complete focusing, the gel strip is sealed on to a conventional

polyacrylamide gel and SDS-PAGE is performed as described above. The

proteins will migrate into the second dimension gel from different horizontal

positions, according to their migration in the first dimension. Thereby, proteins

with same molecular weight but different pI can be separated using this method

(Bjellqvist et al., 1982).

2.1.2 Detection

Proteins containing heme can be detected after SDS-PAGE by using a staining

procedure relying on the peroxidase activity of the heme group (Thomas et al.,

1976). The heme-catalyzed reaction between 3, 3’, 5, 5’-tetramethylbenzidine

(TMBZ) and hydrogen peroxide results in the formation of a deep blue color.

Cytochrome c, in which the heme group is bound covalently by thioether

bonds, is readily detected on SDS-PAGE gels using this method.

Agents used for reduction of disulphides (such 2-mercaptoethanol or DTT)

may affect the coordination state of the heme and either stimulate or inhibit

peroxidase activity (Dorward et al., 1992). Therefore, reducing agents are

specifically omitted from the samples in the electrophoresis. In most cases,

non-covalently bound heme is released from proteins by the denaturation with

SDS, but incomplete denaturation can occur. Consequently, one complication is

that other cytochromes than the c-types may be visualized by heme staining.

Another problem is the possibility of non-specific binding of released heme to

other proteins, resulting in a false signal (Thomas et al., 1976).

2.2 Mass spectrometry

For analysis of larger proteins, peptides can be generated by the enzyme trypsin,

which catalyzes the hydrolysis of peptide bounds. Trypsin cleaves peptide

bonds in which the carbonyl group is contributed by either an arginine or lysine

residue. In-gel digestion is a method for enzymatic cleavage of proteins in

polyacrylamide gels, after SDS-PAGE (Rosenfeld et al., 1992). After protein

identification by staining, the protein band is excised from the gel and destained

20

by ethanol/NH4HCO3. Small gel pieces are incubated with trypsin and peptides

are extracted from the gel pieces by trifluoroacetic acid. The resulting peptides

can be further analyzed by using various mass spectrometry (MS) techniques.

To accomplish mass analysis, the molecules need to be ionized and in gas-phase

state and there are several methods to achieve ionization. One of the methods

in the present work is Matrix Assisted Laser Desorption/Ionization (MALDI)

where the sample is mixed with a matrix compound, capable of absorbing UV-

light (Karas & Hillenkamp, 1987). The matrix will absorb energy from a laser

beam and the embedded peptides are transferred to the gas phase and ionized

by protonation. Another method is Electro Spray Injection (ESI) where an

acidic aqueous solution containing peptides or small proteins is sprayed trough

a needle and a high positive voltage is applied. This high voltage produces a

cone-shaped drop (Taylor cone) at the end of the needle, from which small

drops (< 10 nm) are released as a spray. The solvent will be evaporated from

the small droplets by heat, leaving protonated and multiply charged ions in the

gas phase (Sihlbom, 2006; de Hoffman & Stroobant, 2001).

For further analysis the ions need to be separated according to their masses.

The ions are separated by a mass analyzer according to their mass-to-charge

ratio (m/z). There are several types of mass analyzers based on different

principles. TOF is an analysis method, often in combination to MALDI, where

strong electric field is applied and the ions accelerate, followed by travel in a

field-free flight tube. The flight time to reach the MS detector is proportional to

the square root of the m/z ratio (Guilhaus et al., 2000). Another method, used

in the present work, is a combination of linear trap quadrupole (LTQ) mass

spectrometer and an Orbitrap mass analyzer (LTQ-Orbitrap). Ions from the

ionization source are trapped into a quadrupole made up by four parallel

hyperbolic rods and when the ions collide with an inert gas, they slow down

and will be accumulated at the end of the quadrupole. Pulses of ions are then

injected into the Orbitrap for mass analysis (Hu et al., 2005). The masses

obtained by mass spectrometry can be compared to predicted masses of

peptides or whole proteins, by data base search. Combination of liquid

chromatography (LC) and MS produces efficient separation and sensitive

identification of peptides and proteins, especially when the sample is a complex

mixture of several proteins (Mann & Pandey, 2001).

21

To obtain more specific information of a single peptide, tandem mass

spectrometry (MS/MS) can be used. This is an instrument consisting of two MS

analyzers where a single peptide ion can be selected out of the spectrum

obtained from the first mass spectrometer. This peptide ion is further

fragmented by collision with inert gas and the fragments are separated by the

second mass spectrometer. Cleavage at the peptide bond is the most common

mode of fragmentation and the resulting ions, in this case called b- and y- ions,

retain their charge on the N or C terminus, respectively. Fragmentation is

random and the process generates a series of fragments, differing by a single

amino acid residue (Mann et al., 2001). In case of unknown proteins, with no

corresponding sequences in data bases, fragmentation is the only possibility to

obtain information about the protein. It is possible to reconstruct the sequence

of a single peptide by studying its fragmentation pattern. This method, called de

novo peptide sequencing, is based on knowledge about peptide decomposition

mechanisms (Cañas et al., 2006).

2.2 Protein purification

The isolation and capture of one particular protein from a complex mixture of

several proteins, (e.g. periplasmic extract of bacteria) practically always includes

more than on step. The choice of purification methods is depending on several

factors, for example the condition of the starting material and the characteristic

of the target protein. It is also important to consider the goal of the

purification. Sometime the purity is the most important aspect, but the yield can

also be the more central priority. To achieve both aspects, it is important to

design and optimize the purification steps by a well-chosen approach (Protein

Purification Handbook). The protein purification described in present thesis

involves chromatographic techniques combined in different order, depending

on the target protein.

One method is Ion Exchange Chromatography (IEX), based on different net

surface charge of proteins at a given pH. A column is packed with charged

stationary phase (matrix) and the proteins with opposite net surface charge will

bind to the surface of the IEX matrix. The pH and the ionic strength of the

buffer is selected to provide a protein-matrix interaction to ensure binding of

the target protein when the sample is loaded on to the column. The proteins

22

bound to the column can be eluted by changing the buffer condition by alter

ionic strength or pH of the buffer. Usually proteins are eluted by adding salt

ions (often Na+ or Cl-) which compete with the bound proteins for the charge

of the matrix. It is important to reduce ion strength in the sample before the

application on to the column. Otherwise the ions in the sample will prevent

binding of the proteins.

Another method is Hydrophobic Interaction Chromatography (HIC) which

separates proteins according to difference in surface hydrophobicity. The

column is packed with a porous material composed of ligands containing alkyl

or aryl groups. In high concentration of salt (usually 1-2 M ammonium sulphate

or 3 M sodium chloride) hydrophobic proteins interact with the packing bed

and the elution is achieved by decreasing the salt concentration. This method is

a suitable step if the sample is at high salt concentration, e.g. after a salt

precipitation.

Separation can also be based on the size of the proteins by Gel Filtration (GF).

The column is packed with a porous matrix of spherical gel particles. When a

protein mixture is applied, small proteins can diffuse in and out of the gel

particles whereas large molecules move down in the column as the buffer is

pumped through the system. Thus, smaller proteins will have longer distance to

move and are retained longer in the column. This separation method is often

used as a final polishing step.

The methods described above are applicable to all proteins. In addition, there

are several methods based on specific protein features. One example is

immobilized metal ion affinity chromatography (IMAC), often used for

proteins expressed with an N- or C- terminal poly-His extension (paper II).

Histidine has high affinity for metal ions (e.g. Ni2+) immobilized on the column

matrix. The protein is eluted by increasing concentration of imidazole, which

competes with proteins for binding.

23

3. Results and discussion

The aim of the present study was to elucidate the route of electron transfer

between the inner membrane and the periplasmic chlorate reductase. Soluble c

cytochromes have been isolated from I. dechloratans by extraction of periplasm

from the cells (paper I) and purified and characterized (paper III and VI). Also,

a gene encoding a candidate soluble c-type cytochrome, located in association

with the gene cluster for chlorate reduction, was cloned and expressed

heterologously (paper II).

3.1 Native periplasmic c cytochromes in Ideonella dechloratans [Paper I]

Periplasmic proteins were extracted from chlorate-grown I. dechloratans by

osmotic lysis. The periplasmic extract was first reduced by dithionite and then

exposed to chlorate in the presence of chlorate reductase. Spectrophotometric

analysis showed a sharp peak at 551 nm, indicating reduction of c-type

cytochromes. After addition of chlorate partial reoxidation of the cytochromes

was observed, demonstrated the presence of at least one soluble c cytochrome

capable of supplying electrons for chlorate reduction.

In order to identify c cytochromes the extracted proteins were separated by

SDS-PAGE and stained by the heme-detecting procedure, described in section

2.1.2. Several bands were observable and the result is shown in figure 4. Since

the soluble forms of c-type cytochromes usually occurs in the range 8-14 kDa

(Thöny-Meyer, 1997) further attention was focused on proteins with molecular

weights in that range. Five candidate c cytochromes, with apparent molecular

weights of 20-, 10-, 9-, 8-, and 6 kDa, were observed. The ones most abundant,

according to the heme staining, were the 10- and 6 kDa cytochromes. Among

the minor components, the bands at 8 and 9 kDa were not always observed. As

discussed below, it appears that the 8 kDa protein derives from the 9 kDa

cytochrome. Several high-molecular weight proteins with low stain intensity

were visible, but this could be an artifact caused by unspecific binding of heme

or by incomplete denaturation, as discussed in section 2.1.2.

24

Figure 4. Periplasmic cytochromes separated by SDS-PAGE (NuPAGE™). Lane 2 shows heme-

stained proteins in the range 20-6 kDa. SeeBlue® Plus2 Pre-Stained Standard is used as marker in

lane 1.

For further investigation, an attempt was made to fractionate the c

cytochromes. Using ion-exchange chromatography, we were able to obtain one

fraction containing the 6 kDa cytochrome, one fraction containing the 10 kDa

cytochrome, and a flow-through fraction containing all cytochromes except the

10 kDa.

The redox activities of the partially fractionated cytochromes were investigated

with periplasmic extract or cell homogenate as sources of catalyst. The latter

serves as a source for both chlorate reductase and membrane-bound

components of the respiratory chain, including the terminal oxidase. Reduction

of chlorate is expected to result in production of oxygen due to decomposition

of chlorite by chlorite dismutase. Therefore, chlorate-dependent oxidation of

cytochromes observed when membrane-bound components are present can be

due to electron transfer to both chlorate and oxygen. Periplasmic extract,

lacking membrane-bound oxidase, was used for investigation of the chlorate-

dependent oxidation in the flow-through fraction which contained a mixture of

several cytochromes.

The cytochromes in the different fractions were first reduced by a slight excess

of dithionite. Then, oxidant (chlorate or oxygen) was added and the UV/VIS-

spectra were recorded in the region 450-650 nm. Table III summarizes the

kDa

188

98

62

49

38

28

17

14

6

3

20

109/8

6

kDa

188

98

62

49

38

28

17

14

6

3

20

109/8

6

25

results obtained. The 6 kDa cytochrome was completely reoxidized by both

chlorate and oxygen in the presence of cell homogenate. This result suggests

that the cytochrome is able to donate electrons both to chlorate reductase and

to terminal oxidase. The spectrum obtained from the fraction containing the

10-kDa cytochrome showed two peaks at 552 and 558 nm respectively. When

oxygen was added, spectral changes dominated by a decreased absorbance at

558 nm occurred. This suggests reoxidation of a heme b-containing component.

As the spectral change appears dominated by the 558-nm component, it is

difficult to interpret the reoxidation of the 552-cytochrome c component. No

spectral changes were observed after addition of chlorate.

In the periplasmic extract, partial reoxidation was observed after the addition of

chlorate as judged by the decrease at 551 nm. Also, in the flow-through

fraction, a complete reoxidation was observed when a catalytic amount of

periplasmic extract was used as source of chlorate reductase. No terminal

oxidase was present. The extent of reoxidation in both cases were about 40%,

which most likely can be accounted for by the major 6 kDa cytochrome since

this component was shown to donate electrons to chlorate reductase. In

presence of cell homogenate, the flow-through fraction was completely

reoxidized both by addition of chlorate and oxygen. This suggests that

components which were not oxidized by chlorate can be oxidized by oxygen,

produced as the result of chlorite dismutase activity, in presence of terminal

oxidase. Taken together, these results suggest the minor components (that give

rise to the band at 8-, 9-, and 20 kDa) as candidates as electron donors to the

terminal oxidase but most likely not to chlorate reductase. However, the

individual roles of these cytochromes cannot be assigned.

Table III Reoxidation of dithionite-reduced fractions with different oxidants, as judged by decrease of

absorbance at 551 nm.

Substrate Flow-through fraction 6 kDa

Clr-source Periplasmic extract Cell homogenate Cell homogenate

Oxidant Chlorate partial (40 %) complete complete

Oxygen complete complete complete

26

For further investigation of the c cytochromes peptides were generated by

trypsin in-gel digestion of bands excised from SDS-PAGE gels. Mass

spectrometric analyzes of peptides were performed by MALDI-TOF MS and

MS/MS. One of the peptides from the 6 kDa cytochrome was identical in

mass-to-charge ratio (m/z = 1094.56) to that predicted for the sequence

YAGQKDAVDK from a cytochrome c (class I) in Acidovorax avenae subsp.

citrulli AAC00-1, [YP_969867]. A majority of the theoretical b- and y-ions,

expected from this fragment, could be matched to signals in the MS/MS-

spectrum. Further, the sequence KLVGPSQDVAAR was generated by de novo

from of a peptide with m/z = 1403.76. Also this sequence can be matched to

Acidovorax avenae subsp. citrulli, [YP_969867] located next to YAGQKDAVDK

(1094.56). Furthermore, the sequences show similarity to sequences in three

different cytochrome c (class I), of the chlorate reducer Dechloromonas aromatica.

For the 8- and 9 kDa proteins, no matches were obtained from database

searches. However, the presence of same peptide masses in digest obtained

from these proteins supports the suggestion that the 8 kDa is a degradation

product of the 9 kDa cytochrome. Several of the peptides obtained from the 10

kDa band were, unexpectedly, identified as originating from chlorite dismutase,

suggesting contamination by degradation products of this protein.

3.2 Characterization of a candidate cytochrome c gene associated with

the gene cluster for chlorate respiration [Paper II]

In close proximity to the gene cluster for the chlorate reduction, downstream

from the C subunit of chlorate reductase, two open reading frames were found

by further sequencing of a clone from the GenomeWalker™ library (Karlsson

& Nilsson, 2005). In the ORF closest to the C subunit, the highly conserved

heme-binding motif CXXCH was found in the corresponding translated amino

acid sequence. This finding suggests a gene encoding c-type cytochrome. The

sequence showed similarity to soluble c cytochromes, class I, in Pfam database

and by BLASTP search. The hydrophobic N-terminal sequence is suggested by

SignalP to be a 19-residue signal peptide, and the molecular weight of the whole

apoprotein is predicted to 10,9 kDa after processing. The second ORF further

downstream was identified as a mobB gene encoding a protein functional for the

maturation of molybdopterin as a co-factor for chlorate reductase A subunit.

27

Figure 5 shows comparison of the gene cluster for chlorate metabolism in I.

dechloratans with the corresponding genes in other chlorate reducers. In addition

to those discussed in paper II, this includes the gene arrangement in A.

denitrificans which was described recently (Weelink et al., 2008). This bacterium is

able to degrade benzene under anaerobic conditions in conjunction with

chlorate reduction. The oxygen produced in the degradation of chlorate is

rapidly reduced by the oxidation of benzene. The organization of the gene

cluster for chlorate reduction in A. denitrificans is very similar to that of I.

dechloratans. This cluster includes genes encoding both mobB (YP_004124055)

and a c-type cytochrome, class I (YP_004124056.1) with 100 % identity of the

nucleotide sequences compared to I. dechloratans. Also, the genes encoding, clrB,

and clrD show 100 % identities, but for clrA, clrC, and cld the identity are 99%.

Comparison of the 16S rRNA from both organisms shows 92 % identities,

which excludes the possibility that I. dechloratans and A. denitrificans is the same

organism. Most likely, this similarity of the gene cluster for chlorate metabolism

is caused by lateral gene transfer. However, there are small differences between

the clusters. There is a larger distance between cytC and clrC in A. denitrificans

compared to I. dechloratans. Furthermore, the region between cld and clrA lacks

the insertion element ISIde1, which is located in this region in I. dechloratans.

Figure 5 Comparison of the gene cluster for chlorate metabolism in I. dechloratans, A. denitrificans,

D. aromatica and D. agitata. The sequence downstream pcrD in D. agitata is not known.

28

The results presented in paper I suggest an electron transfer route where

soluble c-type cytochromes serve as mediators. A 6 kDa c cytochrome was

shown to serve as electron donor to chlorate reductase, whereas a 10 kDa

protein did not. However, none of the peptide masses or sequences obtained

from the soluble periplasmic c cytochromes in paper I matched the predicted

sequence of the gene above. In order to assess the role of this gene in chlorate

reduction, the gene was expressed heterologously and the resulting protein was

investigated. The gene, excluding the part encoding the 19-residue signal

peptide, was amplified by PCR and the product was ligated into the expression

vector pET-26b. This vector contributes a pelB leader for export to the

periplasm and a C-terminal six-residue His-tag for subsequent purification by

IMAC. The construct was co-transformed with plasmid pEC86, which includes

the cytochrome c maturation system (ccm), into the E. coli expression host

BL21(DE3). The use of the ccm genes was based on the finding that E. coli

expression hosts usually not express c cytochromes under aerobic conditions

(Thöny-Meyer et al., 1996). For the purpose of synthesizing active holo-c-type-

cytochromes the heme needs to be attached to the apo-protein after export to

the periplasm, which is done by the ccm system.

In transformant BL21(DE3), the target protein was produced at high level but

it was not exported as apo-protein into the periplasm. Instead, insoluble

inclusion bodies were found in the cytosol, lacking the heme-group. In E. coli

NovaBlue(DE3) the construct generated a very low yield of the recombinant c

cytochrome. From these results we decided to produce inclusion bodies for

purification, refolding, and heme reconstitution. The inclusion bodies were

solubilized with urea and the protein was purified in the denatured state by

IMAC. After refolding, reduced heme was added to the protein in the ratio 1:1.

The incorporation of heme was followed spectrophotometrically. At first it was

detected by the appearance of a distinct peak in the α-band at 556.5 nm,

indicating a b-type cytochrome, but within seven days the α-peak was shifted to

553 nm. The formation of covalently bound heme, with two thioether bonds

produced, was confirmed by pyridine hemochrome analysis (Berry &

Trumpower, 1987). The pyridine hemochrome showed a peak in the α-band at

551 nm, which is characteristic for c-type cytochromes.

29

The recombinant cytochrome was investigated by peptide mass fingerprinting

using MALDI-TOF MS, as in the previous paper I. Peptides from the

recombinant c cytochrome could be identified and verified (by MS/MS) with a

coverage of 56 % of the amino acid sequence.

Spectrophotometric studies of the redox activity were performed using the

same approach as for the native proteins, described in paper I. The reduced

form of the recombinant cytochrome c did not reoxidize after addition of

chlorate in presence of chlorate reductase. This result suggests that the gene

product does not serve as an immediate electron donor for chlorate reductase.

However, we cannot exclude that the inability of donating electrons for

chlorate reduction is a result of incorrect refolding or incorporation of heme.

Since the protein was very sensitive to air oxidation, we were not able to test

enzyme-dependent reoxidation by oxygen.

3.3 Purification and characterization of soluble cytochrome c capable of

delivering electrons to chlorate reductase in Ideonella dechloratans

[Paper III]

In the periplasmic extract of I. dechloratans, there is at least one soluble

cytochrome c capable of serving as electron donor to chlorate reductase in the

chlorate reduction reaction (paper I). This paper deals with the purification of

cytochrome c-Id1, denoted as the 6 kDa cyt c in paper I.

If the knowledge of the target protein is limited, the standard protocol for a

Tree Phase Strategy of chromatography techniques is the use of IEX as a

starting step, followed by HIC and GF (Protein Purification Handbook). The

use of IEX is suitable when the sample has low ionic strength, as the case of

periplasmic extract. c-type cytochromes usually have a high pI (~10), which

consequently leads to the choice of a cation exchange column. However, in the

attempt to separate the cytochromes into single fractions in paper I, this

cytochrome did not bind sufficient to the column matrix, but was eluted as a

delayed flow-through fraction.

In the purification of chlorite dismutase (Stenklo et al., 2001) we have earlier

observed the elution of a c-type cytochrome slightly before chlorite dismutase

30

during HIC. Due to the unsuccessful attempt to purify the cytochrome by IEX,

we tried to start with HIC as was preceded by ammonium sulfate precipitation,

as described previously (Stenklo et al., 2001). The collected fractions containing

the desired cytochrome, together with a majority of chlorite dismutase, were

pooled and further applied on to a gel filtration column and pure cytochrome c

was eluted using sodium phosphate at a very low flow rate. The overall yield

was poor, about 0.18 mg from an 8-L culture, but the purity was satisfactory, as

shown in figure 6. The heme content was estimated to 0.9 mol mol-1 protein by

pyridine hemochrome analysis and the absorption maximum of the reduced-

minus-oxidized difference spectrum of 549.7 nm, confirms the notion of a c-

type cytochrome.

(a) (b)

Figure 6 SDS-PAGE of the fractions collected from gel filtration, stained with TMBZ (a) and

Coomassie Brilliant Blue (b). The arrows indicate the fractions of the purified cytochrome c-Id1.

To determine the accurate molecular weight of the protein with the apparent

molecular weight of 6 kDa (according to SDS-PAGE) a sample of the protein

was analyzed by MS (Proteomics Core Facilities, Göteborg) with 9.4 kDa as the

result. To confirm the identity of the protein, tryptic peptides from in-gel

31

digestion were analyzed by MS/MS. The peptides KLVGPSYQDVAAR (m/z

=1403.76) and YAGQKDAVDK (m/z = 1094.56) described in paper I, were

also detected from this digest. Furthermore, peptides with the suggested

sequences AVDQYSPGV (m/z =1276.76) and APNAQALATSK (m/z =

1071.57) was identified by de novo sequencing. These masses have been obtained

earlier and are listed in table 1 (paper I) with the exception of the difference of

one mass unit, for the peptide 1276.76. The difference can be a result of

protonation/de-protonation of the peptide. None of these sequences resulted

in any identity or similarity of other c-type cytochromes from data base search.

The redox midpoint potential of the cytochrome c-Id1 was estimated to 0.261

V. The value for midpoint potential at pH 7 of the chlorate/chlorite couple was

calculated to 0.708 V, which is considerably higher and thereby makes chlorate

as a thermodynamically favorable acceptor in the electron transfer between

cytochrome c-Id1 and chlorate, catalyzed by chlorate reductase.

For kinetic study of the reaction, chlorate reductase was purified as previously

described (Danielsson Thorell et al., 2003; Karlsson & Nilsson, 2005).

Dithionite-reduced cytochrome c was mixed with a catalytic amount of purified

chlorate reductase. When chlorate was added, the chlorate-dependent oxidation

was studied by recording spectra at 1 min intervals. The amount of cytochrome

c was varied in the range 0.6 - 4 µM. First-order reactions were observed for all

concentrations of cytochrome c, and initial rates increased linearly with the

substrate concentration. We were not able to calculate a KM value since this

seemed to be substantially higher than the highest substrate concentration of 4

µM. The poor yield of the cytochrome lead to very limited supply of substrate.

From the results we could calculate a kcat/KM value of 7×102 M-1s-1, but this

value is probably underestimated since chlorate reductase loses activity during

purification (Danielsson Thorell et al., 2003).

In a study by Creevy and coworkers (2008), the interaction between

cytochrome c2 and DMS dehydrogenase of R. sulfidophilum was investigated, and

a KM value of 21 µM was obtained. DMS dehydrogenase and chlorate reductase

of I. dechloratans is close relatives, and it is of interest to compare the kinetic

behavior in the case of R. sulfidophilum with that observed in present study. The

observation with chlorate reductase is consistent with a KM value in the same

range, observed for DMS dehydrogenase. An explanation of a high KM value

could be a low affinity as a result of fast off-rate for the substrate, or a fast

32

electron transfer subsequent to substrate binding. To get a more clear

understanding of the reaction mechanism between the cytochrome c-Id1 and

chlorate reductase, a more detailed kinetic characterization is required.

However, the present study confirms the observation of a soluble cytochrome c

as electron donor to the periplasmic chlorate reductase in the reduction of

chlorate. This is similar to the electron transfer routes reported for DMS

dehydrogenase in R. sulfidophilum and selenate reductase in T. selenatis (Creevey et

al., 2008; Lowe et al., 2010), but different from that suggested in the

(per)chlorate respiring bacteria Dechloromonas species and A. oryzae strain GR-1

(Bender et al., 2005).

3.4 Purification and characterization of a cytochrome c' in the chlorate

respiring bacterium Ideonella dechloratans [Paper IV]

In paper I and III, we concluded that the soluble cytochrome c- Id1 serves as an

electron carrier, capable to deliver electrons in reduction of both chlorate and

oxygen. According to heme-stained SDS-PAGE there is one additional major c

cytochrome present in the periplasmic extract, with an apparent molecular

weight of 10 kDa. The role of this cytochrome was not clarified in paper I.

However, a fraction enriched in this component did not appear to serve as

electron donor for chlorate reduction. On the addition of oxygen, spectral

changes were observed but could have been due to the oxidation of other

heme-containing components, also present in the fraction. The present paper

deals with the purification and characterization of the presumed 10 kDa c-type

cytochrome, in order to clarify its function and to investigate the possibility that

this protein is a product of the cytochrome c gene characterized in paper II.

In paper I we observed that the 10 kDa cytochrome was retained on a cation

exchange column at acidic pH. To get further information of the pI and the

occurrence of other heme containing proteins in this region, the periplasmic

extract was analyzed by 2-dimensional IEF-SDS-PAGE, followed by heme

staining. The isoelectric point of c-type cytochromes is generally high (~10) and

thus an IPG-strip with pH in the range 9-12 was used. The result is shown in

figure 7. Heme staining components with apparent molecular weights at 6- and

10 kDa are clearly visible, confirming that these are the major components. In

33

this case, the sample loading is too low to detect the minor components

discussed in paper I.

Figure 7 Analysis of the periplasmic extract with 2D IEF-SDS-PAGE, followed by heme staining.

For further purification of the 10 kDa cytochrome c, a cation exchange column

was chosen as the initial step. Periplasmic extract was dialyzed against starting

buffer, applied to a HiTrap SP FF column and heme-containing proteins were

eluted by increasing concentration of NaCl. The second purification step was

performed by hydrophobic interaction chromatography resulting in improved,

but not sufficient purity. A substantial amount of chlorite dismutase and several

non-heme proteins in the range 35-60 kDa were still present. In most cases, a

polishing step by gel filtration would be preferred as a following purification

step. However, we have observed that the target protein was eluted together

with other proteins with molecular weights of 30-50 kDa. Instead, we used a

strong cation exchange column (Mono S HR). With very shallow gradient of

increasing NaCl the purity of the target protein was sufficient. The purification

is summarized in figure 8, with the final result shown in lane 6.

34

Figure 8 The Coomassie-stained SDS-PAGE as a summary of the purification. Lanes: (1)

Novex®Sharp Pre-Stained Protein Standard, Invitrogen, (2) periplasmic extract, (3) periplasmic

extract after dialysis, (4) pooled fractions from the first ion exchange chromatography step, (5)

pooled fractions from the hydrophobic interaction chromatography step, (6) pooled fractions from the

second ion exchange chromatography step (MonoS)

Figure 9 shows optical spectra of the purified protein. These are characteristic

for c-type cytochromes class II, also referred to cytochrome c'. They contain

pentacoordinated heme in the high-spin state (Weiss et al., 2006; Barbieri et al.,

2008). In the oxidized state at neutral pH a distinct peak at about 640 nm is

observed, indicating a high-spin heme, and the Soret band at 401 nm exhibits a

shoulder at about 377 nm, as shown in figure 9 A. In the reduced state the

Soret band exhibits a maximum at 428 nm with a shoulder at 435 nm. At pH 9

the maximum shifts to 407 nm with the shoulder at 371 nm in the oxidized

state, figure 9B.

Cytochromes c' are found in a variety of bacteria with different kinds of

metabolism but its function is, with a few exceptions, not well understood. In

Methylococcus capsulatus Bath, it has been demonstrated that a low-spin

cytochrome c' acts as an electron carrier (Weiss et al., 2006) and in Rhodobacter

capsulatus PSA100, it is shown that cytochrome c' protects the bacterium against

NO by binding NO as a sixth ligand (Cross et al., 2000).

35

Figure 9 (A) Optical spectra of the purified cytochrome c in the oxidized (solid line) and reduced

(dashed line) state at neutral pH. (B) Optical spectra in the oxidized state at pH 9.

36

In order to clarify whether this cytochrome participates in electron transport

during chlorate respiration, a slight excess dithionite was added to a nitrogen-

flushed mixture containing the purified protein and cell homogenate. Addition

of chlorate did not produce any spectral changes. Addition of oxygen led to

oxidation, as judged from the spectrum. However, this spectral change was

observed even in absence of cell homogenate, indicating an uncatalyzed

reaction with oxygen. Accordingly, we cannot evaluate a possible function as

electron donor for terminal oxidase. However, we conclude that this

cytochrome c' does not serve as electron donor for chlorate reductase.

For further characterization, a more accurate molecular weight analysis of the

SDS-PAGE was performed, resulting in 13 kDa for a monomer. Using Blue

native PAGE followed by heme staining, a molecular weight of 55 kDa was

found for the native form of the protein. These results suggest that the native

form of the protein is a homotetramer. Most class II c cytochromes described in

the literature appear as mono- or dimers. A tetrameric protein in this family has

not been reported earlier. The heme content was found to be 1.0 mol/mol

protein, by pyridine hemochrome analysis.

De novo sequencing of tryptic peptides by mass spectrometry gave peptide

sequences AGNXDQXK or GAXNDQXK or QNXDQXK (X denoting I or

L in this case). None of these sequences does match the predicted sequence of

the cytochrome c gene located downstream from the chlorate reductase genes

(Paper II), excluding this gene as the source of the protein.

37

5. Conclusion

The aim of this study was to clarify the electron transport pathway in the

chlorate reduction in I. dechloratans, in particular the route between the

membrane-bound components and the periplasmic chlorate reductase. We have

demonstrated that electron transport is mediated by soluble electron carriers. A

c-type cytochrome with a molecular weight of 9.3 kDa denoted cytochrome c-

Id1 is the major donor to chlorate reductase when cells are grown anaerobically

in presence of chlorate. This protein can also serve as donor for the reduction

of oxygen. The periplasmic extract contains several other minor heme-

containing proteins. According to their low concentration, it is difficult to

purify and investigate their function as electron donors. However, the result

described in paper I suggest that these proteins can serve as electron donors in

the reduction of oxygen by the membrane-bund terminal oxidase, but do not

deliver electrons to chlorate reductase.

Another major c cytochrome found in the periplasm was shown to belong to

the class II subgroup of c cytochromes, (cytochrome c'), containing high-spin

pentacoordinated heme. The function of this protein is not clear, but it does

not appear to serve as electron donor, at least not in the chlorate reduction. A

function as NO-protector, as have been suggested in denitrifying bacteria, does

not appear likely in I. dechloratans.

Furthermore, a gene found downstream from the gene encoding the C subunit

of chlorate reductase has been characterized. The gene was identified as a

soluble cytochrome c (class I) which is defined as a soluble electron transfer

protein. The gene was expressed heterologously for the purpose of investigating

its role as electron carrier in the chlorate reduction mechanism. However, the

reconstituted protein was not able to serve as electron donor to chlorate

reductase. We could not identify any product of this gene in the periplasmic

extract and thus, it appears likely that expression of this gene does not occur

under the present conditions. The significance of the location of this gene still

remains to be elucidated.

Since the anaerobic respiration involves production of molecular oxygen, the

reduction of both chlorate and the produced oxygen (with the overall reaction

ClO3- → Cl- + H2O as the result), requires transfer of six electrons. The

38

e-

ClO3-

ClCl

O2- 2

A

2e-

O2

Cl-

B

cbb3 oxidase

chlorate reductase

chlorite dismutase

Q

periplasm

cytoplasm

4e-

H2

O

IV

V

III

dehydro-

genases

ClO2-

reduction of chlorate to chlorite is a two-electron reaction whereas reduction of

oxygen, produced by the subsequent decomposition of chlorite, requires four

electrons. This requires a branched electron pathway, with about one third of

the electrons serving as reductant for chlorate, and two thirds as reductant for

oxygen. Based on the results presented in this thesis, I suggest in figure 10 an

arrangement containing two branches. One is at cytochrome c-Id1 which can

serve as electron donor for both chlorate reductase and for terminal oxidase.

The presence of additional cytochromes, which can denote electrons to oxygen

but not to chlorate reductase, suggests a second branch point earlier in the

electron transport chain. In figure 10, this is depicted at the level of complex

III. With this arrangement, all six electrons required for the complete reduction

of chlorate pass through complex III and contribute to the formation of a

proton gradient across the cell membrane.

Figure 10 The proposed electron-transfer pathways in Ideonella dechloratans.

(A) cytochrome c-Id1, (B) minor soluble heme-containing components.

39

6. Tack

Jag vill rikta stort och hjärtligt tack till min handledare Prof. Thomas Nilsson,

som har varit ett oersättligt stöd under min tid som doktorand. Din entusiasm,

men även ditt lugn har inspirerat och gjort att jag velat gå vidare i arbetet även

när motgångarna varit som störst. Tack!

Jag vill även rikta ett stort tack till mina biträdande handledare Birgitta

Sundström och Maria Rova. Birgitta, du har inte haft möjlighet att följa mitt

arbete så ingående men du har alltid tagit tig tid och engagerat dig när jag

behövt tips och råd. Dessutom har du varit ett stöd i din roll som avdelningens

prefekt. Maria, du är själv aktiv i projektet och har varit ett mycket bra stöd. Du

har i alla lägen ställt upp med både tid och råd på ett mycket lugnt och

pedagogiskt sätt. Tack till er båda!

Jan Bohlin, du var min doktorandkollega när jag började i projektet. Jag vill

tacka dig för att du tog emot mig som ny kollega på allra bästa sätt och för allt

samarbete i både med- och motgång. Med ett ständigt glatt humör tog du de

flesta problem med en axelryckning. Positivt tänkande ger energi. Tack Jan!

Det finns många på min avdelning som jag vill tacka, helt enkelt för att ni finns

där. Men det finns speciellt två personer som är värda ett särskilt stort tack,

nämligen Elisabeth Björk och Mikael Andersén. Ni är två klippor

(laboratorieingenjörer) som ställer upp med det mesta i alla lägen. Många

guldstjärnor till er!

Tyvärr är det några som inte längre är kvar eftersom ni har disputerat och (eller)

fått andra jobb. Jag tänker särskilt på Sandra, Rozbeh (Robban), Cecilia, Sofia,

Tina, Björn, Jeanette och Stina. Vi hade mycket roligt tillsammans och jag

saknar er verkligen. Robban, jag väntar fortfarande på en J.D. & S.A.

Alla ni andra som fortfarande ”plaskar omkring i Möckeln” om dagarna, tack

för att ni bidrar till att vi är ett trivsamt gäng. Särskilt vill jag nämna mina

närmsta doktorandkollegor Miriam, Hanna, Maria, Christina, Sara, Ola och

Alexander, samt doktorerna Gunilla, Patrik och Marcus. Jag vill tacka för allt

roligt vi haft ihop, men jag hoppas att vi ska få ha kul ihop ett tag till. För övrigt

ska ingen känna sig glömd. Tack alla på avdelningen!

40

Jag vill även tacka Prof. Jan van Stam. Tack var dig har jag under den senare

delen av min doktorandtid fått uppdraget att jobba halvtid som

Kemilektorslänk. Jag vill även tacka Kungliga vetenskapsakademien och

Ljungbergsfonden som gett mig uppdraget.

Till hela min familj; tack för att ni finns! Livet består ju tack och lov av mer än

bara bakterier och även om jag periodvis har varit väldigt upptagen av dessa

små ”baggar” är det ändå ni som gör livet värt att leva. Bosse, du har ställt upp

oerhört mycket för mig och skött marktjänsten hemma under de tuffaste tider.

Älskling, vad ska jag nu skylla på för att slippa städa?

41

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Karlstad University StudiesISSN 1403-8099

ISBN 978-91-7063-375-1

c Cytochromes as Electron Carrier in Microbial Chlorate Respiration

Microbial respiration of oxochlorates is important for the biotreatment of effluents from industries where oxochlorates are produced or handled. Several bacterial species are capable to use perchlorate and/or chlorate as an alternative electron acceptor in absence of oxygen. The present study deals with the electron transport from the membrane-bound components to the periplasmic chlorate reductase, in the gram-negative bacterium Ideonella dechloratans. Both chlorate reductase and the terminal oxidase of I. dechloratans were found to utilize soluble c cytochromes as electron donors. For further investigation, two major heme-containing components were purified and characterized. The most abundant was a 9 kDa c-type cytochrome (class I), denoted cytochrome c-Id1. This protein was shown to serve as electron donor for both chlorate reductase, and for a terminal oxidase. The other major component was a 55 kDa homotetrameric cytochrome c’, (class II). A function for this cytochrome could not be demonstrated but it does not appear to serve as electron donor to chlorate reductase. A gene predicted to encode a soluble c cytochrome was found in close proximity to the gene cluster for chlorate reduction. The predicted sequence did not match any of the cytochromes discussed above. The gene was cloned and expressed heterologously, and the resulting protein was investigated as a candidate electron donor for chlorate reductase. Electron transfer from this protein could not be demonstrated, suggesting that the gene product does not serve as immediate electron donor for chlorate reductase.