(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
MeSO as respiratory oxidant by expressing an electron
transfer chain terminating with Me
SO 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) .
DmsABC is a member of a family of
molybdenum-containing oxidoreductases with highly conserved
sequences(1, 6, 7, 8) . These are
enzymes that reduce MeSO, 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.
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 = -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.
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
MeSO. 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.
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 at 100
KHz.
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(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.
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 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.
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 () and F36/pC38S (
) recorded at a microwave
power of 2 milliwatts.
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 (,
) 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.
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 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.
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 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) .
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 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.
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.