Báo cáo khoa học: Properties and significance of apoFNR as a second form of air-inactivated [4Fe-4S]ÆFNR of Escherichia coli pot

Properties and significance of apoFNR as a second form
of air-inactivated [4Fe-4S]ÆFNR of Escherichia coli
Stephanie Achebach
1,
*, Thorsten Selmer
2,
* and Gottfried Unden
1
1 Institut fu
¨
r Mikrobiologie und Weinforschung, Johannes Gutenberg-Universita
¨
t Mainz, Germany
2 Laboratorium fu
¨
r Mikrobiologie, Philipps-Universita
¨
t, Marburg, Germany
Fumarate nitrate reductase regulator (FNR) is a cyto-
plasmic O
2
sensor and regulator present in Escherichia
coli and other bacteria [1–7]. The protein is required
for transcriptional regulation of many genes of faculta-
tive anaerobic metabolism in response to O
2
availabil-
ity [8] and responds to O
2
concentrations in the
medium as low as 1–5 mbar O

2
(about 1–5 lm O
2
)
[7,9,10]. The rate of O
2
diffusion into the bacteria is
high compared with the low rates of O
2
consumption
at the cytoplasmic membrane or within cytoplasm
[3,7,9]. Therefore O
2
tension within the bacterial cyto-
plasm appears to be similar to that in the external
medium under oxic and microoxic conditions. Active
FNR containing a [4Fe-4S] cluster, is found
under anoxic conditions. In the presence of O
2
,
[4Fe-4S]ÆFNR is converted to [2Fe-2S]ÆFNR resulting
in monomerization and inactivation of FNR as a gene
regulator. The [4Fe-4S] ⁄ [2Fe-2S] conversion is respon-
sible for the anaerobic⁄ aerobic switch of FNR and
has been demonstrated in vivo and in vitro [11–14].
Recently it became clear that the FeS cluster of
[2Fe-2S]ÆFNR is also labile in vivo and in vitro in the
presence of air and that FNR loses the [2Fe-2S] cluster
[15,16]. The chemical reactions of O
2

and of peroxide
causing cluster destruction have been studied [15,17].
ApoFNR, which is devoid of FeS clusters, is a
further form of FNR and obtained during isolation of
FNR and by prolonged exposure of [4Fe-4S]ÆFNR to
air [18–20]. It is not known, however, whether apoFNR
is a preparatory artifact, or whether its formation
Keywords
apoFNR; cysteine disulfides; Escherichia
coli; fumarate nitrate reductase regulator;
oxygen sensing
Correspondence
G. Unden, Johannes Gutenberg-Universita
¨
t
Mainz, Institut fu
¨
r Mikrobiologie und
Weinforschung, Becherweg 15,
55 099 Mainz, Germany
Fax: +49 6131 3922695
Tel: +49 6131 3923550
E-mail:
*These authors contributed equally to the
work
(Received 1 June 2005, revised 24 June
2005, accepted 1 July 2005)
doi:10.1111/j.1742-4658.2005.04840.x
The active form of the oxygen sensor fumarate nitrate reductase regulator
(FNR) of Escherichia coli contains a [4Fe-4S] cluster which is converted to

a [2Fe-2S] cluster after reaction with air, resulting in inactivation of FNR.
Reaction of reconstituted [4Fe-4S]ÆFNR with air resulted within 5 min in
conversion to apoFNR. The rate was comparable to the rate known for
[4Fe-4S]ÆFNR ⁄ [2Fe-2S]ÆFNR cluster conversion, suggesting that apoFNR is
a product of [2Fe-2S]ÆFNR decomposition and a final form of air-inacti-
vated FNR in vitro. Formation of apoFNR and the redox state of the
cysteinyl residues were determined in vitro by alkylation. FNR contains five
cysteinyl residues, four of which (Cys20, Cys23, Cys29 and Cys122) ligate
the FeS clusters. Alkylated FNR and proteolytic fragments thereof were
analyzed by MALDI-TOF. ApoFNR formed by air inactivation of
[4Fe-4S]ÆFNR in vitro contained one or two disulfides. Only disulfide pairs
Cys16 ⁄ 20 and Cys23 ⁄ 29 were formed; Cys122 was never part of a disulfide.
The same type of disulfide was found in apoFNR obtained during isolation
of FNR, suggesting that cysteine disulfide formation follows a fixed
pattern. ApoFNR, including the form with two disulfides, can be reconsti-
tuted to [4Fe-4S]ÆFNR after disulfide reduction. The experiments suggest
that apoFNR is a major form of FNR under oxic conditions.
Abbreviations
CM, carboxymethyl; DTNB, 5,5¢-dithiobis(2-nitrobenzoate); FNR, fumarate nitrate reductase regulator; GnHCl, guanidinium hydrochloride;
GST, glutathione S-transferase; TFA, trifluoroacetic acid.
4260 FEBS Journal 272 (2005) 4260–4269 ª 2005 FEBS
is of functional relevance. The recent demonstration
that [2Fe-2S]ÆFNR disappears within few minutes
from cells of E. coli under exposure to air [15,16],
could be an indication that significant amounts of apo-
FNR are found in bacteria. The assumption prompted
our studies to test whether apoFNR is formed at rates
comparable to the air-induced [4Fe-4S] ⁄ [2Fe-2S] clus-
ter conversion and the loss of [2Fe-2S] from FNR.
Comparable rates would indicate that apoFNR is the

product formed from [2Fe-2S]ÆFNR in vivo [15,16]
and in vitro. Therefore the conditions and kinetics
for the formation of apoFNR from active FNR were
analyzed.
Recently it has been shown that various forms of
apoFNR exist in vitro which differ in the redox state
of the cysteinyl residues and their availability for
reconstitution to [4Fe-4S]ÆFNR [18]. Three of the cys-
teine ligands of the FeS cluster of FNR are located at
the N-terminal end (Cys20, Cys23 and Cys29), the
fourth (Cys122) is close to the centre of the protein
[21–23]. The fifth residue (Cys16) is not essential. The
redox state of the cysteinyl residues of apoFNR should
be relevant for function and properties of the protein.
Therefore the redox state of the cysteinyl residues of
the various forms of apoFNR – ‘aerobic’ and ‘anaer-
obic’ apoFNR, and apoFNR obtained by air-induced
inactivation of [4Fe-4S]ÆFNR – was determined in
order to identify cysteinyl residues which are sensitive
to oxidation and disulfide formation. ‘Aerobic’ and
‘anaerobic’ apoFNR are obtained by the isolation
of the protein under oxic or anoxic conditions
[18,22,24,25]. The cysteine disulfides in the three forms
of apoFNR showed a distinct pattern of disulfides,
whereas [4Fe-4S]ÆFNR (and [2Fe-2S]ÆFNR) contained
no disulfides. Thus the disulfides in apoFNR can be
used as a specific indicator for apoFNR formation
from [4Fe-4S]ÆFNR during air inactivation. In this way
it was possible to demonstrate that reaction of
[4Fe-4S]ÆFNR with air generates apoFNR in vitro with

rates comparable to [4Fe-4S] ⁄ [2Fe-2S] cluster conver-
sion [16,17,26] suggesting that apoFNR is the final
form of FNR under oxic conditions.
Results
Redox state of the cysteinyl residues in apoFNR
and in reconstituted [4Fe-4S]ÆFNR
ApoFNR that was devoid of Fe and acid-labile sulfur
(not shown) was obtained by isolation of FNR from
E. coli under oxic and anoxic conditions (‘aerobic’ or
‘anaerobic’). In SDS gel electrophoresis without redu-
cing agents, anaerobic apoFNR formed one band with
the typical molecular mass of FNR ( M
r
30 000). Aero-
bic apoFNR showed an additional band of M
r
27 000
(not shown) as described earlier [22]. The latter form
disappeared when the sample was incubated with
dithiothreitol or 2-mercaptoethanol, whereas the
M
r
30 000 protein was not affected. In aerobic and
anaerobic apoFNR 2.8 and 4.0 from the total of five
cysteinyl residues reacted with 5,5¢-dithiobis-nitro-
benzoate (DTNB) (Table 1) or iodoacetate after dena-
turing by guanidinium hydrochloride (GnHCl), and
were thus in the thiol state. This suggests that aerobic
apoFNR contains a portion with an (additional) disul-
fide which has a more condensed structure and higher

mobility in SDS gel electrophoresis.
After reaction with iodoacetic acid, the carboxy-
methyl derivatives were analyzed by MALDI-TOF
mass spectroscopy. Aerobic and anaerobic apoFNR
showed a molecular mass of 28 112 ± 14 Da before
modification (not shown) which is close to the predic-
ted molecular mass of the recombinant FNR of
28112 Da (FNR plus N-terminal Gly-Ser extension).
The alkylated aerobic apoFNR produced three major
peaks of 28 169 Da, 28 284 Da, and 28 398 Da
(Fig. 1A), corresponding to one-, three- and fivefold
alkylated FNR, as each carboxymethyl (CM) residue
increases the molecular mass by 58 Da. No unmodified
apoFNR was detected. Threefold alkylated apoFNR
showed the most intense signal. The MALDI-TOF
spectrum of anaerobic apoFNR consisted of one major
signal at 28 408 Da after alkylation equivalent to five-
fold alkylated apoFNR, and a minor signal of three-
fold alkylated FNR (Fig. 1B). Therefore, aerobic and
anaerobic apoFNR are mixtures of FNR with one,
three and five, and five and three cysteine thiol resi-
dues, respectively.
In reconstituted [4Fe-4S]ÆFNR up to four cysteinyl
residues reacted with DTNB (Table 1). The residues
Table 1. Thiol and disulfide cysteinyl residues in apoFNR (aerobi-
cally or anaerobically prepared), reconstituted [4Fe-4S]ÆFNR, and air-
inactivated [4Fe-4S]ÆFNR. The contents of cysteine thiols were
determined with DTNB or [
14
C]iodoacetate. The number of cystei-

nyl residues present in the oxidized state (‘Disulfide S’) were calcu-
lated as the difference to the total of five cysteinyl residues
present in FNR. The thiol contents are the mean of three experi-
ments (max. deviation 25%).
Thiol (mol ⁄ mol FNR) Disulfide S
Aerobic apoFNR 2.8 2.2
Anaerobic apoFNR 4.0 1.0
Reconstituted [4Fe-4S]ÆFNR
a
4.0 1.0
Air-inactivated [4Fe-4S]ÆFNR
a
2.2 2.8
a
Fig. 7.
S. Achebach et al. Disulfides of apoFNR
FEBS Journal 272 (2005) 4260–4269 ª 2005 FEBS 4261
were accessible to labeling only after denaturing of the
protein. In MALDI-TOF analysis, the alkylated pro-
tein gave one major signal of 28 395 Da corresponding
to fivefold alkylated FNR (FNR-CM
5
, theoretical
molecular mass 28 402 Da) (not shown). Four- and
threefold alkylated FNR was found only in traces.
In this way, [4Fe-4S]ÆFNR (and presumably [2Fe-
2S]ÆFNR) differ clearly from apoFNR by the lack of
cysteinyl disulfides which can be used for differenti-
ation of apoFNR and [4Fe-4S]ÆFNR in vitro by labe-
ling of cysteinyl thiol residues with iodoacetate after

denaturing the protein.
Kinetics of cysteinyl residue oxidation during
inactivation of [4Fe-4S]ÆFNR by air. Reconstituted
[4Fe-4S]ÆFNR was exposed to air, and after various
times samples were analyzed for the number of
reduced cysteinyl residues by reacting the protein with
[
14
C]iodoacetate (Fig. 2). GnHCl was included at the
same time to destabilize the FeS clusters and to make
the cysteinyl residues accessible to alkylation. In anae-
robic, reconstituted FNR about four cysteinyl residues
were labeled by this method. After addition of air, the
amount of reactive cysteinyl residues increased slightly
for about 2 min. Then the amount decreased to about
two residues within the next 3 min, corresponding to
an average of three cysteinyl residues in the oxidized
state. Under anoxic conditions no change in the num-
ber of reactive cysteine residues was observed. Exten-
ded incubation with air gave no further decrease of
reactive thiols. The result demonstrates that, after a
lag phase of about 2 min, air causes formation of
disulfides in FNR which is finished within about
3 min. Formation of the disulfides has to be accom-
panied by the loss of the FeS cluster of FNR due to
the loss of two or four ligands of the FeS cluster. This
type of kinetics was only obtained when excess Fe ions
were removed by EDTA, presumably due to reaction
of Fe ions with cysteinyl residues.
Defined cysteine disulfides in aerobic and

anaerobic apoFNR
Alkylated forms of aerobic and anaerobic apoFNR
were digested by proteases, and the peptides were iso-
lated and characterized by MALDI-TOF. Modified
trypsin cleaves the protein at the C-terminal site of
arginine and lysine, and the tryptic digest of FNR is
predicted to contain the four N-terminal Cys residues
in the peptide comprising amino acids 11–48
(Pep11 ⁄ 48). Cys122 is found in a peptide of amino
acids 78–135 (Pep78 ⁄ 135). The tryptic digest of aerobic
apoFNR contained unmodified Pep11 ⁄ 48 (4258 Da)
and two derivatives with two and four carboxymethyl
groups, respectively (Fig. 3A). Pep78 ⁄ 135 with Cys122
Fig. 1. MALDI-TOF spectra of aerobically (A) and anaerobically (B)
prepared and carboxymethylated apoFNR. The samples of apoFNR
were alkylated with 10 m
M iodoacetate under denaturing conditions
in 4
M GnHCl, purified by solid phase extraction and subjected to
MALDI-TOF MS analysis. The spectra were taken using sinapinic
acid as matrix in the linear mode of the instrument (for details see
Experimental procedures).
Fig. 2. Measurement of reactive cysteinyl residues during air-inacti-
vation of reconstituted [4Fe-4S]ÆFNR. Aerobic apoFNR was reconsti-
tuted, transferred to a Petridish and incubated under air (0 min). At
the given time-points, samples were removed and denatured in a
solution of GnHCl (4
M)and[
14
C]iodoacetate (12 mM and 43 BqÆ

mmol
)1
iodoacetate). The protein was precipitated and washed
with methanol + chloroform [31] and used for measurement of pro-
tein and radioactivity (mean of three independent experiments).
Disulfides of apoFNR S. Achebach et al.
4262 FEBS Journal 272 (2005) 4260–4269 ª 2005 FEBS
was found only as the alkylated form. No adduct of
Pep11 ⁄ 48 with Pep78 ⁄ 135 was present, ruling out the
presence of a disulfide bridge between both peptides.
Thus aerobic apoFNR is a mixture of proteins with
zero, one or two disulfide bonds within the four
N-terminal cysteinyl residues. When anaerobic apoFNR
was digested with trypsin, only four- and twofold
alkylated, but no unmodified Pep11 ⁄ 48 was found
(Pep11 ⁄ 48-CM
4
and Pep11 ⁄ 48-CM
2
) (Fig. 3B).
Protease AspN cleaves N-terminal to aspartate
By this cleavage, Pep1 ⁄ 21 containing Cys16 and Cys20,
Pep22 ⁄ 39 with Cys23 and Cys29, and Pep102 ⁄ 129 con-
taining Cys122 were generated (Table 2). When the
peptides were produced from aerobic apoFNR after
alkylation, Pep22 ⁄ 39 was found as the unmodified
and, in small amounts, also as the twofold alkylated
species. Pep1 ⁄ 21 was present in the unmodified state,
as well as modified with two alkyl residues. Thus
Cys16 ⁄ Cys20 and Cys23 ⁄ Cys29 formed disulfides in

aerobic apoFNR. Pep102 ⁄ 129, on the other hand, was
found only in the alkylated form, demonstrating again
that Cys122 was not involved in disulfide formation.
No peptides corresponding to the combination of
Pep1 ⁄ 21, Pep22 ⁄ 39 or Pep102 ⁄ 129 were found, and
thus only disulfides Cys16 ⁄ Cys20 and Cys23 ⁄ Cys29
were present within aerobic apoFNR.
Pep1 ⁄ 21 and Pep22 ⁄ 39 were detected from anaerobic
apoFNR in the unmodified as well as the twofold
alkylated form and, therefore, Cys16 ⁄ Cys20 and
Cys23 ⁄ Cys29 could also be responsible for apoFNR
with one disulfide. Cys23 ⁄ 29, however, was found at
significantly lower levels than in aerobic apoFNR, sug-
gesting that the Cys16 ⁄ 20 disulfide is formed preferen-
tially to the Cys23 ⁄ 29 disulfide.
Quantification of disulfide and thiol containing
peptides after HPLC separation
The peptides of the AspN digest with the cysteinyl
residues in the disulfide or thiol state were quantified
Fig. 3. MALDI-TOF spectra of peptides derived from carboxymeth-
ylated aerobic (A) and anaerobic (B) apoFNR (trypsin digest). The
samples (carboxymethylated tryptic digest of aerobic or anaerobic
apoFNR) were purified by solid phase extraction and analyzed by
MALDI-TOF MS in the reflector mode of the instrument using
a-cyano-4-hydroxycinnamic acid as matrix. Masses (m ⁄ z)of
cysteinyl residues containing tryptic peptides Pep11 ⁄ 48 (with
Cys16, Cys20, Cys23, Cys29) and Pep78 ⁄ 135 (with Cys122):
Pep11 ⁄ 48-CM
0
4258 in (A) (predicted 4259); Pep11 ⁄ 48-CM

2
4375
in (A) and 4374 in (B) (predicted 4375); Pep11 ⁄ 48-CM
4
4494 in (A)
and 4493 in (B) (predicted 4491); Pep78 ⁄ 135-CM
1
6248 in (A) and
6244 in (B) (predicted 6244). The predicted masses were obtained
from the mass of the peptide plus the mass increase of 58 after
alkylation per cysteinyl residues by iodoacetic acid.
Table 2. Mass of fragments of aerobically and anaerobically prepared apoFNR after treatment with iodoacetic acid and digestion with prote-
ase AspN. The numbering of the peptides gives the first and the last amino acid residue according to numbering in FNR. The mass of the
fragments and of their alkylated forms were determined by MALDI-TOF spectroscopy. Pep1 ⁄ 21 (with Cys16 and Cys20) contains in addition
the two N-terminal Gly-Ser residues derived from the GST¢-¢FNR fusion. Pep22 ⁄ 39 contains Cys23 and Cys29, Pep102 ⁄ 129 Cys122. The cal-
culated masses were obtained from the mass of the peptide and a mass increase of 58 after alkylation by iodoacetic acid per cysteinyl resi-
due. ND, not detected.
Cys-containing fragments Thiol Disulfide
Mass for alkylated peptides of apoFNR
Calculated (m ⁄ z) Aerobic (m ⁄ z ± 3) Anaerobic (m ⁄ z±3)
Pep1 ⁄ 21-CM
2
2 0 2671 2674 ND
a
Pep1 ⁄ 21-CM
0
0 1 2555 2556 ND
a
Pep22 ⁄ 39-CM
2

2 0 2179 2181 2181
Pep22 ⁄ 39-CM
0
0 1 2063 2062 2062
Pep102 ⁄ 129-CM
1
1 0 3047 3049 3050
a
Fragments can be detected after reversed phase HPLC (Table 3).
S. Achebach et al. Disulfides of apoFNR
FEBS Journal 272 (2005) 4260–4269 ª 2005 FEBS 4263
after alkylation and proteolytic digestion. The peptides
were separated by reversed-phase HPLC chromatogra-
phy (Fig. 4), and the peptides in the peaks were identi-
fied by MALDI-TOF. The amount of peptide was
quantified from the peak area of the HPLC eluate
(Fig. 4). The peptides Pep11 ⁄ 21 (Cys16, Cys20) and
Pep22 ⁄ 39 (Cys23, Cys29) from aerobic and anaerobic
apoFNR were found in the noncarboxymethylated
(disulfide) form as well as with two carboxymethyl
groups (Fig. 4). The peptide with Cys16 ⁄ Cys20 was
present to 65% in the oxidized (disulfide) form in aero-
bic and in anaerobic apoFNR (Table 3). The 23 ⁄ 29
disulfide in Pep22 ⁄ 39 was significantly increased in
aerobic compared with anaerobic apoFNR (Table 3)
which represented the only distinct differences between
both forms of apoFNR. Generally, a smaller portion
of the Cys23 ⁄ Cys29 than of the Cys16 ⁄ Cys20 residues
was in the disulfide state, suggesting that the Cys16 ⁄ 20
disulfide is formed preferentially.

Redox state of cysteinyl residues during
inactivation of [4Fe-4S]ÆFNR with air
The redox state of the cysteinyl residues of
[4Fe-4S]ÆFNR after inactivation by air (Fig. 2) was
determined. After reaction with iodoacetate, FNR of
molecular mass 28 262 Da was found which most prob-
ably represents FNR with one disulfide and three thiol
residues (theoretical molecular mass 28 286 Da). Devia-
tion of experimental and expected molecular mass by
24 Da is due to a variation in absolute mass (± 28 Da)
by nonspecific absorption of ions. Using [
14
C]iodoace-
tate, FNR with two labeled cysteine thiols was identified
after inactivation with air (Fig. 2 and Table 1). Alto-
gether, the experiments show that air-inactivated
[4Fe-4S]ÆFNR is a mixture of apoFNR with one disul-
fide plus three cysteine thiol residues, and FNR with
two disulfides and one cysteine thiol.
The air inactivated and alkylated FNR of the experi-
ment from Fig. 2 was digested with AspN or trypsin
and analyzed by MALDI-TOF for the presence of
alkylated peptides. In AspN digests, peptides Pep1 ⁄ 21
and Pep22 ⁄ 39 with 2555 and 2062 Da were found
which are characteristic of the presence of disulfides
Cys16 ⁄ Cys20 and Cys23 ⁄ Cys29, respectively (not
shown). In the tryptic digest, peptides with masses
indicative for disulfides were found (Fig. 5), suggesting
that up to two disulfides are formed by air-inactivation
of FNR in vitro. This again shows that the air-inacti-

vated FNR is a mixture of apoFNR with one and two
disulfides, and the disulfides are the same as in aerobic
apoFNR (Cys16 ⁄ Cys20 and Cys23 ⁄ Cys29).
Discussion
Significance of apoFNR
[4Fe-4S]ÆFNR is converted rapidly to apoFNR after
exposure to air and a lag phase of about 2 min. The
lag phase presumably includes the [4Fe-4S] ⁄ [2Fe-2S]
conversion, which has no direct effect on the redox
state of the cysteinyl residues. The rate of apoFNR
formation is similar to that of [4Fe-4S] ⁄ [2Fe-2S] con-
version, indicating that apoFNR is a significant prod-
uct of FNR inactivation by air.
Fig. 4. Peptides of aerobic apoFNR after carboxymethylation, tryp-
sin and AspN digestion and reversed-phase chromatography. The
sample buffer was changed by gel filtration on a Nap10 column
and then subjected to trypsin digestion. The digest was separated
by gel filtration on a Superdex Peptide column. The fraction contain-
ing peptide 11 ⁄ 48 (containing Cys16, Cys20, Cys23, Cys29) was
digested with AspN and separated by reversed phase chromatogra-
phy. To identify the peptides in the individual chromatographic frac-
tions, each fraction was subjected to MALDI-TOF analysis. The
corresponding peptides in the fractions are indicated.
Table 3. Quantitative evaluation of thiol and disulfide containing pep-
tides of aerobically and anaerobically prepared apoFNR. Aerobically
or anaerobically prepared apoFNR were incubated with GnHCl +
iodoacetate and digested with trypsin, and after separation on Sepha-
dex Peptide column, by AspN. The desalted peptides were separated
by reversed phase HPLC chromatography (AquaPore RP300) (Fig. 4).
Relevant peak fractions were subjected to MALDI-TOF to identify

the peptides and the degree of alkylation. The relative contents of
the disulfide and the dithiol forms of one peptide from one chromato-
gram was evaluated from the absorption of the corresponding
peaks and the peak areas from three independent experiments and
samples. The data are the mean of three independent experiments
(max deviation 10%).
Peptide
Aerobic apoFNR Anaerobic apoFNR
Pep-SS Pep-CM
2
Pep-SS Pep-CM
2
Pep11 ⁄ 21 (Cys16, Cys20) 65 35 65 35
Pep22 ⁄ 39 (Cys23, Cys29) 36 64 22 78
Pep102 ⁄ 129 (Cys122) 0 100 0 100
Disulfides of apoFNR S. Achebach et al.
4264 FEBS Journal 272 (2005) 4260–4269 ª 2005 FEBS
[4Fe-4S]ÆFNR shows a complex response to the pres-
ence of air (Fig. 6 gives an overview), and the response
differs in vitro and in vivo. The reactions of the FeS
cluster-containing forms of FNR were studied by
Mo
¨
ssbauer spectroscopy, whereas information on apo-
FNR was obtained via the redox state of the cysteine
residues. [4Fe-4S]ÆFNR is converted to [2Fe-2S]ÆFNR
under oxic conditions within few minutes [13,16,17,26],
and the slower reaction in vivo is due to experimental
conditions [13]. It has been estimated from expression
studies that inactivation of FNR by air (which compri-

ses the [4Fe-4S]ÆFNR ⁄ [2Fe-2S]ÆFNR conversion) takes
place within 4 min 30 s [16], which supports the more
rapid in vitro data. [2Fe-2S]ÆFNR was shown to be
rather stable in vitro in the presence of air, whereas
H
2
O
2
caused a rapid disintegration of the cluster
[15,16; Fig. 6]. In vivo, the [2Fe-2S] cluster disappeared
within 15 min (or less) in the presence of air. As a
result, aerobically grown E. coli contains no [2Fe-
2S]ÆFNR or ([4Fe-4S]ÆFNR) [16]. Aerobically grown
E. coli contains FNR protein in amounts which are
similar to, or even higher than, anaerobically grown
bacteria [15,27]. It is suggested that the FNR protein
present under these conditions consists mainly of apo-
FNR. In aerobically grown E. coli,O
2
tension appears
to be similar to that in the external growth medium
[7,9,10]. Therefore, with respect to O
2
supply and
effective O
2
tension, the in vivo conditions are compar-
able to the in vitro conditions used here for FNR
inactivation.
Fig. 5. Redox state of Cys residues in

Pep78 ⁄ 135 (A) and Pep11 ⁄ 48 (B) of recon-
stituted [4Fe-4S]ÆFNR after air-inactivation
trypsin digestion. Reconstituted and subse-
quently air-inactivated [4Fe-4S]ÆFNR was
denatured in 4
M GnHCl and alkylated with
iodoacetate. After solid phase extraction the
protein was digested by trypsin, separated
by gel filtration, and analyzed by MALDI-
TOF MS. (A) Pep78 ⁄ 135, containing
Cys122, with one CM-group (predicted
mass 6244 Da) (B) Pep11 ⁄ 48 in the unmodi-
fied form (predicted mass 4259 Da) contain-
ing Cys16 ⁄ Cys20 and Cys23 ⁄ Cys29 as the
disulfides. Peptide of mass 4411.9 corres-
ponds to Pep10 ⁄ 48 which contains Arg10
in addition (predicted mass 4416) which
is obtained with low yield under some
conditions.
Fig. 6. Reaction steps and time required for conversion of
[4Fe-4S]ÆFNR to apoFNR. The scheme shows species of FNR
involved in the conversion to apoFNR, species not directly meas-
ured for the respective reaction are given in broken lines. The
quoted times are either t
1 ⁄ 2
values or the times required for con-
version between both forms. The corresponding references are
given in brackets.
S. Achebach et al. Disulfides of apoFNR
FEBS Journal 272 (2005) 4260–4269 ª 2005 FEBS 4265

ApoFNR is a product of the reaction of
([4Fe-4S]ÆFNR) with air
The in vitro rate for the conversion of [4Fe-4S]ÆFNR
to apoFNR (via [2Fe-2S]ÆFNR) is either similar to the
rates shown for the cluster conversions or more rapid
(Fig. 6). ApoFNR formation is the counterpart to the
disappearance of [2Fe-2S]ÆFNR in the bacteria [16].
The presence of significant amounts of apoFNR in the
bacteria has to be demonstrated, but preliminary
results indicate that aerobically grown E. coli contain
significant amounts of apoFNR (S. Achebach and
G. Unden, unpublished results). ApoFNR containing
high levels of disulfides can be used successfully for
reconstitution of [4Fe-4S]ÆFNR in vitro [18], further
supporting the physiological significance of apoFNR
in bacteria.
Formation of apoFNR represents a second step of
FNR inactivation and transfers FNR to a functional
state which is less sensitive to reactivation by short-
term or partial anoxic conditions than [2Fe-2S]ÆFNR.
Desensitizing of FNR would be significant for E. coli
growing on a long-term basis under oxic conditions to
avoid production of anaerobic metabolic systems in
response to short-term air depletion.
Different forms of apoFNR
The forms of apoFNR obtained during aerobic or
anaerobic preparation and by in vitro inactivation by
air differed by the redox state or contents of
Cys16 ⁄ Cys20 and Cys23 ⁄ Cys29 disulfides (Fig. 7). The
cysteinyl residues were not oxidized by air to sulfenic

or sulfonic acids, as peptides with the corresponding
mass increases were not detected. Formation of sulfen-
ic and sulfonic acid to a larger extent is also unlikely
as the protein can be reconstituted to active FNR
using sulfhydryl reducing agents like dithiothreitol.
The cysteinyl residues play a central role in the func-
tion of FNR, and their redox state was tested by
chemical modification and MALDI-TOF mass spec-
troscopy which gave consistent results. The cysteine
disulfides could be used as indicators for apoFNR
formation, for which other good biochemical markers
are not available. In the presence of reducing agents
such as dithiothreitol, or glutathione as in the cyto-
plasm of E. coli, the cysteine disulfides are expected to
exist in the thiol state.
Knowledge of the type of cysteinyl disulfides in
FNR provides important information about the bio-
chemistry and properties of the protein, the function
of which relies mostly on the cysteine residues and the
FeS clusters bound by the cysteine residues. Cysteinyl
disulfides were formed only between distinct pairs of
residues (Cys16 ⁄ 20 and Cys23 ⁄ 29), whereas Cys122
was never part of a disulfide (Fig. 7). This could be
related to a specific role of Cys122 which is also
essential for function and cluster assembly [28].
FNR-SS-(SH)
3
, which contains one disulfide, was a
mixture of proteins with either the Cys16 ⁄ Cys20 or the
Cys23 ⁄ Cys29 disulfide. The Cys16 ⁄ Cys20 disulfide is

formed preferentially, but there is no clear order in the
formation of the two disulfides. Formation of the pairs
might reflect suitable spatial vicinity, lacking accessibil-
ity of Cys122, or functional differences of the cysteinyl
residues. Aerobic apoFNR with high disulfide content
[presumably due to the presence of FNR-(SS)
2
-SH]
was shown earlier [18] to have an extended lag phase
Fig. 7. Schematic presentation of FNR and
of cysteinyl thiol and disulfide residues in
different forms of FNR. The scheme shows
the cysteine thiols and disulfides in different
forms of FNR, and their approximate contri-
bution to different functional forms of FNR
(active [4Fe-4S]ÆFNR, air-inactivated FNR,
aerobic and anaerobic apoFNR). The cystei-
nyl residues (Cys16, Cys20, Cys23, Cys29,
Cys122) are presented by their thiol (–SH) or
disulfide (–SS–) residues; FNR-SS-(SH)
3
, e.g.
stands for apoFNR with one disulfide and
three cysteine thiol residues. –, not pre-
sent; +, present; ++, major component.
Disulfides of apoFNR S. Achebach et al.
4266 FEBS Journal 272 (2005) 4260–4269 ª 2005 FEBS
in the reconstitution of [4Fe-4S]ÆFNR from apoFNR
in vitro. The increased lag phase could be overcome
by the addition of dithiothreitol and protein disulfide

reductases.
Experimental procedures
Isolation and reconstitution of FNR
FNR was produced and isolated as a GST¢-¢FNR fusion
protein from E. coli CAG627pMW68 [24]. GST¢-¢FNR
bound to glutathione-Sepharose 4B (1.5 mL bed volume)
was digested for 2 h at 20 °C with 20 U thrombin in 1 mL
buffer C (50 mm Tris ⁄ HCl, pH 7.6) and then eluted from
the column in 3 mL buffer C (without addition of glutathi-
one). For the isolation of anaerobic apoFNR, anoxic buff-
ers prepared and maintained in an anaerobic chamber were
used and the whole procedure starting with incubation of
the bacteria was performed under anoxic conditions [24].
Aerobic apoFNR was prepared in the same way, but all
buffers were air saturated and all steps were performed
under air. FNR obtained from GST-FNR in this way con-
tains a Gly-Ser extension in front of the N-terminal Met-
Ile-Pro of wild-type FNR. Azotobacter vinelandii (NifS
AV
)
was isolated from E. coli BL21(DE3) containing a nifS
AV
expression plasmid for NifS
AV
production [24,29].
[4Fe-4S]ÆFNR was reconstituted under anoxic conditions
[3,24] in a mixture containing isolated apoFNR, 0.3 mm
Fe(II), 2 mm cysteine and NifS
AV
(1 lg ⁄ 20 lg FNR). The

number of thiol groups was determined with 5,5¢-dithiobis-
nitrobenzoate (DTNB) [30] or by the incorporation of
[
14
C]iodoacetate.
Carboxymethylation of FNR
For alkylation of anaerobic apoFNR, 250–350 lg FNR
were anoxically incubated for 30 min with 4 m guanidinium
hydrochloride (GnHCl) and 10 mm iodoacetate, buffer
(25 mm Tris ⁄ HCl, pH 7.6), at room temperature in the
dark. Prior to MALDI-TOF MS, low molecular mass con-
taminations were removed by solid phase extraction. Alky-
lation of aerobic apoFNR was performed in the same way,
but all buffers were air saturated and the experiments were
done under air.
AspN digest
Carboxymethylated protein was desalted by gel filtration
using Nap10 columns (Amersham Pharmacia Biotech, Pis-
cataway, NJ, USA) equilibrated with 1 m GnHCl in 50 mm
ammonium acetate, pH 8.0. The sample was added and frac-
tions of 500 lL each were collected. Fractions containing
the protein were pooled and digested with 1% (w ⁄ w) endo-
proteinase AspN (sequencing grade, Roche, Indianapolis,
IN, USA) for 5 h at 37 °C which cleaves at the N-terminal
end of aspartyl residues. The samples were desalted by
solid phase extraction and subsequently dried by vacuum
centrifugation.
Trypsin digest
The carboxymethylated protein was desalted by gel filtra-
tion on a Nap10 column equilibrated with 10% (v ⁄ v) aceto-

nitrile in 50 mm ammonium acetate, pH 8.6. The eluted
protein was combined and digested with 7–9% (w ⁄ w) modi-
fied trypsin (sequencing grade, Roche) for 8 h at 37 °C
which cleaves at the C-terminal end of arginyl or lysyl resi-
dues. After digest, the peptides were dried by vacuum cen-
trifugation and stored at )20 °C.
Time dependent cysteinyl accessibility during
air-inactivation of [4Fe-4S]ÆFNR
Aerobically prepared apoFNR was reconstituted under
anoxic conditions as described above. Reconstitution was
followed spectroscopically at 420 nm using the absorbance
at 280 nm as reference. After complete reconstitution,
5mm EDTA was added. After 30–50 min the sample
(0.9 mL) was placed into a Petri dish which was transferred
to an atmosphere of air with gentle shaking. Samples
(50 lL) were withdrawn prior and after 2 min, 5 min,
10 min and 20 min of aeration, mixed immediately with
50 lLof8m GnHCl (final concentration 4 m) and
[
14
C]iodoacetate (final concentration 12 mm, specific radio-
activity 43 BqÆmmol
)1
, etc.). After 10 h the protein was pre-
cipitated and washed carefully with methanol–chloroform
[31]. The protein concentration was determined using the
Bradford assay [32] and the radioactivity was measured in
a scintillation counter. Radioactivity was corrected for not
incorporated radioactivity by measuring and subtracting
the radioactivity in identically treated samples lacking apo-

FNR.
Solid phase extraction
Protein or peptide samples were applied to C
18
-Sep Vac 1cc
50 mg cartridges (Waters, Milford, MA, USA) equilibrated
with 0.1% trifluoroacetic acid (TFA). The columns were
washed with 0.1% TFA and proteins or peptides were eluted
with 60% (v ⁄ v) acetonitrile in 0.1% (v ⁄ v) TFA.
HPLC separation and quantification of the
peptides
The carboxymethylated protein was subjected to solid
phase extraction, eluted, dried by vacuum centrifugation
and dissolved in 0.1% (v ⁄ v) TFA and 4 m GnHCl. The
samples were desalted by gel filtration on NAP 10 columns
S. Achebach et al. Disulfides of apoFNR
FEBS Journal 272 (2005) 4260–4269 ª 2005 FEBS 4267
equilibrated with 10% (v ⁄ v) acetonitrile in 50 mm ammo-
nium acetate, pH 8.6. The protein was then digested with
5% (w ⁄ w) modified trypsin (sequencing grade, Roche) for
11 h at 37 °C. The trypsin digest was subjected to HPLC
separation over a gel filtration column (Superdex
TM
Peptide
10 ⁄ 30) operated in 0.1% (v ⁄ v) TFA in 10% (v ⁄ v) acetonit-
rile in order to separate the large (4 and 6 kDa) cysteine-
containing from the bulk peptides (< 3 kDa). Fractions
containing these peptides were identified by mass spectro-
metry, pooled and dried. The peptides were re-dissolved in
1 m GnHCl in 20 mm Tris ⁄ HCl, pH 8, containing 2 lgof

endoproteinase AspN. The samples were digested for 9 h at
37 °C. The resulting peptides were separated by reversed
phase HPLC on an Aquapore RP 300 C
8
column
(2.1 · 100 mm) and monitored at 215 nm. Individual pep-
tides were collected manually and analyzed by MALDI-
TOF MS.
MALDI-TOF MS
Mass spectra of proteins and peptides were collected using
a Perkin-Elmer Voyager-RP ⁄ DE mass spectrometer (Per-
septive Biosystems, Wiesbaden, Germany). In order to
avoid an interfering binding of salts to FNR, the protein
was carefully desalted by solid-phase extraction prior to
MALDI-TOF MS. Samples thus obtained were either
dissolved in 67% (v ⁄ v) acetonitrile in 0.1% (v ⁄ v) TFA or
matrix solutions yielding final concentrations of 1–10 pmolÆ
lL
)1
protein or peptides. Equal volumes of samples and
saturated solutions of either sinnapinic acid (proteins) or
a-cyano-4-hydroxycinnamic acid in 0.1% (v ⁄ v) TFA, 67%
(v ⁄ v) acetonitrile were mixed on the sample slide and dried
in a stream of air. Proteins were measured in the positive
linear mode of the instrument at an acceleration voltage of
25 kV using a grid voltage of 89% and a delay time of
300 ns. Peptide spectra were collected in the positive reflec-
tor mode at an acceleration voltage of 20 kV using a grid
voltage of 58% and a delay time of 100 ns.
Acknowledgements

The authors thank R. Thauer for the access to
MALDI-TOF MS at the Max-Planck-Institut fu
¨
r Ter-
restrische Mikrobiologie, Marburg. The work was sup-
ported by grants of Deutsche Forschungsgemeinschaft
and of the Fonds der Chemischen Industrie.
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