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INTRODUCTION |
Escherichia coli, when grown anaerobically with
Me2SO as respiratory oxidant, develops a respiratory chain
terminated by a membrane-bound menaquinol:Me2SO
oxidoreductase (Me2SO reductase, DmsABC)1 (1). DmsABC is a
complex [Fe-S]-molybdoenzyme comprising a molybdenum
cofactor-containing catalytic subunit (DmsA, 87.4 kDa) (2, 3), a
[4Fe-4S] cluster-containing electron transfer subunit (DmsB, 23.1 kDa) (2, 4-6), and a menaquinol-oxidizing membrane anchor subunit
(30.8 kDa) (7, 8). DmsA and DmsB comprise a catalytic dimer anchored to
the cytoplasmic membrane by DmsC (9). Cofactor extracts released from
DmsABC have been shown to contain molybdopterin guanine dinucleotide
(MGD) (3). On the basis of sequence comparisons (see below), the
cofactor is almost certainly present in the form of Mo-bisMGD in the
intact holoenzyme. DmsABC is a member of an emerging family of
bacterial complex [Fe-S]-molybdoenzymes that includes the E. coli formate dehydrogenases (FdnGHI and FdoGHI) (10, 11), E. coli nitrate reductases (NarGHI and NarZYV) (12, 13),
Salmonella typhimurium thiosulfate reductase (PhsABC) (14),
and Wolinella succinogenes polysulfide reductase (PsrABC)
(15). Each member of this family has a molybdenum cofactor-containing
catalytic subunit (assumed to be Mo-bisMGD (16, 17)), a four-[Fe-S]
cluster-containing electron transfer subunit, and a hydrophobic
membrane anchor subunit. The various prosthetic groups of these enzymes
catalyze overall reactions that involve the transfer of two electrons
through the electron transfer subunit to or from a quinol-binding site
associated with the membrane anchor subunit (6, 18). The use of EPR spectroscopy to study the interactions between the prosthetic groups in
wild-type and appropriately mutagenized enzymes may yield important
information on the electron transfer pathway through the [Fe-S]
clusters of the electron transfer subunit to the Mo-bisMGD cofactor of
the catalytic subunit.
In DmsABC, two of the [4Fe-4S] clusters of DmsB appear to be
thermodynamically competent to transfer electrons from menaquinol (MQH2, reduced midpoint potential at pH 7 Em,7 =
70 mV) to the Mo-bisMGD of DmsA
(Em,7 values as follows: Mo(IV/V) =
175 mV; Mo(V/VI) =
15 mV (19)), and these have Em,7 values
of
50 and
120 mV, respectively (2, 6). It has been demonstrated
that there is a significant conformational link between the
Em,7 =
50 mV [4Fe-4S] cluster and a single
menaquinol (MQH2) binding site associated with His-65 of
DmsC (6, 20). This was established by using the MQH2 analog
2-n-heptyl-4-hydroxyquinoline-N-oxide to elicit
an EPR line shape change on a genetically engineered [3Fe-4S] cluster
in a DmsB-C102S mutant (4, 6). In this mutant, the
Em,7 =
50 mV cluster is replaced by a high
potential [3Fe-4S] cluster, and therefore a role for the
Em,7 =
50 mV [4Fe-4S] cluster of the wild-type
enzyme can be envisioned in electron transfer from MQH2.
The other cluster that appears to be thermodynamically competent to
participate in catalytic electron transfer is the Em,7 =
120 mV cluster. It has been demonstrated
that there is a strong spin-spin interaction between this cluster and
the Em,7 =
50 mV cluster of the wild-type enzyme,
consistent with both of them being part of an eight-iron (2[4Fe-4S])
ferredoxin motif (2, 6). This pair may provide a conduit for electron
flow through DmsB to the Mo-bisMGD of DmsA. The two remaining low
potential clusters of DmsB (Em,7 =
240 and
330
mV) may play a role in defining the overall structure of this subunit
in a manner similar to that suggested for the low potential [4Fe-4S]
clusters of E. coli nitrate reductase A (21) and fumarate
reductase (22).
Evidence exists to suggest a pathway of electron transfer from DmsB to
the Mo-bisMGD of DmsA. The microwave power saturation properties of the
Mo-bisMGD Mo(V) EPR spectrum are sensitive to the redox state of the
Em,7 =
120 mV [4Fe-4S] cluster of DmsB (2). DmsA
also contains a vestigial [4Fe-4S] cluster binding motif close to its
N terminus, which, when appropriately mutagenized, can be made to bind
an engineered [3Fe-4S] cluster with an Em,7 of
approximately 178 mV (23). This motif has been shown to be involved in
physiological electron transfer from the [4Fe-4S] clusters of DmsB to
the Mo-bisMGD cofactor of DmsA (24). This suggests that the vestigial
[4Fe-4S] cluster binding motif may be on the electron transfer
pathway from the Em,7 =
120 mV cluster of DmsB to
the Mo-bisMGD of DmsA. It would therefore be interesting to determine
if the presence of an engineered DmsA [3Fe-4S] cluster has any effect
on the observed interaction between the Mo-bisMGD and the
Em,7 =
120 mV [4Fe-4S] cluster of DmsB.
The structures of three proteins that have significant sequence
similarity to DmsA have been solved. These are the Me2SO
reductases from Rhodobacter sphaeroides and
Rhodobacter capsulatus (16, 25) and the formate
dehydrogenase H (FdhF) from E. coli (26). In each case, the
molybdenum cofactor is a Mo-bisMGD. Given the sequence similarity
between these proteins and DmsA (35-41% similar, 25-32% identical),
it is almost certain that the latter contains a Mo-bisMGD cofactor as
well. The structure of FdhF is particularly relevant, since it contains
a [4Fe-4S] cluster coordinated by its N-terminal Cys group. This
cluster has been proposed to have an important role in the electron
transfer pathway from formate to the artificial electron acceptor
benzyl viologen (26). Significant differences in the sequence of the
N-terminal Cys groups have been identified between the bacterial
molybdoenzyme catalytic subunits that contain a [4Fe-4S] cluster and
those that do not (23). EPR evidence exists to place E. coli
FdhF and Paracoccus denitrificans (formerly
Thiosphaera pantotropha) periplasmic nitrate reductase
(NapA) in the former group (27, 28), and the catalytic subunits of the
E. coli respiratory Me2SO and nitrate reductases (DmsA and NarG) in the latter group (23, 29).
In the structure of FdhF (26), it is significant that there is a Lys
residue located between the [4Fe-4S] cluster and one of the pterins
of the Mo-bisMGD. This residue is highly conserved in the group of
enzymes that contain a [4Fe-4S] cluster coordinated by the N-terminal
Cys group of the catalytic subunit (23). In the group that has the Cys
group but no [4Fe-4S] cluster, this residue is replaced by an Arg
(Arg-77 of DmsA and Arg-98 of NarG). When DmsA-R77 is mutagenized to
Ser, electron transfer from the [4Fe-4S] clusters of DmsB to the
Mo-bisMGD of DmsA is blocked (24). It would therefore be interesting to
test the effect of the DmsA-R77S mutation on the Mo(V)-[4Fe-4S]
cluster interaction.
In this paper, we describe the interactions observed by EPR that
involve the Mo-bisMGD cofactor, the Em,7 =
120 mV [4Fe-4S] cluster of DmsB, and engineered [3Fe-4S]
clusters of appropriately mutagenized DmsA and DmsB. In the absence of
detailed crystallographic data, these studies provide important
information on the interactions between the prosthetic groups of DmsABC
as well as on the electron transfer pathway from MQH2 to
Me2SO.
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MATERIALS AND METHODS |
Bacterial Strain and Plasmids--
The E. coli strain
and plasmids used in this study are listed in Table
I. pDMS170 bears the wild-type
dmsABC operon behind an fnr-dependent
promoter and was generated by ligating the 4.8-kilobase pair
EcoRI-SalI fragment from pDMS223 (4) into pBR322
that had previously been cut with PvuII and NruI
and self-ligated to destroy these sites. pDMS160-S176A-C102S encoding
DmsAS176ABC102SC was constructed by subcloning
a 3.37-kilobase pair EcoRV-SalI fragment bearing
the dmsBC102S mutation from pDMS160-C102S into
pDMS160-S176A. All routine cloning work was carried out essentially as
described by Sambrook et al. (30). All plasmids were
transformed into E. coli HB101. pDMS170 was used to
express the wild-type DmsABC used in this study and is functionally
equivalent to the plasmid pDMS160 used in previous studies (4, 19, 24).
pDMS160-C38S, pDMS160-R77S, and pDMS160-S176A-C102S were used to
express DmsAC38SBC, DmsAR77SBC, and
DmsAS176ABC102SC, respectively.
Growth of Cells and Preparation of Membrane Vesicles--
Cells
were grown anaerobically in 20-liter batch cultures at 37 °C for
48 h on a glycerol-fumarate minimal medium (4, 31). Cells were
harvested and washed in 100 mM MOPS and 5 mM
EDTA (pH 7.0). Membranes were prepared by French pressure cell lysis
and differential centrifugation as described previously (4, 6).
Preparation of EPR Samples--
Membrane vesicles were suspended
at a protein concentration of approximately 30 mg ml
1 in 100 mM MOPS and 5 mM EDTA (pH 7.0).
Dithionite-reduced (5 mM) samples were incubated under
argon at 23 °C for 5 min. Oxidized samples were prepared by
incubating membranes in the presence of 0.2 mM potassium
ferricyanide for 2 min. Ferricyanide-oxidized, glycerol-inhibited (32,
33) samples were prepared as described in the legend to Fig. 5.
Redox Potentiometry--
Redox titrations were carried out at
25 °C under argon in an anaerobic chamber as described previously
(6, 34). Membranes were used at a protein concentration of
approximately 30 mg ml
1, and the following redox dyes were
added to a final concentration of 50 µM: quinhydrone,
2,6-dichloroindophenol, 1,2-naphthoquinone, toluylene blue, phenazine
methosulfate, thionine, duroquinone, methylene blue, resorufin,
indigotrisulfonate, indigodisulfonate, anthraquinone-2-sulfonic acid,
phenosafranine, benzyl viologen, and methyl viologen. All samples were
prepared in 3-mm internal diameter quartz EPR tubes and were rapidly
frozen in liquid nitrogen-chilled ethanol before being stored under
liquid nitrogen until use.
EPR Spectroscopy--
Spectra were recorded using a Bruker
ESP300 EPR spectrometer equipped with an Oxford Instruments ESR-900
flowing helium cryostat. Instrument conditions and temperatures were as
described in the individual figure legends. Microwave power saturation
data were fitted to the equation,
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(Eq. 1)
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where S is the signal height, K is a
proportionality factor, P is the microwave power,
P1/2 is the microwave power for half-saturation, and
b is the inhomogeneity parameter (35, 36).
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RESULTS |
Mo(V) EPR Spectra of the Wild-type, DmsA-C38S, and DmsA-R77S
Enzymes--
Fig. 1 shows Mo(V) EPR
spectra recorded at 75 K of membranes containing the wild-type (Fig.
1A), DmsA-C38S (Fig. 1B), and DmsA-R77S (Fig.
1C) mutant forms of DmsABC. All three spectra appear to be
essentially identical, with g values of approximately 1.984, 1.980, and
1.960 (g1, g2, and g3), suggesting
that neither the DmsA-C38S nor the DmsA-R77S mutation has any
significant effect on the immediate coordination sphere of the
molybdenum of the Mo-bisMGD cofactor.

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Fig. 1.
Mo(V) EPR spectra of wild-type, DmsA-C38S,
and DmsA-R77S DmsABC. A, wild-type DmsABC poised at
91 mV. B, DmsA-C38S mutant poised at 95 mV.
C, DmsA-R77S poised at 101 mV. Spectra are of HB101
membranes containing overexpressed wild-type and mutant DmsABC in 100 mM MOPS and 5 mM EDTA (pH 7). EPR conditions
were as follows: temperature, 75 K; modulation amplitude, 3.8 Gpp at 100 kHz; microwave frequency, 9.47 GHz; microwave
power, 2 mW.
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Effect of the DmsA-C38S and DmsA-R77S Mutations on the Mo(IV/V) and
Mo(V/VI) Em,7 Values--
Fig.
2A shows Mo(V) potentiometric
titrations of wild-type and DmsA-C38S mutant DmsABC. For the wild-type
enzyme, plots of the intensity of the g = 1.980 peak-trough
versus Eh can be fitted to two
Em,7 values of
3 ± 3 mV
(Mo(V/VI)) and
148 ± 14 mV (Mo(IV/V)) (three determinations),
in reasonable agreement with previously reported values (19). In the
case of the DmsA-C38S mutant, plots of the g = 1.980 peak-trough
versus Eh can be fitted to two
Em,7 values of
5 ± 4 mV (Mo(V/VI)) and
128 ± 13 mV (Mo(IV/V)) (three determinations), indicating that
the mutation and/or the presence of the DmsA [3Fe-4S] cluster has a
minor effect on the Mo(IV/V) couple (a
Em,7 of
approximately +20 mV). Fig. 2B shows the effect of the
DmsA-R77S mutation on the Em,7 values of the
Mo-bisMGD cofactor. The plots can be fitted to two
Em,7 values of
27 ± 3 mV (Mo(V/VI)) and
149 ± 10 mV (Mo(IV/V)) (three determinations). In this case,
the mutation has a minor effect on the Mo(V/VI) couple (a
Em,7 of approximately
24 mV). Given their lack
of effect on the Mo(V) EPR line shape and their minor electrochemical effects, it is unlikely that the two DmsA mutations cause gross changes
in the overall structure of DmsABC.

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Fig. 2.
Potentiometric titrations of the Mo(V)
signals of wild-type, DmsA-C38S, and DmsA-R77S DmsABC.
A, comparison of wild-type and DmsA-C38S DmsABC. The
intensity of the g = 1.98 peak-trough was plotted
versus Eh and fitted to two
Em,7 values of 5 and 140 mV (wild type,
squares) and 10 and 115 mV (DmsA-C38S,
triangles). B, comparison of wild-type and
DmsA-R77S DmsABC. The intensity of the g = 1.98 peak-trough was
plotted versus Eh and fitted to two
Em,7 values of 5 and 140 mV (wild type,
squares), and 30 and 138 mV (DmsA-R77S,
triangles). Spectra were recorded as described in the legend
of Fig. 1.
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The Spin-Spin Interaction between the Em,7 =
120 mV [4Fe-4S] Cluster and the Mo(V) of Wild-type
DmsABC--
Cammack and Weiner (2) originally identified an
enhancement of the spin relaxation of the Mo(V) species of wild-type
DmsABC that was consistent with it being due to an interaction between the Mo(V) and the reduced Em,7 =
120 mV cluster.
Fig. 3A shows microwave power
saturation curves recorded at various redox potentials for the g = 1.98 peak-trough of the Mo(V) spectrum at 30 K. As the potential is
reduced, the data can be fitted to two components, a saturable
noninteracting component (dominant at high Eh) and a
nonsaturable component (dominant at low Eh). The
fraction of noninteracting Mo(V) decreases with decreasing
Eh with an apparent n = 1 Em,7 of
140 mV (Fig. 3B). Given that
the fraction of noninteracting Mo(V) at each Eh is
obtained from log-log plots of the microwave power saturation data, the
observed Eh of
140 mV is in reasonable agreement
with the published Em,7 of
120 mV for
one of the [4Fe-4S] clusters of DmsB (2, 6). These data are
consistent with (i) the interacting [4Fe-4S] cluster being the
Em,7 =
120 mV cluster and (ii) the
Em,7 =
50 mV cluster not contributing
independently to this interaction.

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Fig. 3.
Effect of Eh on the
microwave power saturation properties of the Mo(V) EPR signal of
wild-type DmsABC at 30 K. A, microwave power saturation
curves for the g = 1.98 peak-trough of Mo(V) spectra of
redox-poised samples. Filled squares, Eh = 211 mV, P1/2 = 1.7 mW, b = 1.5 (0.10). Filled circles, Eh = 177 mV,
P1/2 = 2.1 mW, b = 1.6 (0.28).
Open diamonds, Eh = 151 mV,
P1/2 = 1.7, b = 1.5 (0.42).
Open circles, Eh = 117 mV,
P1/2 = 2.1 mW, b = 1.5 (0.72).
Open triangles, Eh = 70 mV,
P1/2 = 1.7 mW, b = 1.5 (0.97).
Open squares, Eh = 21 mV,
P1/2 = 1.2 mW, b = 1.5 (1.00).
Numbers in parentheses indicate the estimated
fraction of noninteracting Mo(V). Where appropriate, fits to
experimental data included a nonsaturable interacting component with a
nominal P1/2 of 10 watts. Spectra were
recorded as described for Fig. 1 but at 30 K with a modulation
amplitude of 6 Gpp. B, effect of
Eh on the fraction of noninteracting
Mo(V). The fraction of noninteracting Mo(V) was determined from the
fits to the microwave power saturation curves of A and
plotted versus Eh. Data were fitted to a single
n = 1 Em,7 of 140 mV.
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Effect of the DmsA-C38S and DmsA-R77S Mutations on the Paramagnetic
Interaction between the Em,7 =
120 mV Cluster of
DmsB and the Mo(V) of DmsA--
Fig. 4
shows microwave power saturation curves for wild-type, DmsA-C38S, and
DmsA-R77S DmsABC at potentials of approximately
20 and
150 mV. In
the DmsA-C38S mutant, the microwave power saturation curve of the
Eh =
20 mV Mo(V) signal is essentially identical
to that of wild-type DmsABC, saturating with a single P1/2 of 1.2 mW. In contrast to the wild type, at
Eh =
155 mV the Mo(V) signal saturates as an
apparent single component with a P1/2 of 2.0 mW and
an inhomogeneity parameter (b) of 0.8. When microwave power saturation curves are recorded using redox poised samples (between Eh =
20 mV and Eh =
180 mV),
b decreases to a lower limit of approximately 0.8 as the
Em,7 =
120 mV [4Fe-4S] cluster becomes reduced
(data not shown). Potentiometric studies indicate that the
Em,7 values of the DmsB [4Fe-4S] clusters
(including the Em,7 =
120 mV cluster) remain
unaltered in the DmsA-C38S mutant (23). Thus, the presence of the
[3Fe-4S] cluster appears to significantly modulate the interaction
between the Em,7 =
120 mV cluster of DmsB and the
Mo(V) of the Mo-bisMGD cofactor of DmsA.

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Fig. 4.
Interaction between the Mo(V) and the
Em,7 = 120 mV [4Fe-4S] clusters in
wild-type, DmsA-C38S, and DmsA-R77S DmsABC. Microwave power
saturation curves are shown for the g = 1.98 peak-trough of Mo(V)
spectra of redox-poised samples. Open triangles, wild-type
DmsABC poised at Eh = 140 mV,
P1/2 = 1.2 mW (60%), and 10 watts (40%),
b = 1.4. Open squares, wild-type DmsABC
poised at Eh = 20 mV, P1/2 = 1.2 mW, b = 1.4. Open diamonds, DmsA-C38S
mutant poised at Eh = 155 mV,
P1/2 = 2 mW, b = 0.8. Open
circles, DmsA-C38S mutant poised at Eh = 20
mV, P1/2 = 1.2 mW, b = 1.4. Closed triangles, DmsA-R77S mutant poised at
Eh = 146 mV, P1/2 = 1.0 mW,
b = 1.6. Closed squares, DmsA-R77S mutant
poised at Eh = 18 mV, P1/2 = 1.4 mW, b = 1.6. Data were collected from spectra
recorded at 30 K with a modulation amplitude of 6 Gpp at
100 kHz and a microwave frequency of 9.47 GHz.
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In the DmsA-R77S mutant, the microwave power saturation curve of the
Eh =
18 mV sample is again very similar to that of
the wild-type enzyme (Fig. 4). In this case, the
P1/2 is estimated to be 1.4 mW. However, at
Eh =
146 mV, the saturation curve is essentially
identical to that observed at
18 mV, indicating that the DmsA-R77S
mutation eliminates the magnetic interaction between the Mo(V) and
Em,7 =
120 mV cluster of DmsB. EPR spectra recorded at 12 K of redox-poised samples indicate that there is no
detectable shift of the midpoint potential of the
Em,7 =
120 mV cluster in the DmsA-R77S mutant
(data not shown). When the potential dependence of the microwave power
saturation curves are investigated, greater than 95% of the Mo(V)
saturates with a low P1/2, even at potentials as low
as
200 mV (data not shown). Because of the disappearance of the Mo(V)
signal at low Eh, it was not possible to investigate the effect of reduction of the two lowest potential [4Fe-4S] clusters (the Em,7 =
240 and
330 mV clusters) on the
microwave power saturation properties or line shape of the Mo(V) spectrum.
Interaction between Glycerol-inhibited Mo(V) and the Oxidized
DmsA-C38S [3Fe-4S] Cluster--
Me2SO reductase from
Rh. sphaeroides can be prepared in a glycerol-inhibited form
by reduction followed by reoxidation in the presence of high
concentrations of glycerol (32, 33). The Mo-bisMGD of this form of the
enzyme remains in the Mo(V) state under oxidizing conditions. In order
to investigate potential interactions between the molybdenum and the
DmsA [3Fe-4S] cluster in the DmsA-C38S mutant, we generated membrane
preparations containing glycerol-inhibited wild-type and mutant DmsABC.
Fig. 5A shows EPR spectra
recorded at 75 K of the glycerol-inhibited forms of both wild-type
(Fig. 5A, i) and DmsA-C38S mutant (Fig.
5A, ii) DmsABC. Glycerol-inhibited enzyme (33)
was prepared by oxidation of dithionite-reduced enzyme in the presence
of 50% (v/v) glycerol. In both cases, the EPR spectrum has g values
(g1, g2, and g3) of 1.986, 1.978, and 1.959. Thus, the spectrum of the glycerol-inhibited form of Mo(V)
is slightly more rhombic than the noninhibited form (cf.
Fig. 1), allowing resolution of the g1 peak from the
g2 peak-trough. Except for a slight broadening of the
g2 peak-trough in the glycerol-inhibited DmsA-C38S mutant (Fig.
5A, ii), there appears to be little effect elicited on the glycerol-inhibited Mo(V) spectrum by the presence of a
[3Fe-4S] cluster in DmsA.

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Fig. 5.
Interaction between the glycerol-inhibited
Mo(V) and an engineered [3Fe-4S] cluster in DmsA. A,
EPR spectra of glycerol-inhibited, ferricyanide-oxidized DmsABC
(i) and DmsAC38SBC (ii). The
glycerol-inhibited form was generated as follows. Membranes at
approximately 40 mg ml 1 protein were reduced with a small
excess of dithionite for 2 min under argon at 23 °C. Glycerol was
then added to 50% (v/v), and following incubation for a further 2 min,
the membranes were oxidized with an excess of ferricyanide
(approximately 0.4 mM). EPR spectra were recorded at 75 K
with a modulation amplitude of 3 Gpp and a microwave
frequency of 9.47 GHz. B, microwave power saturation curves
of glycerol-inhibited Mo(V) signals at 30 K with a modulation amplitude
of 6 Gpp. Triangles, DmsAC38SBC
(P1/2 = 0.9 mW, b = 1.5);
squares, DmsABC (P1/2 = 0.6 mW,
b = 1.6).
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The estimated P1/2 values for the Mo(V) signals of
the glycerol-inhibited wild-type and DmsA-C38S DmsABC are 0.6 and
0.9 mW, respectively (Fig. 5B). These results suggest that
there is no significant enhancement of the spin relaxation of the
glycerol-inhibited Mo(V) caused by the presence of an engineered [3Fe-4S] cluster in DmsA. The redox state of the DmsA [3Fe-4S] cluster was verified by recording spectra at 12 K, and under the conditions used to generate the glycerol-inhibited samples it was found
to be in the oxidized S = 1/2 state.
Interestingly, in the case of the redox-poised data presented in Fig.
4, it is clear that the reduced S = 2 state of the DmsA
[3Fe-4S] cluster also does not significantly enhance the spin
relaxation of the Mo(V) spectrum.
Interaction between the Mo(V) of a DmsA-S176A Mutant and an
Engineered [3Fe-4S] Cluster in DmsB--
We have previously
demonstrated that mutagenesis of the protein-molybdenum ligand of DmsA
(Ser-176) generates a form of the Mo-bisMGD in which the molybdenum
remains in the paramagnetic Mo(V) redox state at high
Eh (19). We have also generated mutants of DmsB in
which the [4Fe-4S] cluster coordinated primarily by Cys group III is
replaced by a high potential [3Fe-4S] cluster (4, 6). By
straightforward subcloning (see "Materials and Methods"), it was
possible to generate a double mutant,
DmsAS176ABC102SC, which at high
Eh contains Mo(V) and the oxidized DmsB [3Fe-4S] cluster.
Fig. 6A shows EPR spectra
recorded at 30 K and 0.2-mW microwave power of ferricyanide-oxidized
membranes containing overexpressed DmsAS176ABC (Fig.
6A, i) and
DmsAS176ABC102SC (Fig. 6A,
ii). In both cases, a Mo(V) signal was observed with g
values of approximately 2.018, 1.982, and 1.961 (g1,
g2, and g3), as previously reported (19). The
minor features in the g = 2.00-2.03 region result from the
incomplete broadening of the DmsB-C102S [3Fe-4S] cluster spectrum at
the temperature and microwave power used to record the spectra of Fig.
6A. Fig. 6B shows microwave power saturation
profiles of the Mo(V) spectra of DmsAS176ABC and
DmsAS176ABC102SC. In both cases, the estimated
P1/2 is 0.8 mW, indicating that the presence of the
oxidized S = 1/2 [3Fe-4S] cluster of DmsB has
no detectable effect on the microwave power saturation properties of
the Mo(V) of DmsA.

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Fig. 6.
Interaction between the Mo(V) of DmsA and an
engineered [3Fe-4S] cluster in DmsB. A,
Mo(V) EPR spectra of ferricyanide-oxidized HB101 membranes
containing overexpressed DmsAS176ABC (i) and
DmsAS176ABC102SC (ii). EPR
conditions were as described for Fig. 5A, except that the
temperature was 30 K, the microwave power was 0.2 mW, and the
modulation amplitude was 6 Gpp. B, microwave
power saturation curves of the Mo(V) signal from
DmsAS176ABC (squares, P1/2 = 0.8 mW, b = 1.5) and
DmsAC176ABC102SC (triangles,
P1/2 = 0.8 mW, b = 1.5).
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DISCUSSION |
We have demonstrated herein the effects of mutations of residues
along the electron transfer pathway of DmsA on the EPR properties and
redox chemistry of the Mo-bisMGD cofactor of DmsABC. Based on their
effects on the Mo(V) EPR spectrum, it is clear that the DmsA-C38S and
DmsA-R77S mutants have little effect on the environment or coordination
sphere of the molybdenum and only minor effects on its redox chemistry.
Given these results, it is unlikely that the mutations result in any
gross modification of the protein structure that may result in
significant differences in intercofactor distances between the
wild-type and mutant proteins. The DmsA-C38S and DmsA-R77S mutations
both have significant effects on the observed Mo(V)-[4Fe-4S]
interaction. These effects provide important insights into the electron
transfer pathway from the [4Fe-4S] clusters of DmsB to the Mo-bisMGD
cofactor of DmsA.
That the DmsA Cys-38 and Arg-77 residues do not contribute to the
coordination sphere of the molybdenum of the Mo-bisMGD cofactor is
corroborated by comparison of DmsA with the structurally characterized FdhF (26). In FdhF, the residue equivalent to DmsA-C38 (FdhF-C11), provides a ligand to the [4Fe-4S] cluster found in this protein. The
residue equivalent to DmsA Arg-77 (FdhF Lys-44) is located between the
[4Fe-4S] cluster and one of the pterins of the Mo-bisMGD cofactor and
may play an important role in the electron transfer mechanism (26). The
effects of the DmsA-C38S and DmsA-R77S mutants on the EPR and redox
properties of the molybdenum contrast with those of a mutation of a
residue whose side chain contributes to the immediate coordination
sphere of the molybdenum. For example, mutation of the
protein-molybdenum ligand of DmsABC, DmsA Ser-176 to Ala has a profound
effect on both the Mo(V) EPR spectrum and the overall redox chemistry
of the Mo-bisMGD cofactor (19). In FdhF, mutation of the selenocysteine
protein-molybdenum ligand also significantly modifies the Mo(V) EPR
spectrum (37).
The DmsA-C38S and DmsA-R77S mutants appear to have minor, but opposite
effects on the redox chemistry of the Mo-bisMGD. The shift elicited by
the DmsA-C38S mutant is a
Em,7 = +20 mV shift of
the Mo(IV/V) couple, whereas the shift elicited by the DmsA-R77S mutant
is a
Em,7 =
24 mV shift of the Mo(V/VI) couple.
The [3Fe-4S] cluster of the DmsA-C38S mutant is reduced at
Eh values where the Mo(V) is visible (it has an
Em,7 of approximately 178 mV (23)) and therefore
does not carry a formal charge. Thus, it is likely that subtle changes
in structure caused by the introduction of a [3Fe-4S] cluster are
responsible for the small change in the Mo(IV/V)
Em,7. In the case of the DmsA-R77S mutant, the
Em,7 =
24 mV shift in the Mo(V/VI)
Em,7 may be rationalized in terms of the loss of the
electron-withdrawing effect of the positive charge of the Arg residue.
This suggests that the residues that contact the pterin ring systems
may have an important role in defining the Mo(IV/V) and Mo(V/VI)
Em,7 values. It should be noted that in the case of
both mutations, the observed effects are on only one of the two
molybdenum Em,7 values. Further mutagenesis studies
will be necessary to confirm the role of pterin contact residues in
defining the electrochemistry of the Mo-bisMGD.
Spin-spin interactions between EPR-visible prosthetic groups can
provide important information on the topographical arrangement of these
centers in multicofactor enzymes. Of relevance to the data presented
herein are the studies carried out on milk xanthine oxidase (36,
38-41). In this enzyme, there is a strong spin-spin interaction
between various accessible forms of the Mo(V) of the molybdo-molybdopterin cofactor and one of the [Fe-S] clusters. This
interaction results in both splitting of the EPR spectrum of the Mo(V)
and in an enhancement of its spin relaxation rate at low temperatures
that manifests itself as a significant increase in the
P1/2. In the case of DmsABC, there is a strong
spin-spin interaction between the Mo(V) species and the
Em,7 =
120 mV [4Fe-4S] cluster that manifests
itself as an increase in the Mo(V) P1/2 (Fig. 3),
but does not result in a line shape change that can readily be
interpreted as arising from a dipolar interaction.
A structure has recently become available of a bacterial protein
similar in structure and sequence to xanthine oxidase. This protein,
Desulfovibrio gigas aldehyde oxidoreductase (42), contains a
molybdo-molybdopterin cytosine dinucleotide cofactor, and two [2Fe-2S] clusters. This enzyme has EPR properties similar to those of
xanthine oxidase (43). It has been suggested that the cluster equivalent to the one interacting with the molybdenum in xanthine oxidase may be located approximately 15 Å from the molybdenum (44). In
the structure of aldehyde oxidoreductase (42), one of the Cys ligands
of this cluster is also hydrogen-bonded to the pterin. These
observations bear interesting comparison with distance estimates for
xanthine oxidase based on EPR analyses that are in the 8-25-Å range
(41). However, it should be noted that the assignment of the
interacting cluster in the structure of aldehyde oxidoreductase remains
controversial (44). In another protein that is much more closely
related to DmsA, E. coli FdhF, a [4Fe-4S] cluster is
located approximately 13 Å from the molybdenum (26). No interaction
between the [4Fe-4S] cluster and the molybdenum of FdhF has yet been reported.
Based on comparisons with xanthine oxidase and FdhF, a position for the
Em,7 =
120 mV cluster of DmsB that is consistent with the observed spin-spin interaction between this cluster and the
molybdenum would be equivalent to that of the interacting [2Fe-2S]
cluster of xanthine oxidase or the [4Fe-4S] cluster of FdhF. However,
it is clear from analyses of mutants of the DmsA Cys group that there
is no EPR-detectable cluster coordinated by DmsA in the wild-type
enzyme (23, 24). Also, mutants of DmsA Cys-38 and DmsA Arg-77 still
contain a potentiometrically identifiable Em,7 =
120 mV cluster, further supporting the assertion that there is no
cluster coordinated by the DmsA Cys group. Based on qualitative
comparisons with D. gigas aldehyde oxidoreductase, it is
therefore likely that the portion of DmsB containing the Em,7 =
120 mV cluster is located within
approximately 15 Å of the molybdenum of DmsA.
One important distinction between the interaction reported herein and
that observed in xanthine oxidase is the lack of apparent line shape
change accompanying the enhancement of the spin relaxation of the Mo(V)
signal. In xanthine oxidase, the line shape change manifests itself
when the temperature of the sample is reduced to sufficiently slow down
the spin relaxation rate of the interacting [2Fe-2S] cluster so that
it is compatible with that of the Mo(V) species (39). The interacting
[2Fe-2S] cluster of xanthine oxidase is a saturable (at 20 K) reduced
[2Fe-2S] cluster (36) that displays readily interpretable behavior
with increasing microwave power. In the case of DmsABC, the interacting
species is the Em,7 =
120 mV [4Fe-4S] cluster of
DmsB, which comprises half of a 2[4Fe-4S] ferredoxin motif with the
Em,7 =
50 mV [4Fe-4S] cluster. At the potentials
where the spin relaxation enhancement is observed, the
Em,7 =
120 mV cluster is itself undergoing a
complex interaction with the Em,7 =
50 mV cluster
(2, 6). This interaction is equivalent to that observed in the
bacterial 2[4Fe-4S] ferredoxins and results in an unsaturable EPR
spectrum indicative of a very rapid spin relaxation rate (40, 45-47).
Thus, in the interaction between the Mo(V) of DmsA and the
Em,7 =
120 mV cluster of DmsB, the latter center
is essentially unsaturable with increasing microwave power, and its
relaxation rate at 30 K is very likely to be too high for the
observation of significant splittings in the Mo(V) spectrum. A lack of
quantifiable splitting of the DmsABC Mo(V) signal by the
Em,7 =
120 mV [4Fe-4S] cluster of DmsB precludes a quantitative estimate of the intercenter distance based on the data
presented herein.
We have demonstrated that incorporation of a [3Fe-4S] cluster
coordinated by the DmsA Cys group modifies the interaction between the
Em,7 =
120 mV cluster and the molybdenum (Fig. 4).
This is consistent with (i) this cluster being located close to or on the vector joining the Mo(V) and the [4Fe-4S] cluster (for a dipolar interaction) and/or (ii) this cluster being on or close to the interaction pathway between the two centers (for an exchange
interaction). The effect of the DmsA-R77S mutation (Fig. 4) clearly
favors the second explanation, since presumably it has little effect on
the distance between the two centers but is still able to eliminate the
detectable interaction. Given that both the presence of a [3Fe-4S]
cluster and the DmsA-R77S mutant both essentially eliminate electron
transfer between DmsB and DmsA (23, 24), the data presented herein are
consistent with the interaction pathway being equivalent to the
electron transfer pathway. In the case of the interaction in xanthine
oxidase, it has also been proposed that it occurs through a specific
pathway through the protein and that it is also primarily exchange in
nature (39).
Given the significant increase in the observed P1/2
for the Mo(V) spectrum observed in the wild-type enzyme, we anticipated
that it would be possible to detect a spin-spin interaction between the
Mo(V) and either the reduced S = 2 or oxidized
S = 1/2 [3Fe-4S] cluster of the DmsA-C38S
mutant. In the case of the glycerol-inhibited, ferricyanide-oxidized
DmsA-C38S enzyme, there appears to be no enhancement of the Mo(V) spin
relaxation and only a minor broadening of its EPR spectrum (Fig. 5).
This result is somewhat surprising, since the [3Fe-4S] cluster of the DmsA-C38S mutant enzyme is, based on comparison with the structurally characterized FdhF (26), very likely to be located approximately 13 Å from the molybdenum.
Given the lack of significant interaction between the
glycerol-inhibited Mo(V) and the engineered [3Fe-4S] cluster in
DmsA, it is not surprising that no interaction is observed between the Mo(V) of the DmsA-S176A mutant and the engineered [3Fe-4S] cluster of
the DmsB-C102S mutant (Fig. 6). In this case, the lack of interaction may simply be due to the extra distance between the centers or perhaps
due to the Em,7 =
120 mV cluster acting as a
"shield," preventing the observation of an interaction. In the
DmsB-C102S mutant, the Em,7 =
50 mV [4Fe-4S]
cluster of DmsB is converted to a [3Fe-4S] cluster (4, 6). As
described above, in the wild-type enzyme, this cluster appears to form
half of an 2[4Fe-4S] ferredoxin motif. Based on the structurally
characterized bacterial 2[4Fe-4S] ferredoxins (48), this cluster is
potentially a maximum of approximately 12 Å further away from the
molybdenum of DmsA than the Em,7 =
120 mV cluster
(this depends on the angle between the vector joining the
Em,7 =
120 and
50 mV clusters and that joining
the Em,7 =
120 mV cluster and the molybdenum
center). Thus, an approximate maximum distance of 27 Å (12 plus 15 Å)
is apparently too great for an interaction to be observed in
DmsABC.
The data presented herein and previously reported data on the topology
and electron transfer pathway of DmsABC (4-6, 9, 20, 23, 24, 49) allow
a tentative model for electron transfer through the enzyme to be
proposed. MQH2 binding and oxidation occur at a single
dissociable site in DmsC (20), which is conformationally linked to the
Em,7 =
50 mV [4Fe-4S] cluster of DmsB (6). This cluster forms half of a 2[4Fe-4S] ferredoxin pair (2, 6) with the
Em,7 =
120 mV [4Fe-4S] cluster. Electrons pass
from MQH2 through these two clusters and continue to the
Mo-bisMGD cofactor via a vestigial [4Fe-4S] cluster binding domain
defined by the N-terminal Cys motif of DmsA (24). This segment of the pathway is sensitive both to the presence of an engineered [3Fe-4S] cluster in the DmsA-C38S mutant and to mutation of a residue (DmsA Arg-77) that is in close juxtaposition to one of the pterins of the
Mo-bisMGD (23, 24, 26). We have presented evidence herein that is
consistent with this segment of the pathway being equivalent to the
pathway of the magnetic interaction between the Em,7 =
120 mV [4Fe-4S] cluster and the molybdenum of the Mo-bisMGD
cofactor. Given the potential position of DmsA Arg-77 in relation to
one of the pterins, it is also very likely that one of the functions of
this pterin is to provide a conduit for electron transfer to the molybdenum.