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(Received for publication, September 28,
1995; and in revised form, December 20, 1995) From the
Dimethyl-sulfoxide reductase (DmsABC) is a complex
[Fe-S] molybdoenzyme that contains four [4Fe-4S]
clusters visible by electron paramagnetic resonance (EPR) spectroscopy.
The enzyme contains four ferredoxin-like Cys groups in the electron
transfer subunit, DmsB, and an additional group of Cys residues in the
catalytic subunit, DmsA. Mutagenesis of the second Cys, Cys-38, in the
DmsA group to either Ser or Ala promotes assembly of a fifth
[Fe-S] cluster into the mutant enzyme. The EPR spectra, the
temperature dependences, and the microwave power dependences
demonstrate that the new clusters are [3Fe-4S] clusters. The
[3Fe-4S] clusters in both of the C38S and C38A mutant enzymes
are relatively unstable in redox titrations and have midpoint
potentials of approximately 178 and 140 mV. Mutagenesis of the DmsA Cys
group to resemble a sequence capable of binding an [4Fe-4S]
cluster did not change the cluster type but reduced the amount of the
cluster present in this mutant enzyme. This report demonstrates that
all four EPR detectable [Fe-S] clusters in the wild-type
enzyme are ligated by DmsB. Wild-type DmsA does not ligate an
[Fe-S] cluster that is visible by EPR spectroscopy.
Escherichia coli grows anaerobically using
Me DmsABC is a member of a family of
molybdenum-containing oxidoreductases with highly conserved
sequences(1, 6, 7, 8) . These are
enzymes that reduce Me Ferredoxins that
contain [4Fe-4S] clusters usually ligate these clusters by
Cys groups consisting of four Cys residues spaced such that the first
two Cys residues are separated by two amino acids, while the spacing
between the second, third, and fourth Cys residues is somewhat
variable. A Cys group from the thermophilic methanogen Methanococcus thermolithotrophicus(25) has four amino
acids separating the first two ligands, but we have not identified a
Cys group with three intervening residues. The first three Cys residues
and one distal Cys, often from a second Cys group elsewhere in the
protein, provide the ligands to the cluster(26, 27) .
Alignment of the amino-terminal regions of the large subunits of the
molybdoenzymes (Fig. 1) shows four conserved Cys residues
arranged in a manner reminiscent of a [4Fe-4S] ferredoxin Cys
group. The sequences can be divided into three types. Type I enzymes
contain three Cys residues spaced similar to a bacterial ferredoxin Cys
group and one other conserved Cys, which could provide the fourth
ligand. The Type II enzymes also have four Cys residues, but the
spacing is such that three amino acids instead of two separate the
first and second Cys residues. DmsABC belongs to this group, as do the
two membrane-bound E. coli nitrate reductases in which the
first Cys is replaced by a His. His can be a ligand to a
[4Fe-4S] cluster, as in the nickel-iron hydrogenase from Desulfovibrio gigas, but the first two ligands of this
cluster, His and Cys, are separated by two amino acids (28) .
The Type III enzymes include biotin sulfoxide reductase (BisC) and TMAO
reductase (TorA) which share sequence identity with the other
molybdoenzymes but do not contain the Cys region.
Figure 1:
Sequence alignment showing the three
types of amino-terminal Cys regions present in this family of bacterial
molybdoenzymes. The enzymes are Synechococcus NarB (11) , Klebsiella pneumoniae NasA(12) , E.
coli FdhF(24) , Methanobacterium formicicum FdhA(21) , Wolinella succinogenes FdhA(19) , E. coli FdnG (18) , E.
coli FdoG(20) , Alcaligenes eutrophus NapA(13) , E. coli DmsA(6) , E. coli NarG(7) , E. coli NarZ(10) , T.
pantotropha NapA(14) , W. succinogenes PsrA(17) , E. coli TorA(9) , Rhodobacter sphaeroides BisC(16) , and E. coli BisC(15) . Conserved Cys residues are shown in boldface, and the DmsA residues that were examined in this
study are underlined.
The periplasmic
nitrate reductase, NapAB, from Thiosphaera pantotropha has
been shown to contain a [4Fe-4S] cluster(29) . This
is a Type I enzyme, and the Cys residues in NapA are the only
candidates to ligate the [4Fe-4S]
cluster(14, 29) . This raises the possibility that the
Cys region may ligate a [4Fe-4S] cluster in other members of
this family. The subunit of E. coli formate hydrogenlyase that
contains the formate dehydrogenase activity, FdhF, may contain an
[Fe-S] cluster based on iron analysis(30) . The
[Fe-S] clusters of DmsABC have been characterized by electron
paramagnetic resonance (EPR) spectroscopy. The enzyme contains four
[4Fe-4S] clusters with midpoint potentials, E The DmsA Cys region has previously
been examined through the use of site-directed
mutagenesis(32) . Cys-38, Cys-42, and Cys-75 were mutated to
Ser, and only the C75S mutant enzyme was able to support growth on
Me
Figure 2:
EPR spectra of reduced F36 membranes. a, F36/pBR322; b, F36/pDMS160; c, F36/pC38A;
and d, F36/pC38S. Samples were incubated at 25 °C under
argon for 2 min. 5 mM dithionite was added, and the samples
were incubated under argon an additional 10 min before freezing in
liquid nitrogen. Spectra were recorded under the following conditions:
temperature, 12 K; microwave power, 20 milliwatts; microwave frequency,
9.45 GHz; modulation amplitude, 10 G
Fig. 2, c and d, shows
the spectra of dithionite-reduced F36 membranes containing the C38A and
C38S mutant enzymes, respectively. The features of these spectra are
very similar to that of F36/pDMS160 membranes, and all of the DmsABC
features are present. The slight difference in the size of some of the
features in the reduced EPR spectrum of the C38A mutant enzyme is due
to the lower amount of enzyme present. Reduction by dithionite at pH 9
did not highlight any difference between the spectra of the wild-type
and mutant enzymes (data not shown).
Figure 3:
EPR
spectra of oxidized F36 membranes. Samples were prepared from membranes
of F36/pBR322 (a), F36/pDMS160 (b), F36/pC38A (c), and F36/pC38S (d). Ferricyanide (150
µM) was added to membrane samples, and the samples were
incubated for 2 min prior to freezing in liquid nitrogen. Instrument
parameters and protein concentrations were as described for Fig. 2.
To identify the nature of the paramagnetic species present
in the oxidized Cys-38 mutants, we studied the microwave power
saturation properties and the temperature dependences of the new
signals. Fig. 4shows the effect of increasing microwave power
on the mutant center signals at 12 K. Microwave power saturation data
obtained from F36/pC38A membranes were fitted to an empirical equation
to obtain the microwave power required for half saturation of the
signal, the P
Figure 4:
Microwave power dependences of the signals
present in oxidized membranes of the DmsA mutants. The saturation
behavior of the g = 2.03 signal was plotted and fitted
to the empirical equation of Rupp et al.(42) to
derive P
Figure 5:
Temperature dependences of the oxidized
signals present in the DmsA mutants. The percent peak height of the g = 2.03 signal was measured from spectra of membranes
of F36/pC38A (
Figure 6:
Redox titration curves showing the change
in signal amplitude of the g = 2.03 signal of F36/pC38A (a) and F36/pC38S membranes (b). Spectra were
recorded under the conditions outlined in Fig. 2, and (
Fig. 7shows spectra of the
ferricyanide-oxidized membranes from the DmsA mutants. The double
mutant ligated a [3Fe-4S] cluster, but the amount of cluster
was reduced to approximately 25% of the amount of cluster ligated by
C38S, estimated by double integration. The line shape is similar to
C38S, but the signal is broader. Spectra of the mutants in whole cells
are identical to that of the membrane preparations, indicating that the
clusters in all three mutant enzymes are [3Fe-4S] clusters in vivo and are not [4Fe-4S] clusters altered upon
oxidation during cell breakage (data not shown). The signal intensity
of the double mutant appeared larger in whole cells than in the
membrane samples. Redox titrations of the double mutant identify four
[4Fe-4S] and one [3Fe-4S] cluster (Table 2)
with the ratio of reduced to oxidized [Fe-S] clusters being
approximately 13:1. The high ratio is likely due to the reduced amount
of [3Fe-4S] cluster present in these membrane preparations.
Figure 7:
EPR spectra of oxidized membranes of the
DmsA mutants. Samples of F36/pC38A (a), F36/pC38S (b), and F36/pC38S,N37C (c) were oxidized with
ferricyanide as described in Fig. 3. Instrument parameters were
as described for Fig. 2.
Wild-type DmsABC has a complex EPR spectrum (Fig. 2b) that has been analyzed as two pairs of
interacting [4Fe-4S] clusters (1, 4) . Our
research has been aimed at identifying which residues ligate the
[Fe-S] clusters in DmsABC through the use of site-directed
mutagenesis and EPR. Cys groups III (31) and I A major new signal is observed in spectra of the oxidized DmsA
mutants with a peak at g = 2.03 and a peak/trough at g = 2.00 (Fig. 3). The line shapes of the new
signals are distinct from the spectrum of fumarate reductase center
FR3(40, 41) . The temperature and power dependences
shown in Fig. 4and Fig. 5are similar to those of the
artificial [3Fe-4S] clusters formed by site-directed
mutagenesis of Cys groups(31, 47) . The multiple
components, hysteresis, and fragility displayed by the DmsA mutant
clusters demonstrate cluster instability in this environment. It is
unlikely that the [3Fe-4S] signal is due to oxidative damage
of the DmsB clusters, as the levels of the [4Fe-4S] clusters
are the same in the wild-type and mutant enzymes, and the C42S and C75S
mutant enzymes do not show this new [3Fe-4S] cluster signal. The possibility exists that the [3Fe-4S] cluster in the
DmsA mutants could be generated from a [4Fe-4S] cluster
present in the wild-type enzyme. Conversion of [Fe-S] cluster
types through site-directed mutagenesis has been demonstrated
previously. In DmsB and Synechocystis photosystem I (clusters
F It appears that
the [3Fe-4S] cluster in DmsA mutants is not formed via
cluster conversion but that mutagenesis of Cys-38 has altered the
protein environment such that a cluster can assemble. This is supported
by the lack of evidence to suggest that a fifth [4Fe-4S]
cluster exists in wild-type DmsA. Reduction of the enzyme by dithionite
gives only four clusters, and increasing the reduction potential of
dithionite by increasing the pH did not reduce any additional clusters.
Photoreduction with proflavin and EDTA did not reduce any additional
centers (data not shown). No changes in the reduced EPR spectrum to
indicate loss of a [4Fe-4S] cluster were visible in the
Cys-38 mutant enzymes. Double integrations of the reduced and oxidized
spectra of DmsA [3Fe-4S]-containing mutants gave ratios of
four to one, indicating that these mutants gained an [Fe-S]
cluster compared with the three to one ratio from DmsB C102 mutants,
which altered one of the existing [Fe-S]
clusters(31) . Extensive EPR characterization of NarGHI (also a
Type II enzyme) has identified four [Fe-S] clusters ligated
by four Cys groups in the electron transfer subunit (NarH), but no
fifth cluster was ligated by
NarG(50, 51, 52) . The spacing of the Cys
residues in DmsA was altered so the first and second Cys residues were
separated by only two amino acids, but the double mutant still ligated
a [3Fe-4S] cluster with a line shape similar to that of the
C38S cluster. The amount of this cluster was reduced, and multiple
components were present in the analysis. [3Fe-4S] clusters
can be generated from [4Fe-4S] clusters that are damaged by
oxidation, but EPR analysis of whole cells expressing the double mutant
demonstrated that the enzyme ligated a [3Fe-4S] cluster in vivo. The natural function of the DmsA Cys group is
unknown, but it could be involved in binding some factor, perhaps a
metal ion, the loss of which may destroy function in the Cys-38 and
Cys-42 mutant enzymes. The role of Cys-38 in DmsA is unique.
Substitution of Ser or Ala for Cys should cause little perturbation of
the protein structure, indicating that the sulfhydryl of the Cys-38 is
important. In the Type I enzymes, there is an abundance of conserved
Gly residues in this region that are not conserved in the Type II
enzymes (Fig. 1). A cluster may be sterically hindered from
assembling in this region until mutation of Cys-38 disrupts normal
function and frees the Cys group to ligate an [3Fe-4S]
cluster. Another possible reason for the loss of function in C38S is
the presence of the [3Fe-4S] cluster. The C38S enzyme has a
block in the electron pathway between the [4Fe-4S] clusters
and Mo-MGD, where the substrate is reduced(32) . The high E We have divided the enzymes into three classes. The Type I enzymes
such as NapAB, FdhF, and perhaps the other members of this type are
likely to have a [4Fe-4S] cluster located in their amino
terminus. The Type II enzymes, DmsABC, and the two E. coli nitrate reductases have a Cys region, but they are not likely to
ligate an [Fe-S]. The Type III enzymes lack this region
altogether, and neither TorA or BisC have been suggested to contain
[Fe-S] clusters. This region in DmsA is likely a degenerate
Cys group that has lost [Fe-S] binding capability upon
evolution of the enzyme, although in DmsA the Cys group retains an
essential role in electron transfer, perhaps interacting with the
Mo-MGD.
Volume 271,
Number 9,
Issue of March 1, 1996 pp. 4620-4626
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
SO as respiratory oxidant by expressing an electron
transfer chain terminating with MeSO reductase,
DmsABC(
)(1) . DmsABC is a complex [Fe-S]-
and molybdenum-containing enzyme located on the cytoplasmic surface of
the inner membrane(2) . DmsA is the largest subunit (87.4 kDa)
and binds a molybdenum-molybdopterin guanine dinucleotide cofactor,
Mo-MGD (3) . DmsB (23.1 kDa) is an electron-transfer subunit
containing four ferredoxin-like Cys groups proposed to ligate
[4Fe-4S] clusters(1, 4) . DmsC (30.8 kDa) is
a membrane-intrinsic subunit that anchors DmsAB to the membrane. DmsC
accepts electrons from menaquinol, transferring them through the
[4Fe-4S] clusters in DmsB to the active site in DmsA (1) . The dmsABC operon has been cloned and sequenced,
and the enzyme can be expressed to high levels in
membranes(5, 6) .SO, trimethylamine N-oxide
(TMAO)(9) ,
nitrate(7, 10, 11, 12, 13, 14) ,
biotin sulfoxide(15, 16) , and polysulfide (17) or enzymes that oxidize
formate(18, 19, 20, 21, 22, 23, 24) .
Each enzyme contains a large catalytic subunit with a noncovalently
bound molybdenum cofactor. The sequence identity is located in segments
throughout the polypeptide. Many of these enzymes are similar to DmsABC
in prosthetic groups and subunit composition.
= -50,
-120, -240, and -330 mV (4) . These clusters
are believed to be ligated by the four ferredoxin-like Cys groups
(I-IV) in DmsB(1, 4) , although the possibility exists
that the Cys region in DmsA might be able to ligate a cluster.
Site-directed mutagenesis of DmsB groups III (31) and I (
)has demonstrated that these Cys groups provide ligands for
two [4Fe-4S] clusters.SO. All three mutant enzymes retained some level of
catalytic activity with an artificial electron donor, reduced benzyl
viologen (BV) and with the quinol
analog, 2,3-dimethyl-1,4-naphthoquinol. The C38S and C42S mutants were
blocked in using electrons from the quinol pool to reduce substrate,
although the [4Fe-4S] clusters responded to the redox state
of the quinol pool. In this manuscript we show that the DmsA Cys group
does not coordinate an EPR visible [Fe-S] cluster, but when
the second Cys of this group is mutated, a [3Fe-4S] cluster
assembles into the mutant enzyme.
Bacterial Strains and Plasmids
E. coli strain F36 is a mutant of E. coli HB101(supE44 hsdS20
(rm
)
recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1), which is
impaired in molybdenum cofactor insertion into DmsABC(4) . E. coli TG1 (supE hsd
5 thi (lac-proAB)
F`[traD36 proABlacI
lacZ
M15]) was used for routine DNA manipulation
(Amersham Corp.) and is the parent strain of DSS301, which carries a
deletion of the entire dmsABC operon(33) . The
plasmids used are the vector pBR322 (Pharmacia Biotech Inc.), pDMS160,
which contains the dmsABC operon cloned into
pBR322(31) , and pC38S and pC38A, which are derivatives of
pDMS160-containing point mutations in the dmsA gene(32) . pTZ18R (Pharmacia) was used as a cloning vector
and to sequence fragments containing mutant DNA.Materials
Synthesis of oligonucleotides and DNA
sequencing were carried out in the Department of Biochemistry DNA Core
facility at the University of Alberta using an Applied Biosystems model
392 DNA synthesizer and a model 373A DNA sequencer (Perkin Elmer).
Restriction endonucleases and modifying enzymes were obtained from Life
Technologies Inc. All other materials were of reagent grade and were
obtained from commercial sources.Site-directed Mutagenesis
Manipulations of strains
and plasmids were carried out as described in Sambrook et al. (34) . The double mutant of dmsA with substitutions in
residues Asn-37 and Cys-38 was generated through PCR mutagenesis of
pC38S using the following mutagenic primers: 5`-GTACAGTTTGCTCTGGTAG-3`
and 5`-CGACTACCAGAGCAAACTG-3`(35) . The mutant product was
cloned into pTZ18R and sequenced. The fragment bearing the double
mutation was subcloned into the wild-type operon to construct the
plasmid pC38S,N37C.Growth of Bacteria
Cultures were grown
anaerobically at 37 °C. F36 (19 liters) and HB101 (1 liter) were
grown on glycerol-fumarate medium for 48 h(36) . F36 for whole
cell preparations (1 liter) were grown on glucose-peptone-fumarate
medium for 16 h(37) . DSS301 was grown on
glycerol-MeSO media(36) . Antibiotics were used at
the following concentrations: ampicillin and streptomycin, 100 µg
ml; kanamycin, 40 µg ml
.
Harvesting of Cells and Preparation of Membrane
Fractions
For whole cell EPR samples, cells were harvested,
washed, and resuspended in degassed 100 mM MOPS, pH 7.0, 5
mM EDTA buffer containing 20 mM succinate(31) . Glycerol-fumarate grown cells were
harvested, washed, and resuspended in 50 mM, MOPS, pH 7.0, 5
mM EDTA. Phenylmethanesulfonyl fluoride (0.2 mM) was
added, and cells were subjected to French pressure lysis and
differential centrifugation to prepare crude membrane
fractions(4) . For EPR experiments, the membranes were washed
and resuspended in 100 mM MOPS, pH 7.0, 5 mM EDTA.
Membranes were stored at -70 °C prior to use.Protein Determination and Polyacrylamide Gel
Electrophoresis
Protein concentrations were estimated by a
modification of the Lowry procedure (38) using a Bio-Rad bovine
serum albumin protein standard. Polyacrylamide gel electrophoresis was
carried out using the Bio-Rad mini-gel system and a discontinuous SDS
buffer system(39) . Gels (12.5%) were stained with Coomassie
Blue and destained, and the relative amount of DmsA protein was
determined using a Joyce-Loebel Chromoscan 3 densitometer.Enzyme Assays
DmsABC activity in crude HB101
membrane preparations was determined by monitoring the TMAO-dependent
oxidation of BV(4) .
EPR Spectroscopy
Samples were prepared as
described by Cammack and Weiner (4) from either whole cells or
washed membranes. Redox titrations were performed using the following
mediators: quinhydrone, 2,6-dichloroindophenol, 1,2-naphthoquinone,
toluylene blue, phenazine methosulfate, thionine, duroquinone,
methylene blue, and resorufin. Spectra were recorded using a Bruker
ESP300 EPR spectrometer equipped with an Oxford Instruments ESR-900
flowing helium cryostat. Instrument conditions and temperatures are
described in the individual figure legends. Spin quantitations were
carried out by double integrations of spectra from redox titrations
(microwave power, 2 milliwatts; temperature, 12 K). The presence of
background signals (primarily fumarate reductase) in the membranes was
partly compensated for by subtracting the base line intensity (at
approximately 0 mV) from the fully oxidized and reduced intensities.
Growth Properties and Enzyme Activities of the Cys-38
Mutants
The ability of the mutant enzymes to support growth on
MeSO was assessed using the dmsABC deletion
strain, DSS301 (Table 1). Only the wild-type enzyme was able to
support growth. DSS301 was not used for further characterization of the
mutant enzymes for reasons previously mentioned (31, 32) . The specific activities of HB101 membrane
preparations were assayed using TMAO as the electron acceptor and the
artificial electron donor, BV (Table 1). Both mutant enzymes showed decreased specific
activities when compared with the wild-type enzyme, as shown
previously(32) . The relative amounts of enzyme present in the
F36 membrane preparations used for the EPR studies were determined by
densitometry (Table 1). The relative percentage of DmsA in the
F36/pC38A membranes is lower than in the wild-type or C38S
preparations.
EPR Characteristics of Dithionite-reduced F36 Membranes
Containing Overexpressed Wild-type and Mutant DmsABC
In F36 the
Mo-MGD cofactor is not inserted into DmsABC and cannot interfere with
the [Fe-S] signals in the redox titrations as occurs when
DmsABC is expressed in HB101(4) . Although the enzyme produced
in F36 is inactive, it assembles the [Fe-S] clusters
normally. Fig. 2shows the EPR spectra of reduced membranes at
12 K. F36/pBR322 membranes contained very little DmsABC, expressed from
the chromosomal copy of the operon. The spectrum from these membranes
shows a peak at g = 2.02 and a peak-trough at about g = 1.94. These features are characteristic of the
reduced [2Fe-2S] cluster of fumarate reductase,
FR1(40, 41) . F36/pDMS160 membranes contained a high
level of DmsABC, and the spectrum of the dithionite-reduced samples (Fig. 2b) is very similar to spectra obtained from both
purified and membrane-bound DmsABC(4, 31) . The
spectrum contains peaks located at g = 2.06, g = 2.02, g = 1.99, and g =
1.95. Two troughs are observed at g = 1.92 and g = 1.87. The peak at g = 2.02 is most likely
due to fumarate reductase, which would also contribute to the
peak/trough at g = 1.95 to g =
1.92(31) . The peaks at g = 2.06 and g = 1.99 and the peak/trough at g = 1.95 to g = 1.92 arise from DmsABC. Repeating the dithionite
reduction at pH 9, to decrease the redox potential, did not change the
spectrum observed.
at 100
KHz.
EPR Characteristics of Oxidized F36 Membranes Containing
Amplified Wild-type and Mutant DmsABC
Fig. 3shows EPR
spectra recorded at 12 K of F36 membranes oxidized with ferricyanide.
The spectrum of F36/pBR322 membranes (Fig. 3a) has a
small peak at g = 2.02 with a broad trough immediately
upfield, characteristic of the oxidized [3Fe-4S] cluster of
fumarate reductase, FR3(40, 41) . The spectrum of
F36/pDMS160 membranes (Fig. 3b) shows similar features
to that of F36/pBR322 membranes, indicating that FR3 is the major EPR
visible species present. Spectra from the mutant enzymes show major new
features. The spectrum of F36/pC38S membranes (Fig. 3d)
is comprised of a sharp peak at g = 2.03 and a
peak/trough centered at g = 2.00. The F36/pC38A
spectrum (Fig. 3c) is similar, but the signal is
broader than in F36/pC38S membranes. The EPR spectra of the oxidized
mutant enzymes can be attributed to centers having axial symmetry with
approximate g values of g
= 2.03
and g = 2.00. Oxidized spectra of HB101
membranes containing the C42S and C75S mutant enzymes were similar to
that of FR3, indicating that these mutants do not assemble significant
amounts of a [3Fe-4S] cluster ligated in DmsA (data not
shown).
(42) . A two-component
model was required to fit the data giving P
values of 1 (40%) and 185 milliwatts (60%). The presence of two
components suggests that the protein conformation around the cluster is
not homogeneous. Microwave power saturation data from the F36/pC38S
membranes were fitted to one component with a P
of 9 milliwatts. Fig. 5shows the effect of temperature on
the intensity of the new signals. The F36/pC38A and F36/pC38S signals
reached a maximum intensity at 9 K and 11 K that decreases until, at 30
K, they were hardly visible. The microwave power saturation and
temperature dependences of the new centers in the C38A and C38S mutants
are typical of the behavior of [3Fe-4S]
clusters(43, 44) . We therefore assign these signals
to new [3Fe-4S] clusters ligated by the DmsA Cys group in the
mutant enzymes.
values. Data obtained from spectra at
12 K of membranes of F36/pC38A (
) and F36/pC38S (
) were
fitted to P values of 1 and 185 milliwatts for
C38A and 9 milliwatts for C38S.
) and F36/pC38S () recorded at a microwave
power of 2 milliwatts.
Redox Titrations of the C38A and C38S Mutant
Enzymes
Redox titrations were carried out to determine the
midpoint potentials for the [4Fe-4S] clusters and the new
[3Fe-4S] clusters in the DmsA mutant enzymes (Table 2).
In the mutant enzymes, four [4Fe-4S] clusters were detected
with midpoint potentials close to those of the wild-type enzyme. The
amount of each cluster present was very similar in the wild-type and
mutant enzymes. To determine the midpoint potential of the
[3Fe-4S] cluster of C38A, F36/pC38A membranes were oxidized,
reduced, and reoxidized (Fig. 6a). Upon reoxidation of
the membranes, most of the [3Fe-4S] clusters were destroyed.
Data obtained from the titration were fitted to a two-component model
of the Nernst equation with E
= 140 (69%) and 75 mV (31%). The F36/pC38S redox titration
data were fitted to one component, but the cluster routinely exhibited
hysteresis (Fig. 6b). The E
in the oxidizing direction was 190 mV, and the E
in the reducing direction was 165
mV. The ratio of reduced [Fe-S] clusters to that of oxidized
[Fe-S] clusters was determined from double integrations of
reduced and oxidized samples. In the oxidized spectrum of membranes
containing wild-type DmsABC, there is only a small amount of
[3Fe-4S] cluster visible, so the ratio of reduced to oxidized
[Fe-S] clusters is very large. The C38A and C38S mutants
contain approximately four reduced clusters for each oxidized cluster,
giving a total of five clusters.
,
) represent data obtained during the addition of ferricyanide,
while () represents data obtained during the addition of
dithionite. C38A data were fitted to two components with E
values of 75 and 140 mV.
C38S data were fitted in the oxidizing direction to an E
of 190 mV and in the
reducing direction to an E
of 165 mV.
Mutagenesis of the DmsA Cys Group to a Consensus
Ferredoxin Cys Group
Sequences known to ligate
[4Fe-4S] clusters usually contain four Cys
residues(26, 27) . The first and second Cys residues
are separated by two amino acids, an exception being M.
thermolithotrophicus ferredoxin, which has four intervening
residues(25) . We altered the sequence of the DmsA Cys group so
that the first two Cys residues would only be separated by two amino
acids. The plasmid, pC38S, was further mutated to produce pC38S,N37C,
which contains the sequence, CTVCSGSNC. This
spacing of Cys residues occurs in Cys group II of DmsB and other
electron transfer subunits of enzymes belonging to this family (1) and in Azotobacter vinelandii ferredoxin
I(45, 46) . Expression and specific activity of the
double mutant, C38S,N37C, are similar to that of C38S, and this enzyme
is also unable to support growth on MeSO in DSS301 (Table 1).
of
DmsB each ligate a [4Fe-4S] cluster. In this report, the Cys
region of DmsA was mutated so that it is unlikely to bind a
[4Fe-4S] cluster, but the EPR spectra of reduced membranes
containing DmsA mutant enzymes was essentially identical to the
wild-type enzyme (Fig. 2). All four previously characterized
[4Fe-4S] clusters are present in the mutant enzymes in the
correct amounts and with midpoint potentials similar to those of the
wild-type enzyme (Table 2). We conclude that the four EPR visible
[4Fe-4S] clusters are all ligated by the Cys groups of DmsB. 
and F
), [4Fe-4S] clusters were
converted to [3Fe-4S] clusters, although the conversion was
incomplete for F(31, 47, 48) .
The conversion of a [3Fe-4S] to a [4Fe-4S] cluster
was generated in fumarate reductase(49) . of the [3Fe-4S] cluster
in C38S relative to the potentials of the Mo(VI)/(V) and Mo(V)/(IV)
couples (-75 and -90 mV,(4) ) suggests that the
[3Fe-4S] cluster may act as an ``insulating
cluster'' (28, 53) between the [4Fe-4S]
clusters and the Mo-MGD to decrease the rate of electron transfer.
),
reduced benzyl viologen, EPR, electron paramagnetic resonance; Mo-MGD,
molybdenum-molybdopterin guanine dinucleotide cofactor; MOPS,
4-morpholinepropanesulfonic acid; TMAO, trimethylamine N-oxide.
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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