(Received for publication, November 13, 1995; and in revised form, January 19, 1996)
From the
Carbon-monoxide dehydrogenase (CODH) from Rhodospirillum
rubrum contains two metal centers: a
Ni-X-[FeS
]
cluster (C-center) that serves as the COoxidation site and a standard
[Fe
S
]
cluster (B-center) that mediates electron flow from the C-center
to external electron acceptors. Four states of the C-center were
previously identified in electron paramagnetic resonance (EPR) and
Mössbauer studies. In this report, EPR-redox
titrations demonstrate that the fully oxidized, diamagnetic form of the
C-center (C
) undergoes a one-electron reduction to the
C
state (g
= 1.87) with a
midpoint potential of -110 mV. The reduction of C
to
C
is shown to coincide with the reduction of an
[Fe
S
]
cluster in redox-titration experiments monitored by UV-visible
spectroscopy. Nickel-deficient CODH, which is devoid of nickel yet
contains both
[Fe
S
]
clusters, does not exhibit EPR-active states or reduced
Fe
S
clusters at potentials more positive than
-350 mV.
Carbon-monoxide dehydrogenase (CODH) ()from the
purple, nonsulfur bacterium Rhodospirillum rubrum is an
oxygen-labile, nickel-containing enzyme that reversibly catalyzes the
oxidation of CO to CO
(1) . Unlike CODHs from
acetogenic and methanogenic bacteria, R. rubrum CODH is unable
to catalyze the synthesis or degradation of
acetyl-CoA(2, 3, 4, 5, 6, 15) .
As purified, CODH is a 62-kDa monomer with 1 nickel atom and
8
iron atoms/ monomer(7) . A nickel-deficient form of CODH, which
contains all of the iron components of holo-CODH yet has no
CO-oxidation activity, is obtained by growing R. rubrum cultures on nickel-depleted medium(8, 9) .
Treating nickel-deficient CODH with NiCl
produces an
activated form of the enzyme that contains 1 mol of nickel/mol of
enzyme and exhibits the same level of CO-oxidation activity as CODH
purified from cells grown on nickel-supplemented medium
(holo-CODH)(9) .
Holo-CODH contains only two metal centers,
which have been designated as the B-center and C-center. Similar metal
centers are also found in CODHs from methanogenic and acetogenic
bacteria, including the well studied CODH from Clostridium
thermoaceticum(10, 11, 12, 13, 14, 15) .
The C-center, which serves as the CO-oxidation
site(16, 17, 18, 19, 20) ,
is composed of an
[FeS
]
cluster (22) that is linked to nickel by an unidentified
bridging ligand, X (Ni-X-[Fe
S
]
) (19, 20, 21) . The B-center consists solely
of a standard
[Fe
S
]
cluster (22) which mediates electron flow between the
C-center and external electron
acceptors(8, 17, 18) . Nickel-deficient CODH,
which can only be obtained in R. rubrum, has the B-center and
a [Fe
S
]
cluster (referred to as the C*-center) that is a precursor to the
C-center of holo-CODH. The Fe
S
clusters of
nickel-deficient CODH cannot be reduced by CO(20) .
Four
states of the C-center of holo-CODH have been characterized (Table 1, Fig. 1) ( (8) and references within).
C and C
have electronic spins S = and S = , respectively, and result from the reduction of
holo-CODH with CO or dithionite. C
and C
are replaced by a second S = form, C
, and a diamagnetic form,
C
, by the oxidation of CO- or dithionite-reduced holo-CODH
with the redox dye indigo carmine (E`
=
-125 mV). Oxidation with thionin (E`
= 56 mV) fully converts the C-center to the C
form which is EPR silent. If all states of the C-center contain
Ni(II)(20) , then C
, C
and C
appear to be
one-electron reduced forms of C
(22) .
C
, however, has been proposed to be three electrons more
reduced than C
, therefore C
may contain a
more reduced form of nickel(11) .
Figure 1:
Model for the redox
states of the B- and C-centers of R. rubrum CODH. Approximate
midpoint potentials of the redox couples are centered between their
vertical positions in the figure. The C/C
couple undergoes a reversible one-electron redox reaction with a
midpoint potential of -110 mV. C
/C
appear to be separated by one
electron with a midpoint potential below -400 mV. C
may be one or three electrons more reduced than C
.
All forms of the C-center are thought to contain Ni(II), with the
exception of C
which may contain a more reduced form of
nickel in the event that C
is three electrons more
reduced than C
.
The C*-center of
nickel-deficient CODH is stable in two forms (Table 1, Fig. 1)(22) . The C* state, with
electronic spin S = , is a one-electron reduced form of
the C*-center observed following reduction of nickel-deficient CODH
with dithionite. The EPR signal originating from C*
has
broad resonances in the g = 4-6 region. This
signal is very similar to the C
EPR signal of holo-CODH. Following oxidation of reduced,
nickel-deficient CODH with indigo carmine or thionin, the C*
state is completely converted by a one-electron oxidation to the
diamagnetic C*
form.
The properties of the B-center in
holo-CODH and nickel-deficient CODH are very similar(22) . The
B-center is stable in only two forms (Table 1, Fig. 1)(22) . The B state develops
following the reduction of nickel-deficient CODH with dithionite or the
reduction of holo-CODH with CO or dithionite. By oxidizing either form
of reduced CODH with indigo carmine or thionin, B
is
completely converted to B
. The reversible oxidation of
B
to B
in holo-CODH was previously shown to
be a one-electron process with a midpoint potential of -418
mV(23) .
A significant problem in studying the
nickel-containing CODHs has been that the spin concentrations of the
C (g
= 1.87) and C
(g
= 1.86) EPR-signals vary from one
sample preparation to another (e.g. 0.05-0.5 mol of
spin/mol of C-center), and are much lower than expected for isolated S = systems. Hu et al.(22) concluded
that this observation derives from heterogeneity in the C-center
population resulting from a mixture of spin and oxidation states; EPR
and Mössbauer studies demonstrate a combination of
C
and C
states in CO- or dithionite- reduced holo-CODH and a combination
of C
and C
states in indigo
carmine-oxidized holo-CODH. Interestingly, the ability to observe the
C
or C
EPR signal does not correlate with
enzyme activity; all preparations of purified CODH have similar
specific activities. The factors affecting the states of the C-center
are incompletely understood; therefore, this report investigates the
effect of redox potential on the states of the C-center.
In this
report, we have combined potentiometric redox titrations with
UV-visible and EPR spectroscopies to study the redox characteristics of
the metal centers in holo- and nickel-deficient CODH. The midpoint
potential of the C/C
redox couple has been
determined and correlated with the reduction of an
[Fe
S
]
cluster in holo-CODH. Finally, this work demonstrates
nickel-deficient CODH, which does not exhibit a C
state,
does not contain an
[Fe
S
]
cluster that can be reduced over the range of potentials that
afford the C
state in holo-CODH.
The electrochemical cell and redox solutions
were prepared in an anaerobic glove box (Vacuum/Atmospheres Dri-Lab
glovebox model HE-493) with an N atmosphere containing less
than 1 ppm O
. The buffer used in all experiments was 100
mM MOPS, pH 7.2. Buffer solutions and the electrochemical cell
were stored in the glove box prior to use. In a typical experiment,
approximately 15-30 mg of CODH (with a specific activity of 4,200
units/mg) in a buffer solution containing 400 mM NaCl and 1
mM dithionite was chromatographed through a Sephadex G-25
gel-filtration column to remove dithionite. The dithionite-free CODH
eluent was diluted to 3 ml with anaerobic buffer containing mediators
(approximately 0.04 mM final concentration each) and KCl (0.1 M final concentration). This solution was added to the
electrochemical cell, which was assembled and removed from the glove
box in order to perform the potentiometric titrations. Once outside the
glovebox, a continuous flow of scrubbed argon was passed through the
electrochemical cell to maintain oxygen-free conditions. The redox
potential was established with an Electrosynthesis model 410
Potentiostatic Controller, and once equilibrated (i.e. drift
< 2 mV/min), the redox-poised solution was anaerobically transferred
into an EPR tube, and frozen in liquid N
. Fully reduced
samples of holo-CODH and nickel-deficient CODH were prepared by
exposure to CO, or by adding an excess of dithionite. EPR spectra were
recorded on a Bruker ESP 300 or a Varian E-15 spectrometer using an
Oxford Instruments ER910A cryostat. Relative signal intensities were
fitted to a linearized form of the Nernst equation (), and
the midpoint potential (E
) and the number of
electrons (n) involved in a redox reaction were determined
according to published
methods(13) .
The data were plotted as signal intensity versus the
measured potential (E), and a theoretical curve
was generated with n and E
values
determined by Nernst analysis. Redox potentials are reported in
reference to the normal hydrogen electrode.
CO- or dithionite-reduced CODH exhibited a minimum absorbance at
420 nm (A (minimum),
= 20.1 mM
cm
), and thionin-oxidized CODH exhibited a
maximum absorbance at 420 nm (A
(maximum),
= 35.6 mM
cm
)(8) .
Figure 2:
Upper panel, representative EPR spectra of
holo-CODH from R. rubrum at different redox potentials.
Purified CODH (6 mg/ml) was in 100 mM MOPS, pH 7.2, and 0.1
mM KCl. Redox potentials of samples were set as described
under ``Experimental Procedures.'' Spectrometer conditions
were: microwave power, 5 mW; modulation amplitude, 5 G; frequency,
9.236 GHz; temperature, 10 K. Spectra shown were recorded for samples
poised at -33 mV, spectrum A; -315 mV, spectrum B; and
-425 mV, spectrum C. Lower panel, EPR
spectroelectrochemical titration of the C-center. The amplitude of the g = 2.03 resonance was followed as the redox potential
was varied. Analysis by the Nernst equation yielded an E of -110 mV and a slope of -62
mV.
The
EPR-redox titration data presented in Fig. 2indicate that
C (g
= 1.87) results from a
one-electron reduction of the C
with a midpoint potential
of -110 mV. The maximum spin concentration measured in this
experiment for the g
= 1.87 EPR signal is
0.2 mol of spin/mol of CODH; therefore, the formation of the
C
state appears to be influenced by factors other than
redox potential alone. This observation is consistent with the findings
of Hu et al., who report a mixture of C
and
C
states in Mössbauer spectra of
holo-CODH poised at potentials as low as -300 mV. Hu et al. concluded that, due to sample heterogeneity, a fraction of
holo-CODH molecules may contain C-centers that are unable to form
C
. This fraction, therefore, is proposed to remain in
the C
state until potentials low enough to result in the
formation of C
and C
are achieved.
Nickel-deficient CODH did not exhibit EPR-active
states in the g = 2 region (Fig. 3A) or g = 4-6 region (data not shown) at potentials
more positive than -350 mV. Initial development of the B signal (g
= 1.94) was observed at
-400 mV (Fig. 3B). At -510 mV, however,
nickel-deficient CODH exhibited a two-component spectrum consisting of
B
and a similar and overlapping signal with g values at 2.07, 1.93, and 1.86 (Fig. 3C). The
nature of the second signal is unknown and is currently under
investigation.
Figure 3: Representative EPR spectra of nickel-deficient CODH from R. rubrum at different redox potentials. Purified nickel-deficient CODH (10 mg/ml) was in 100 mM MOPS, pH 7.2, and 0.1 mM KCl. Redox potentials of samples were set as described under ``Experimental Procedures.'' Spectrometer conditions were as described in Fig. 1, except that the microwave power was 20 mW and the frequency was 9.460 GHz. Spectra shown were recorded for samples poised at -100 mV (spectrum A), -400mV (spectrum B), and -510 mV (spectrum C).
The C*-center does not appear to be redox active over
the range of potentials that afford the C state. This
observation is consistent with the proposal that an interaction between
the nickel and
[Fe
S
]
components in the C-center strongly influences the redox behavior
of the Fe
S
cluster. In the absence of nickel,
therefore, reduction of the
[Fe
S
]
cluster of
C*
can only occur at potentials significantly lower than
the E
= -110 mV of the
C
/C
redox transition.
The B-centers in
holo- and nickel-deficient CODH exhibited similar redox behavior. The
reduced form of the B-center, B, is not observed at
potentials more positive than -350 mV in EPR-redox titrations of
either form of CODH ( Fig. 2and Fig. 3). The redox
properties of the B-center, therefore, appear to be unaffected by
nickel in holo-CODH. The absence of B
at potentials above
-350 mV is also consistent with the finding of Smith et
al.(23) that the B-center in holo-CODH undergoes
reduction with a midpoint potential of -418 mV.
The corresponding EPR and
UV-visible spectra of fully oxidized holo-CODH had no significant EPR
signals (Fig. 4A, upper panel) and a maximum
absorbance at 420 nm (Fig. 4A, lower panel).
This shows that all C-centers were in the C form and all
[Fe
S
]
clusters were oxidized. Reduction of 33% of the
Fe
S
clusters in holo-CODH, as determined by A
measurement (Fig. 4B, lower panel), was accompanied by the appearance of the
C
state (g
= 1.87) in the
corresponding EPR spectrum (Fig. 4B, upper
panel). (The
[Fe
S
]
clusters of the B- and C-centers are assumed to contribute
equally to the optical spectrum at 420 nm). As the percentage of
reduced Fe
S
clusters increased beyond 33%, the
EPR signal intensity of C
(g
= 1.87) did not increase. With 56% of the
Fe
S
clusters reduced in holo-CODH, partial
development of the B
state (g
= 1.94) was evident (Fig. 4C, upper and lower panels). Fully reduced holo-CODH exhibited EPR
signals originating from the B
and C
(g
= 1.86) states (Fig. 4D, upper panel) and a minimum
absorbance at 420 nm (Fig. 4D, lower panel).
Figure 4:
EPR and UV-visible spectra of holo-CODH
samples at various levels of Fe-S cluster reduction determined by A. A dithionite-free sample of
``as-isolated'' CODH (3 mg/ml) in 100 mM MOPS, pH
7.2, was obtained as described under ``Experimental
Procedures.'' The C
state and 100% oxidation of Fe-S
clusters were obtained by adding a slight excess of thionin (traces
A); the C
state (g = 2.03, 1.88 and
1.71) and 33% reduction of Fe-S clusters in ``as-isolated''
CODH were obtained without addition of oxidant or reductant (traces
B); the C
and B
(g =
2.04, 1.97 and 1.88) states and 66% reduction of Fe-S clusters were
obtained following the addition of a small amount of sodium dithionite (traces C); the B
and C
(g = 1.97, 1.88 and 1.75) states and full reduction of Fe-S
clusters were obtained after incubation under CO (traces D).
EPR conditions were as described in Fig. 1.
The data in Fig. 4correlates the development of the
C EPR signal (g
= 1.87)
with a decrease in A
, which is consistent with
the proposal that C
results from the one-electron
reduction of the [Fe
S
]
component of C
. The development of the
lower-potential B
state was accompanied by additional
loss of absorbance at 420 nm, and C
was not observed
until all [Fe
S
]
clusters became reduced. Although present in holo-CODH, nickel
does not contribute to the optical spectrum at 420 nm(8) .
Figure 5:
UV-visible spectroelectrochemical
titration of the Fe-clusters in CODH. The intensity of the absorbance
at 420 nm was followed as the redox potential was varied. Solid
circles (holo-CODH) and open circles (nickel-deficient
CODH) represent the fractional absorbance changes at 420 nm expressed
in percentages. The theoretical lines drawn through the points are
Nernst n = 1 curves, E =
-415 mV for nickel-deficient CODH; E
= -415 mV, and E
=
-125 mV for holo-CODH.
Beginning at approximately -350
mV, decreases in A were observed for holo-CODH
and nickel-deficient CODH, and the minimum A
absorption value for each sample was achieved at -500 mV,
indicating full reduction of all
[Fe
S
]
clusters (Fig. 5). Notably, significant reduction of
Fe
S
clusters in nickel-deficient CODH was not
observed at potentials greater than -350 mV. This observation,
coupled with the absence of the C*
EPR signal at similar
potentials, shows that the fully oxidized form of the C*-center
(C*
) is not redox active at potentials greater than
-350 mV.
Although this work focuses primarily on the redox
properties of
[FeS
]
clusters that in part define the various states of the B- and
C-centers, the role of nickel in the C-center should not be overlooked.
The presence of nickel dramatically increases the midpoint potential of
the C-center
[Fe
S
]
cluster by over 200 mV. Moreover, Hu et al.(22) found evidence in Mössbauer
studies that the [Fe
S
]
component of the C
state contains a unique
pentacoordinate iron subsite, called ferrous component II(29) ,
which was not observed in the C*-center; the spectroscopic properties
of the C*-center Fe
S
cluster were consistent
with all iron atoms having tetracoordination(22) . Treating
nickel-deficient CODH with nickel converts the C*-center to the
C-center coincident with the development of the unique ferrous
component II subsite; therefore, the incorporation of nickel appears to
substantially alter the coordination environment of one iron atom in
the C-center. The reduced
[Fe
S
]
cluster in the
C
state, therefore, may be stabilized by electrostatic
interactions from the nickel cation or from an alteration of the
ligands and protein environment of the C-center Fe
S
cluster when nickel is bound to CODH.
It is interesting to
note that the activation of nickel-deficient CODH by NiCl occurs only at potentials lower than -350 mV(9) .
From the results presented here, this suggests that the C*-center must
be in the C*
state (and perhaps the B-center in the
B
state) before the appropriate ligand environment is
accessible to the nickel cation.
The nickel ion appears to remain as
Ni(II) in the various states of the C-center studied here. Evidence of
a Ni(III) EPR signal was not observed at any potential. Furthermore,
the correlation established between the optical changes monitored at
420 nm and the development of the C (g
= 1.87) EPR signal demonstrates that the one-electron
reduction of C
that affords C
is localized
on the [Fe
S
]
component of the C-center. The redox behavior of the C-center at
potentials more negative than -350 mV is unclear, as is the
relationship between CO-oxidation activity and the appearance of the
C
, C
and C
states. Both the C
and C
forms of the
C-center must be competent to bind and be reduced by CO as holo-CODH
containing either form of the C-center can be fully reduced by CO. It
is possible that multiple ``routes'' of reduction exist among
the four states of the C-center, but data presented here do not allow
us to distinguish such possibilities. Finally, the number of electrons (i.e. one or three) separating C
from C
is unknown.
In summary, the C/C
redox couple has a midpoint potential of -110 mV. The
formation of C
from C
is concomitant with
the one-electron reduction of a
[Fe
S
]
cluster in holo-CODH. The nickel cation, proposed to be Ni(II) in
the C
and C
states, appears to strongly
affect the redox behavior of the
[Fe
S
]
component of the C-center; reduction of the C*-center
[Fe
S
]
cluster in nickel-deficient CODH is not observed at potentials
more positive than -350 mV.