Báo cáo khoa học: Comparing the substrate specificities of cytochrome c biogenesis Systems I and II docx

Spontaneous periplasmic assembly of cytochrome c552 appears to occur, as some holocytochrome is detected in the absence of any biogenesis system Fig.. Cytochrome c550 containing an AXXCH

Comparing the substrate specificities of cytochrome c

biogenesis Systems I and II

Bioenergetics

Alan D Goddard*, Julie M Stevens*, Arnaud Rondelet, Elena Nomerotskaia, James W A Allen and Stuart J Ferguson

Department of Biochemistry, University of Oxford, UK

Introduction

Nature employs at least five distinct systems for the

biogenesis of c-type cytochromes [1–3]; this

post-trans-lational modification process covalently links the heme

cofactor to, normally, two cysteines in a CXXCH

motif System I is found in many Gram-negative

bacte-ria and various mitochondbacte-ria, including from plants

[4,5]; System II appears in Gram-positive and some

Gram-negative bacteria, and chloroplasts [6]; System

III occurs in many non-plant mitochondria [5]; System

IV is specific for the unusual cytochrome b6 involved

in photosynthesis [7], and a fifth system, which remains

to be characterized, exists in trypanosomatids [8] Very

unusually, some thermophilic cytochromes c are able

to form spontaneously in vitro or in the cytoplasm of Escherichia coli[9], although it is believed that they are naturally matured by one of the biogenesis systems above

The experimental amenability of E coli has allowed the heterologous replacement of its own cytochrome

c maturation (Ccm) machinery (encoded by the ccmABCDEFGH operon, called System I, Fig 1) with systems from other organisms to facilitate their analy-sis The enzyme heme lyase (System III) has been shown to function in E coli cytoplasm [10] and to

Keywords

cytochrome c; cytochrome c maturation;

heme; heme provision; System II

Correspondence

S J Ferguson, Department of

Biochemistry, University of Oxford, South

Parks Road, Oxford OX1 3QU, UK

Fax: +44 1865 613201

Tel: +44 1865 613299

E-mail: stuart.ferguson@bioch.ox.ac.uk

*These authors contributed equally to this

work

(Received 28 July 2009, revised 12 October

2009, accepted 25 November 2009)

doi:10.1111/j.1742-4658.2009.07517.x

c-Type cytochromes require specific post-translational protein systems, which vary in different organisms, for the characteristic covalent attach-ment of heme to the cytochrome polypeptide Cytochrome c biogenesis System II, found in chloroplasts and many bacteria, comprises four subun-its, two of which (ResB and ResC) are the minimal functional unit The ycf5 gene from Helicobacter pylori encodes a fusion of ResB and ResC Heterologous expression of ResBC in Escherichia coli lacking its own bio-genesis machinery allowed us to investigate the substrate specificity of Sys-tem II ResBC is able to attach heme to monoheme c-type cytochromes

c550 from Paracoccus denitrificans and c552 from Hydrogenobacter thermo-philus, both normally matured by System I The production of holocyto-chrome is enhanced by the addition of exogenous reductant Single-cysteine variants of these cytochromes were not efficiently matured by System II, but System I was able to produce detectable amounts of AXXCH variants; this adds to evidence that there is no obligate requirement for a disulfide-bonded intermediate for the latter c-type cytochrome biogenesis system In addition, System II was able to mature an AXXAH-containing variant into

a b-type cytochrome, with implications for both heme supply to the peri-plasm and substrate recognition by System II

Abbreviations

Ccm, cytochrome c maturation; IPTG, isopropyl thio-b- D -galactoside; MESA, 2-mercaptoethane sulfonate.

produce mitochondrial holocytochrome c System II

from Helicobacter pylori has been substituted for the

E coli Ccm machinery, enabling a comparison of the

heme delivery activities of the two systems towards a

diheme cytochrome c [11] In E coli, natural

cyto-chrome c biogenesis requires at least 10 proteins,

con-trasting with the single protein constituting System III

System II is of intermediate complexity and comprises

four proteins in, for example, Bacillus subtilis and

Bordetella pertussis, namely ResA, ResB, ResC and

CcdA [12,13] (Fig 1) Notably, the genomes of some

Bordetella species, e.g B parapertussis, encode both

Systems I and II [1]

In common with System I, it is not clear whether

heme is transported across the membrane by System II

itself or by some other process Heme must then be

attached to the CXXCH apocytochrome motif, the

cys-teine side-chains of which need to be in the reduced

state CcdA (or, in some organisms, DsbD) is a

mem-brane protein that provides the required reducing power

to the thioredoxin-like protein ResA, which is thought

to reduce the apocytochrome [14] ResB (also called

Ccs1 and CcsB) is a membrane protein of unknown

function with a large lumenal⁄ extracytoplasmic domain

[15,16] ResC (also called CcsA) is also membranous

with a soluble domain, and contains a tryptophan-rich

signature motif found in various cytochrome c

biogene-sis proteins (the System I proteins CcmC and CcmF),

which has been proposed to function in heme handling

[17–19] The biogenesis machinery from H pylori

appears to be a single protein that is a fusion of the

pro-teins ResB and ResC, making it a useful minimal

System II model The expression of H pylori ResBC

in E coli allowed the heterologous maturation of

B pertussis cytochrome c4, a cytochrome normally matured by System II [11] In addition, this approach allowed the identification of essential histidine residues within ResBC proposed to act as axial ligands to heme iron [20] However, little is known about the substrate recognition or specificity of System II

A particularly notable point for examination is the ability of Systems I and II to mature single-cysteine-containing c-type cytochromes (XXXCH or CXXXH motifs) XXXCH cytochromes occur in nature in the mitochondria of Euglenozoan organisms, such as Cri-thidia fasciculata, and it has been demonstrated that the overall structure of cytochrome c from this organ-ism bears remarkable similarity to yeast cytochrome c [21] It is believed that organisms which possess such single-cysteine c-type cytochromes exhibit an as yet unidentified, novel biogenesis system All fully sequenced genomes of organisms expressing such sin-gle-cysteine cytochromes lack identifiable homologues

of known c-type cytochrome biogenesis proteins The ability (or otherwise) of System I or II to mature such cytochromes may shed light on the mechanism of heme attachment in these systems To date, System I has not been clearly observed to attach heme to such single-cysteine variants [22,23] Contrastingly, System

II has been proposed to attach heme to an SXXCH motif in NrfH [24]

In this work, we have cloned the ResBC-encoding gene from H pylori (ycf5) into the backbone plasmid (pACYC184) of pEC86 which contains the E coli ccm operon [25] and which has been very widely used for heterologous cytochrome c production Heterologous expression of H pylori ResBC from this new plasmid (pHP86) in E coli allowed us to explore the substrate

Heme handling/ligation

System I

System II

CcmE

CcmF

ResA

DsbD D

CcmB

CcmA

CcmC

p-side

p-side

Disulfide isomerization Fig 1 Schematic representation of

cytochrome c biogenesis Systems I and II.

The systems illustrated are System I (the

Ccm system) from E coli and System II

from B subtilis Note that ResBC is a fusion

protein in H pylori The two systems can

each be subdivided into proteins which

contribute to the handling of heme and

ligation of heme to the apocytochrome, and

those involved in the provision of reductant

to the apocytochrome in order to reduce a

potential disulfide bond in the CXXCH

heme-binding site CcmH, which in some

organisms is two separate proteins CcmH

and CcmI [50], appears to be involved in

both heme handling⁄ ligation and reductant

provision [2].

specificity of System II in direct comparison with

System I

Results

Various c-type cytochrome proteins were used to probe

different aspects of the specificity of the expressed

cytochrome c biogenesis systems Experiments were

conducted in a strain of E coli lacking all known

endogenous cytochrome c biogenesis proteins, EC06

In each case, control experiments were performed for

spontaneous heme attachment to exogenous

cyto-chromes [using the biogenesis system plasmid

back-bone containing no cytochrome c biogenesis genes

(AD377)] In addition, correction was performed for

the formation of any endogenous E coli cytochromes

catalyzed by the products of the different biogenesis

plasmids in the absence of a gene for an exogenous

cytochrome

System II can mature monoheme c-type

cytochromes in E coli

Cytochrome c550 from Paracoccus denitrificans is well

characterized as a heterologous holocytochrome

pro-duced by the E coli Ccm system [26] The ability of

System II to attach heme to this monoheme bacterial

cytochrome in the periplasm of EC06 cells was tested

Holocytochrome c550 was detected in periplasmic

extracts of cells containing pHP86 (H pylori ResBC)

and pKPD1 (cytochrome c550) and was quantified

spectroscopically (Fig 2) The yield was approximately

0.6% of that with System I, which produces very large

amounts of the holocytochrome (Table 1) SDS-PAGE

analysis followed by heme staining (Fig 2, right-hand

inset) shows a band of the expected mass ( 15 kDa

for P denitrificans holocytochrome c550 cleaved of its

signal peptide) for the cytochrome produced by System

II, the intensity of which is consistent with the amount

of cytochrome determined spectroscopically compared

with System I The a-band of the pyridine

hemo-chrome spectrum, which is indicative of the saturation

of the heme vinyl groups to which the cysteine residues

attach, was found to be at 550 nm for the System

II-matured cytochrome c550, as expected for the

forma-tion of two thioether bonds (Fig 2, left-hand inset)

Cytochrome c550made by System II (Fig 2) was

there-fore indistinguishable from that made by System I, its

natural biogenesis machinery This is the first

demon-stration that System II can mature a cytochrome

nor-mally matured by System I

We also examined the biogenesis of

Hydro-genobacter thermophilus cytochrome c552 This System

0.035

0.030

0.025

550 560 570 540

530 0.020

6

Wavelength (nm) Fig 2 Maturation of P denitrificans cytochrome c 550 by System II Visible absorption spectra reflecting the formation of P denitrificans cytochrome c 550 and variants in periplasmic extracts of E coli EC06 catalyzed by H pylori ResBC: wild-type cytochrome c 550 (full line), AXXCH-containing variant (broken ⁄ dotted line) and CXXAH-contain-ing variant (broken line) The vertical scale bar represents 0.01 absorbance units The spectra are vertically offset for clarity Sam-ples were reduced by the addition of a few grains of disodium dithi-onite The absorbance maxima for wild-type cytochrome c550are at

415, 521.5 and 550 nm The inset spectrum shows the reduced pyridine hemochrome spectrum of cytochrome c 550 produced by

H pylori ResBC The vertical line indicates 550 nm The inset gel illustrates the detection of c-type cytochromes via SDS-PAGE analysis, and subsequent heme staining of the gel, of periplasmic fractions from cells expressing P denitrificans cytochrome c550and the indicated biogenesis system (I or II) The periplasmic fraction from cells expressing System I and cytochrome c 550 was diluted 20-fold before analysis (equating to 0.25–0.5 lg protein loaded, compared with 5–10 lg for the undiluted System II-produced sam-ple) The left-most lane (M) contains See-Blue Plus 2 protein mark-ers of the indicated molecular weights (kDa).

Table 1 Levels of holocytochrome production for biogenesis Systems I and II expressed in E coli strain EC06 These values have been corrected to account for any spontaneous formation of the respective cytochromes and for background levels of endoge-nous cytochrome production The units are milligrams of holocyto-chrome per gram of wet cell pellet The values in parentheses are standard deviations ND, not detectable.

Cytochrome c550

Cytochrome c552

I-matured thermophilic cytochrome has also been used

as a substrate to test the properties of the E coli

cyto-chrome c biogenesis system [22] System I is able to

produce large quantities of the c552 holocytochrome

(Table 1 and Fig 3) Co-expression of cytochrome c552

with the System II plasmid resulted in approximately

16% of the level produced by System I, a much higher

proportion than that observed with the mesophilic

cytochrome c550 The spectroscopic features and

mobil-ity on SDS-PAGE of the System II-produced

cyto-chrome c552 are identical to those of the same

cytochrome produced by System I (Fig 3 and inset)

Spontaneous periplasmic assembly of cytochrome c552

appears to occur, as some holocytochrome is detected

in the absence of any biogenesis system (Fig 3) The

data presented in Table 1 have been corrected for the

mean level of spontaneous heme attachment

Uncata-lyzed heme attachment to cytochrome c552is known to

occur in the E coli cytoplasm [9], and a small amount

of cytoplasmic contamination of periplasmic extracts

can occur during preparation [22] However,

SDS-PAGE analysis of the periplasmic fractions in this study demonstrated that the spontaneous holocyto-chrome formation detected was essentially all periplas-mic, as the observed mass was consistent with that of the cytochrome polypeptide cleaved of its periplasmic targeting sequence The mass of H thermophilus holo-cytochrome c552cleaved of its signal peptide is approx-imately 9 kDa, whereas the uncleaved product has a mass of approximately 11 kDa

Maturation of single-cysteine holocytochromes There are natural examples of cytochromes in which heme is attached via a single thioether bond to a cys-teine in the protein [8,27], which raises questions about the purpose of covalent heme attachment via two bonds [21,28] The determination of whether the pres-ence of two cysteine thiols is essential could also address the requirement for an intramolecular disulfide bond, known to occur within apocytochromes [29], in the heme attachment reaction It has been argued that System II can attach heme to one SXXCH motif gen-erated by site-directed mutagenesis in the tetraheme cytochrome NrfH from Wolinella succinogenes [24] However, this is an important point requiring further investigation

Cytochrome c550 containing an AXXCH motif (C35A) acquired approximately 1.1% of the level of heme attachment observed for the wild-type CXXCH protein when expressed with System I (Table 1) The values in Table 1 are based on the absorption values

at single wavelengths However, they are only taken to indicate the presence of the specific holocytochrome under investigation if the features of the spectrum, in terms of wavelength maxima, and the position and intensity of heme-staining bands on SDS-PAGE gels, also appropriately demonstrate holocytochrome forma-tion The AXXCH variant produced by System I has spectroscopic features indicative of single-cysteine holo-cytochrome formation, and a band of the expected mass is observed on heme-stained gels (data not shown) The values in Table 1 imply that a small amount of the C38A (CXXAH) variant may have undergone heme attachment by System I However, using the criteria described above (spectra and heme staining), we conclude that the single-wavelength absorption intensity is not in fact indicative of C38A holocytochrome Effectively, therefore, the value in Table 1 for the C38A variant of cytochrome c550

matured by System I represents the lower limit of detectability and the experimental error Notably, the production of the AXXCH variant of cytochrome c550 was detected by western blotting using anti-cytochrome

6 14

Wavelength (nm)

Fig 3 Maturation of H thermophilus cytochrome c 552 by Systems

I and II Visible absorption spectra reflecting the formation of

H thermophilus cytochrome c552in periplasmic extracts of E coli

EC06 catalyzed by E coli System I (full line), H pylori System II

(broken line) and in the absence of any biogenesis system (AD377)

(broken ⁄ dotted line) (showing the level of spontaneous, i.e

uncata-lyzed, holocytochrome formation) The vertical scale bar represents

0.2 absorbance units The spectra are vertically offset for clarity

and normalized for wet cell weight Samples were reduced by the

addition of a few grains of disodium dithionite The absorbance

maxima are at 417, 521 and 552 nm The inset gel illustrates the

detection of c-type cytochromes via SDS-PAGE analysis of

periplas-mic fractions from cells expressing H thermophilus cytochrome

c 552 and the indicated biogenesis system (I or II), and subsequent

heme-staining of the gel Loading was normalized for total protein

content The left-most lane (M) contains See-Blue Plus 2 protein

markers of the indicated molecular weights (kDa).

c550 serum, whereas the CXXAH variant was not

(Fig S1, see Supporting Information)

Although it is possible that the CXXAH variant of

P denitrificanscytochrome c550 is unstable and cannot

be made, the two single-cysteine-containing (and

AXXAH) variants of H thermophilus cytochrome c552

can form stably in the E coli cytoplasm [30,31]

Sys-tem I was unable to produce the CXXAH variant

cytochrome c552, but some System I-dependent

forma-tion of the AXXCH variant (approximately 3%

rela-tive to CXXCH) was detected (this is the value after

subtraction to account for the level of spontaneous

holocytochrome formation) Figure 4 shows that the

heme-staining band corresponding to holo-c552

AXXCH matured by System I is significantly more

intense than the band observed when no biogenesis

genes were co-expressed (i.e with spontaneous

holocy-tochrome formation) This is a significant observation

regarding the substrate specificity of the Ccm system

Neither single-cysteine holocytochrome c550 was

detected with the coexpression of the System II

plas-mid, as shown in the spectra of the periplasmic

extracts in Fig 2, which have no features indicative of

holocytochrome formation It should be noted that

pEC86 (System I) complements the Ccm deletion of

E coli strain EC06, whereas pHP86 (System II) does

not; thus our experimental errors as a result of

back-ground (endogenous) cytochrome production are much

larger with System I than with System II Although

cultures grown in this work are considered to be

aero-bic, some microanaerobicity can occur, which causes

low-level expression of the endogenous E coli c-type

cytochromes System II appears to produce a very low

level of the AXXCH holocytochrome c552 (Table 1),

compared with the CXXCH form The analysis of 12

independent experiments revealed the production of

spectroscopically detectable AXXCH above the level

of spontaneous cytochrome formation in two of the

cultures These data are responsible for the apparent

formation of AXXCH by System II when compared with AD377 (reported as mean values in Table 1) It is possible that in the majority of our observations single-cysteine cytochromes were formed by System II

at such low levels that they were undetectable either by spectroscopic analysis of periplasmic fractions or heme staining of appropriate SDS-PAGE gels

System II mediates the formation of a b-type cytochrome

Unexpectedly, the spectra of periplasmic extracts of cells containing the System II plasmid and the double-alanine cytochrome c550(AXXAH motif, C35A⁄ C38A) indicated the presence of small amounts of a typical low-spin, b-type cytochrome (Fig 5) The Soret band

is red shifted by 4 nm and the a-band by 5 nm com-pared with the wild-type cytochrome c550, as would be expected for noncovalently bound heme (saturation of each heme vinyl group on formation of a c-type cyto-chrome causes a blue shift of 2–3 nm in the a-band of the absorption spectrum) To confirm the presence of variant cytochrome c550, we performed a western blot

of periplasmic extracts from this strain and a strain containing only pKK223-3 (i.e no cytochrome)

A band consistent with the molecular weight of

14 6

-Fig 4 Maturation of H thermophilus cytochrome c552AXXCH

vari-ant SDS-PAGE analysis and subsequent heme staining of

periplas-mic extracts from E coli EC06 cells containing the H thermophilus

cytochrome c552 AXXCH variant and the indicated biogenesis

sys-tem [I or II (with the lane marked - being periplasm from cells

containing empty vector, AD377)] Loading was normalized for total

protein content The left-most lane (M) contains See-Blue Plus 2

protein markers of the indicated molecular weights (kDa).

Wavelength (nm)

Fig 5 Maturation of a b-type cytochrome AXXAH variant of

P denitrificans cytochrome c 550 Visible absorption spectra of peri-plasmic extracts from E coli EC06 cells expressing H pylori ResBC and P denitrificans cytochrome c550 (broken-dotted line), cyto-chrome c 550 AXXAH variant (full line) and no cytochrome (pKK223-3) (dotted line) The vertical scale bar represents 0.005 absorbance units Samples were reduced by the addition of a few grains of disodium dithionite The Soret and a-band absorbance maxima are

at 415 and 550 nm, respectively, for wild-type cytochrome c 550 , and at around 419 and 555 nm for the AXXAH-containing variant The spectrum of the wild-type cytochrome c550has been reduced

by a factor of seven for clarity, and the spectra are vertically offset The vertical line indicates 550 nm.

cytochrome c550 was evident in the strain expressing

AXXAH-containing cytochrome c550, but not in the

control strain containing pKK223-3 (Fig S1, see

Sup-porting Information) It was not possible to detect low

levels of b-type complexes (if they exist) made with

System I because of the relatively high levels of

endog-enous cytochromes that are produced (see above)

would mask the spectroscopic features of the b-type

cytochrome However, we were unable to detect the

formation of a b-type AXXAH variant by System I

using western blot analysis and anti-cytochrome c550

serum (Fig S1, see Supporting Information) In

addi-tion, no b-type AXXAH cytochrome was detected

when no System II biogenesis proteins were present

These observations have implications for the provision

of heme to the periplasm by ResBC, and suggest that

it may facilitate heme delivery from the cytoplasm, in

agreement with a recent study by Frawley & Kranz

[20]

Provision of reductant increases significantly

System II-mediated c-type cytochrome formation

As the H pylori biogenesis system expressed in this

study lacks the thiol-disulfide oxidoreductase

compo-nents that are thought to reduce the cysteine thiols in

the cytochrome heme-binding motif (neither ResA of

System II nor CcmG of System I are present), the

effect of the addition of a chemical reductant to the

growth medium was tested: 5 mm 2-mercaptoethane

sulfonate (MESA) was added to cells containing

wild-type (CXXCH) P denitrificans cytochrome c550, and

the System II plasmid and holocytochrome contents

were determined The added reductant caused a

two-to three-fold increase in holocytwo-tochrome formation

(data not shown) Exogenous chemical reductant has

been used to recover the phenotypes of strains lacking

other oxidoreductases [32,33] The addition of 5 mm

MESA to cells expressing the single-cysteine c550C35A

variant did not result in the formation of detectable

holocytochrome, implying that the augmentation in

wild-type holocytochrome maturation with the

addi-tion of reductant is a result of the reducaddi-tion of a

disul-fide in the apocytochrome

Production of endogenous E coli cytochromes

Escherichia coli contains a number of endogenous

c-type cytochromes that it expresses under different

anaerobic growth conditions Some of these are

observed at low levels in periplasmic extracts when the

Ccm deletion strain EC06 is complemented with System

I (pEC86), but not with System II (pHP86), as shown in

Fig 6 The two bands observed correspond to the masses of the soluble cytochromes NapB (around

15 kDa) and NrfA (around 50 kDa) However, given the relative maturation levels of exogenous cytochromes c (see above), it may be that any endogenous cytochrome matured by System II would be present below the lower limit of detection in our experiments We have determined that the limit of detection for heme on a heme-stained SDS-PAGE gel is 1 nmol per lane (A D Goddard & S J Ferguson, unpublished observations)

E coliNapB has two hemes and NrfA five Therefore,

we would expect to detect 0.5 and 0.2 nmol of these cytochromes, respectively

Discussion

The complex and somewhat unpredictable natural distribution of cytochrome c biogenesis systems does not correlate specifically with the types of cyto-chrome produced by the organisms concerned [5,34] Cytochromes c vary widely in terms of overall fold, heme iron ligands, number of hemes per polypeptide, the presence of other cofactors, number of subunits, being membrane-bound or soluble, as well as the way

in which the heme is linked to the protein (a few cyto-chromes have single thioether bonds to heme) The latter group includes the unusual cytochrome b6 and the trypanosomatid cytochromes c The specificity of

E coli System I has been studied extensively It can produce cytochromes c from a wide variety of organ-isms, with many hemes per polypeptide, and even attach heme to peptides as short as 12 residues in length [35,36] The specificity of some heme lyases (System III) has also been determined; some organisms

62 49 38

14

28 17

6

Fig 6 Analysis of endogenous cytochrome production SDS-PAGE analysis and subsequent heme staining of periplasmic extracts from E coli EC06 cells containing pKK223-3 (no exogenous cyto-chrome) and the indicated biogenesis system (I or II) The left-most lane (M) contains See-Blue Plus 2 protein markers of the indicated molecular weights (kDa) Equal amounts of total protein were loaded in each lane.

contain a heme lyase for each mitochondrial

cyto-chrome c (e.g yeast cytocyto-chromes c and c1), whereas

others (e.g animals) contain a single such enzyme that

apparently catalyzes heme attachment to both

cyto-chromes [37] No study has examined the specificity of

System II, which in nature produces an array of

mono- and multiheme cytochromes

System II produces monoheme bacterial

cytochromes

In this work, we have shown that coexpression of the

plasmid pHP86 (expressing System II) in E coli with

the mesophilic cytochrome c550 from P denitrificans

and the thermophilic cytochrome c552from H

thermo-philus, both naturally matured by System I, results in

heme attachment to these cytochromes, yielding

prod-ucts that are indistinguishable from those produced by

System I This suggests that System II, in common

with System I and in contrast with System III [38], has

a broad substrate specificity and is able to mature

c-type cytochromes from a variety of sources, including

those that it does not naturally encounter A relatively

high level of holocytochrome c552 was produced (16%

relative to System I) considering that the System II

fusion protein is expressed heterologously and without

the remaining (disulfide oxidoreductase) components

of the biogenesis system A previous study has

inter-preted a reduced level of cytochrome production by

System II compared with System I as the former

hav-ing a lower affinity for heme [11]; as no measurement

of the relative abundance of the biogenesis proteins

was shown, and there is no reason to believe that they

would be equally stable in E coli, we have reservations

about this interpretation

That higher levels of thermophilic cytochrome c552

are produced by System II compared with a mesophilic

cytochrome (c550) is possibly a result of the higher

sta-bility of the apocytochrome c552 when it is delivered

by the Sec system to the periplasm Proteolytic

degra-dation of apocytochromes might compete with the

heme attachment machinery In addition,

apocyto-chromes are susceptible to periplasmic oxidation of the

heme-binding cysteine residues In our heterologous

System II, the oxidoreductase that would normally

reduce such a disulfide bond, ResA, is absent; the

oxi-dation would also slow heme attachment Our

observa-tion that added reductant results in a substantial

increase in cytochrome c550 production indicates that

oxidation of the heme-binding motif can reduce the

efficiency of heme attachment by System II

Neverthe-less, it is becoming increasingly clear from this work

and others [20] that dithiol⁄ disulfide oxidoreductases

are not strictly necessary for cytochrome c maturation

in the periplasm of E coli

Maturation of single-cysteine-attached cytochromes c

We found no detectable heme attachment to the sin-gle-cysteine-containing variants of P denitrificans c550 with coexpression of the System II plasmid A very low, variable level of heme attachment was observed with the AXXCH variant of cytochrome c552, but none with the CXXAH form If there is a capability to attach heme to a single-cysteine cytochrome then, in common with System I, it is to a very low extent com-pared with heme attachment to CXXCH, below the level of detection of the analysis conducted in this study It is notable that, in the work of Simon et al [24], evidence was found for heme attachment to only one SXXCH heme-binding motif of the four possible (and investigated) in NrfH, and that no heme attach-ment to CXXSH was reported [24] It may be that the observed heme attachment to one SXXCH motif was not in fact catalyzed by System II, e.g it was instead spontaneous, perhaps facilitated by substantial folding

of the protein around the three other hemes attached

to CXXCH motifs by System II

It has been reported previously that System I cannot produce detectable levels of single-cysteine-containing holocytochrome c552 [22] In that work, the lower level

of detectability was estimated as 2% of the wild-type (CXXCH) holocytochrome yield Control experiments performed in the present work, to allow a direct com-parison of System I and II plasmids (which are identi-cal but for their encoded operons), permit a refinement

of the conclusion of the former work We found here low but detectable levels of the holo-forms of AXXCH variants of both cytochromes c550and c552 (1 and 3% relative to the wild-type CXXCH cytochromes, respec-tively) when expressed with pEC86 The difference is presumably partly a result of the different E coli strains used Here, we used EC06, a ccm deletion strain, which had a significant effect on the amount of background cytochrome c matured (producing no detectable c-type cytochromes in the absence of a plas-mid-borne biogenesis system) The sensitivity of the analytical methods used (e.g the former work did not use heme-stained gels) may also contribute to the dif-ferences That System I can attach some heme to sin-gle-cysteine-containing cytochromes is significant, particularly in the context of a possible relationship between heme attachment and a disulfide bond in the CXXCH motif The fact that two cysteines are not absolutely essential for the heme attachment reaction

demonstrates that the chemistry of such a reaction is

not necessarily via an obligate disulfide-containing

intermediate Breaking a disulfide bond could be

envis-aged as providing the driving force for formation of

the thioether bonds to heme However, successful

in vitro thioether bond formation using a phosphine

(in the absence of thiol reagents) to reduce the

apocy-tochrome disulfide also indicated that disulfide-linked

chemistry is not involved in the heme attachment

reac-tion [39] The present work implies that the formareac-tion of

the two thioether bonds is not thermodynamically

neces-sary to release heme from the covalent heme-binding

chaperone CcmE We observe, as might be anticipated

[21,31,40], that the single-cysteine variant in which the

heme-binding cysteine is directly adjacent to the heme

iron-ligating histidine (i.e XXXCH) is the more likely to

be recognized by the system and to undergo heme

attach-ment than is CXXXH Nevertheless, it remains clear that

System I is far more effective and efficient at attaching

heme to apocytochrome c with two cysteines, rather than

one, in the heme-binding motif

System II facilitates the formation of a b-type

cytochrome in the periplasm

In the absence of any biogenesis proteins, it was not

possible to detect the b-type forms (i.e containing

non-covalently bound heme) of c-type cytochromes lacking

the heme-binding cysteine residues (i.e the AXXAH

variants) Apocytochromes c appear to be

proteolyti-cally degraded when heme is not attached [41] Because

of the clean background observed with the System II

plasmid (i.e the lack of any endogenous c-type

cytochromes), it was possible to detect some b-type

cytochrome when cytochrome c550 AXXAH was

coex-pressed with pHP86 It is possible that an equivalent

cytochrome is produced by System I, but that it is

ren-dered undetectable as a result of the production of

endogenous cytochromes c which mask the b-type

spectra [b-type cytochromes generally lose heme in

SDS-PAGE and therefore cannot be detected by the

heme staining of gels; western blotting with

anti-cyto-chrome c550serum failed to detect the presence of any

cytochrome (Fig S1, see Supporting Information)]

Alternatively, it is possible that, as a result of a

cova-lent intermediate (CcmE–heme) [42], System I is

unable to pass heme to an AXXAH variant

apocyto-chrome; System II (ResBC) from Helicobacter

hepati-cus does not appear to covalently bind heme [20]

However, a recent study with Bacillus subtilis ResB

and ResC (unfused proteins in that organism) revealed

covalent binding of heme to the cytoplasmic side of

ResB [43] It is therefore possible that an initial

cova-lent attachment of heme to ResB occurs, followed by trafficking through ResC, before insertion of heme into the periplasmic cytochrome However, the residue covalently bound to the heme of ResB was found to

be nonessential for cytochrome c biogenesis The func-tion, if any, of System II proteins covalently binding heme therefore remains to be resolved

That expression of the System II protein in E coli allows the formation of a b-type cytochrome suggests that heme provision from the cytoplasm to the peri-plasm might be performed by ResBC, concurrent with recent observations [20] The study by Frawley & Kranz [20] also demonstrated the essentiality of H858

of H hepaticus ResBC in holocytochrome formation, and proposed that this residue, together with H77, is involved in supplying heme to the periplasm We note that a H857E mutant in H pylori ResBC (equivalent

to H858 in the H hepaticus protein) is unable to mature the b-type cytochrome described above (A D Goddard & S J Ferguson, unpublished observations) This is consistent with H857 playing a role in heme transport It is not known how heme is transported across the inner membrane by System I, but it has been shown conclusively that, contrary to earlier sug-gestions, CcmA and CcmB are not involved in heme transport in E coli [44,45] Notably, maturation of an AXXAH-containing variant b-type cytochrome c550 in the present study indicates a nascent heme-binding site, even in this mesophilic apocytochrome c (see also [46]), as well as possible recognition features in the apocytochrome, at least for cytochrome c biogenesis System II, other than the CXXCH heme-binding motif These data also suggest that heme delivery to apocytochrome and thioether bond formation by System II are independent processes

Materials and methods

Strains, plasmids and culture conditions

deletion of the ccm operon and was used to examine holocy-tochrome formation in the presence of the plasmid-encoded biogenesis systems E coli strain DH5a (Invitrogen, Paisley, UK) was used for routine molecular biology KOD poly-merase (Merck Chemicals Ltd, Nottingham, UK) was used for PCRs All oligonucleotides used in this study are listed

in Table S1 (see Supporting Information)

Biogenesis plasmids The plasmids used in this work are listed in Table S2 The

from pEC86 [25] To create a comparable plasmid lacking

any biogenesis system, inverse PCR was performed on

pEC86 using the primers AG234 and AG235, and the

prod-uct was self-ligated This removed the entire ccm operon,

and the plasmid created is AD377 (no biogenesis system)

To create a suitable plasmid for the expression of other

bio-genesis systems, a XhoI site was introduced immediately

after the initiating ATG of ccmA in pEC86 via Quikchange

mutagenesis using the primers WC1 and WC2 The

resul-tant construct is pEC86x, in which the entire ccm operon

can be excised by digestion with XhoI and StuI The ycf5

gene was amplified from H pylori (strain 26695) genomic

DNA using oligonucleotides HelF and HelR The PCR

resultant plasmid for the expression of H pylori ResBC is

pHP86 (System II)

Cytochrome c plasmids

iso-propyl thio-b-d-galactoside (IPTG)-inducible promoter of

pKPD1 [26] Mutations within the CXXCH heme-binding

AX-XCH, AXXAH and CXXAH variants were expressed from

the plasmids pEST210, pEST211, pEST212 and pEST213,

respectively [22]

In each case, the plasmid bearing the biogenesis system

confers resistance to chloramphenicol and the expression of

the proteins is constitutive The plasmids bearing the

cyto-chromes are inducible with IPTG and confer resistance to

carbenicillin All constructs were sequenced before use

Routine cell growth was conducted using Luria–Bertani

Growth on solid medium used Luria–Bertani medium

sup-plemented with 1.5% bacteriological agar For the

prepara-tion of periplasmic fracprepara-tions, single colonies containing

appropriate plasmids were picked into 500 mL 2· TY

NaCl), supplemented with 1 mm IPTG and appropriate

with shaking at 200 r.p.m for 16–20 h before harvesting

Analysis of cytochrome production

Periplasmic extractions were performed as described

previ-ously [22] Extracts were analyzed by SDS-PAGE

(Invitro-gen pre-cast 10% Bis-Tris gels), followed by heme staining

[48], which stains proteins with covalently bound heme

Samples were normalized for wet cell weight, and equal

amounts of protein were loaded per lane (5–10 lg)

were performed according to the manufacturer’s

secondary IgG raised in goat The marker used was See-Blue Plus 2 (Invitrogen)

UV–visible spectroscopy was performed using a Perkin-Elmer (Waltham, MA, USA) Lambda 2 spectrophotometer; samples were reduced by the addition of a few grains of disodium dithionite (Sigma-Aldrich Company Ltd, Poole,

according to the method described by Bartsch [49] The normalized cytochrome content of each extract is pre-sented as the number of milligrams of holocytochrome c per gram of cell pellet The data are averages of at least five growths The extinction coefficients used to calculate the yields of holocytochromes were as follows: wild-type

are unknown; the wild-type value was therefore used Cor-rections of the average normalized values for each dataset were performed by subtracting the value observed when no biogenesis genes were expressed (i.e with plasmid AD377 and the relevant cytochrome plasmid, to correct for sponta-neous holocytochrome production) and subtracting the value observed when no heterologous cytochrome gene was expressed (i.e with plasmid pKK223-3 and the relevant bio-genesis plasmid, to correct for the production of endoge-nous E coli cytochromes) Finally, the values for cells expressing the two empty vectors AD377 and pKK223-3 were added back, so that any corrections were for endoge-nous or spontaneous cytochrome c production only Cultures for the corrections were grown and analyzed at least three times, and the mean values were used for the corrections

Acknowledgements

This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC; grant numbers BB⁄ D523019 ⁄ 1, BB ⁄ E004865 ⁄ 1 and BB ⁄ D019753⁄ 1) J.W.A.A is a BBSRC David Phillips Fellow A.D.G gratefully acknowledges the E P Abra-ham Cephalosporin Fund We thank Professor David Kelly (University of Sheffield) for kindly providing

H pylorigenomic DNA

Since the submission of this manuscript Kern et al [50a] have also shown that System II cannot attach heme to a single-cysteine motif in a cytochrome at detectable levels [sentence added at proof stage]

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