From the Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799
Received for publication, October 13, 2002, and in revised form, November 27, 2002
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ABSTRACT |
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The acetyl-CoA decarbonylase/synthase (ACDS)
complex catalyzes the central reaction of acetyl C-C bond cleavage in
methanogens growing on acetate and is also responsible for synthesis of
acetyl units during growth on C-1 substrates. The ACDS The methanogenic Archaea utilize a unique metabolic pathway for
degradation of acetate under anaerobic conditions, and cleavage of
acetate thereby accounts for a major proportion of the methane formed
in the environment. The central reaction in this pathway is carried out
by an unusual multienzyme complex, designated acetyl-CoA decarbonylase/synthase
(ACDS),1 which contains five
different polypeptide subunits and accounts for as much as 25% of the
soluble protein in species such as Methanosarcina thermophila and Methanosarcina barkeri growing on
acetate. The ACDS complex catalyzes cleavage of the acetyl C-C bond
using the substrates acetyl-CoA and tetrahydrosarcinapterin
(H4SPt), a tetrahydrofolate analog which serves as methyl
acceptor, and yields the products CoA,
N5-methyltetrahydrosarcinapterin,
CO2, and two reducing equivalents, as given in Reaction 1 (1).
subunit
contains nickel and an Fe/S center and reacts with acetyl-CoA forming
an acetyl-enzyme intermediate presumably directly involved in acetyl C-C bond activation. To investigate the role of nickel in this process
two forms of the Methanosarcina thermophila
subunit were overexpressed in anaerobically grown Escherichia coli.
Both contained an Fe/S center but lacked nickel and were inactive in acetyl-enzyme formation in redox-dependent
acetyltransferase assays. However, high activity developed during
incubation with NiCl2. The native and nickel-reconstituted
proteins both contained iron and nickel in a 2:1 ratio, with
insignificant levels of other metals, including copper. Binding of
nickel elicited marked changes in the UV-visible spectrum, with intense
charge transfer bands indicating multiple thiolate ligation to nickel.
The kinetics of nickel incorporation matched the time course for enzyme
activation. Other divalent metal ions could not substitute for nickel
in yielding catalytic activity. Acetyl-CoA was formed in reactions with
CoA, CO, and methylcobalamin, directly demonstrating C-C bond
activation by the
subunit in the absence of other ACDS subunits.
Nickel was indispensable in this process too and was needed to form a characteristic EPR-detectable enzyme-carbonyl adduct in reactions with
CO. In contrast to enzyme activation, EPR signal formation did not
require addition of reducing agent, indicating indirect catalytic
involvement of the paramagnetic species. Site-directed mutagenesis
indicated that Cys-278 and Cys-280 coordinate nickel, with Cys-189
essential for Fe/S cluster formation. The results are consistent with
an Ni2[Fe4S4] arrangement at the
active site. A mechanism for C-C bond activation is proposed that
includes a specific role for the Fe4S4 center
and accounts for the absolute requirement for nickel.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
This overall reaction is made up of a series of partial reactions
catalyzed by different protein subcomponents of the ACDS complex as
shown in Scheme 1 (2). Acetyl-CoA binds
to the
subunit, and under low redox potential conditions, as
required for activity, transfers the acetyl group to a nucleophilic
center on the enzyme forming an acetyl-enzyme species and releasing CoA (Scheme 1, acetyl transfer) (2, 3). The acetyl intermediate then undergoes C-C bond cleavage by a reaction that is presumed to
involve metal-based decarbonylation and/or methyl group migration (Scheme 1, cleavage). The nascent methyl group is then
transferred to a corrinoid cofactor present on the
subcomponent,
which catalyzes subsequent methyl transfer to the substrate
H4SPt (Scheme 1, methyl transfer) (2). The
carbonyl group is oxidized to CO2 by a process involving
the
CO dehydrogenase subcomponent, with regeneration of the
reduced form of the
subunit. Previous studies on the
subunit
have focused on a C-terminally truncated form of the protein purified
from the native ACDS complex following partial proteolytic digestion
(2-4).
View larger version (18K):
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Scheme 1.
ACDS subcomponents and partial reactions in
overall cleavage of acetyl-CoA.
The genes encoding the five ACDS subunits are arranged together in an
operon along with one additional open reading frame in all species of
Methanosarcina and in certain other methanogens as well
(5-7, 9).2 The operon
structure is shown in Scheme 2, with the
designated genes and corresponding subunit molecular masses indicated
for M. thermophila TM-1. The additional open reading frame
encodes an accessory protein thought to be involved in nickel
insertion, and nickel is present in both the large CO dehydrogenase
subunit (CdhA) and in the
subunit (CdhC) containing the active
site for acetyl-enzyme formation. In addition to nickel, CdhA and CdhC also contain iron in the form of Fe/S clusters. Early indications that
the
subunit contains a unique Ni-Fe/S center, the spectroscopically designated A cluster, came from studies on detergent dissociation of
the clostridial CO dehydrogenase/acetyl-CoA synthase enzyme (CODH/ACS), an
2
2 heterodimer, with
subunit homologous to the
subunit from methanogens (10, 11). When
reacted with CO in the presence of a reducing agent, the isolated
clostridial
subunit exhibited an EPR spectrum similar to the A
cluster NiFeC signal found in earlier studies on the intact ACDS
complex and the native clostridial CODH/ACS enzyme (11).
Characterization of the ability of the ACDS
subunit to bind
acetyl-CoA and CoA, and to catalyze acetyl group transfer reactions by
way of a high energy acetyl-enzyme intermediate formed on the enzyme at
low redox potentials (3), and other studies (4), further implicated the
subunit A center as the site where acetyl C-C bond activation takes place. Functional preparations of the clostridial
subunit have now been obtained that catalyze acetyl-CoA formation from CoA and
CO in the presence of methylated clostridial corrinoid iron-sulfur
protein (12). Recently, a crystallographic structure of the clostridial
CODH/ACS enzyme was published in which the A center was shown to
contain an Fe4S4 cluster in close proximity to
a binuclear metal site containing nickel and another metal ion
suggested to be copper (13).
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The purpose of the present study was to characterize the role of the
subunit in cleavage of the acetyl C-C bond and to better understand the role of nickel at the active site. Therefore, we developed methods to generate large quantities of the protein using
recombinant techniques to overexpress the ACDS
subunit in
Escherichia coli under anaerobic growth conditions.
UV-visible spectroscopic analyses were employed to investigate the
kinetics and stoichiometry of nickel binding to the
subunit
apoprotein. The requirements for nickel were established for activity
in acetyltransferase, and for the ability to form the C-C bond of
acetyl-CoA in the absence of all other protein components. EPR analyses
were used to examine the role of nickel in forming a paramagnetic
A-center enzyme-CO adduct, with the non-requirement for reducing agent indicative of the oxidation state of nickel in the NiFeC species, relevant to indirect involvement of this species in the mechanism of
C-C bond activation. In efforts to define the nickel and Fe/S coordination environments site-directed mutagenic analysis was employed
to identify amino acid residues providing ligands to nickel and iron.
Additional insight into the structure and catalytic function of the
Ni-Fe/S active center was obtained from spectroscopic and enzymological
characterization of the mutants. The absolute requirement for nickel
and a defined role for the Fe/S center are incorporated in a mechanism
proposed for C-C bond activation that is consistent with the
properties of the Ni-Fe/S center described here and in accordance with
findings from previous investigations.
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MATERIALS AND METHODS |
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General Procedures-- Restriction enzymes were obtained from New England Biolabs. Unless otherwise indicated, all other chemicals were from Sigma and of the highest purity grade offered. All solutions were prepared using Milli-Q deionized water (Millipore Corp.). Anaerobic procedures were performed under an atmosphere of nitrogen containing 1-3% H2 using a Coy-type anaerobic chamber. Oxygen levels were monitored by a Teledyne model 3190 trace oxygen analyzer and were typically in the range of 0.5-2 ppm O2.
Overexpression and Purification of Wild Type and Mutant Forms of
the ACDS Subunit--
Previously we determined the complete
sequence of the ACDS operon from M. thermophila strain TM-1
(GenBankTM accession number AF173830). The gene for the
full-length ACDS
subunit (cdhC) and a truncated form of
the gene coding for a protein lacking 75 amino acids at the C terminus
(cdhC*) were overexpressed using a modified version of the
pQE60 (Qiagen) vector from which the His6 tag was deleted,
designated pQE60
His. Modifications to remove the His6
tag were as described previously (14). PCR amplifications of
cdhC and cdhC* were performed using genomic DNA
as template isolated from M. thermophila strain TM-1 with forward primers incorporating an NcoI site and reverse
primers containing a BamHI site. PCR products were initially
subcloned into the plasmid pCRII-TOPO (Invitrogen). After digestion
with NcoI and BamHI and purification by agarose
gel electrophoresis, the eluted fragments were cloned into the
pQE60
His vector, previously cut with NcoI and
BamHI. E. coli strain M15 [pREP4] was then
transformed with the pQE60
His expression constructs and used for
overexpression as described below. Site-directed mutagenesis of
cdhC* was performed using the QuikChange Multisite-directed
Mutagenesis kit, according to instructions provided by the manufacturer
(Stratagene). The sequences of all genetic constructs and bordering
regions were verified by sequencing of isolated plasmid DNAs using the
method of dideoxynucleotide termination with the ABI PRISM Big-Dye
Terminator cycle sequencing kit version 3.0 with AmpliTaq DNA
polymerase FS (Applied Biosystems).
Overexpression of the wild type and all mutant forms of the ACDS subunit was carried out by growth at 31 °C under strictly anaerobic
conditions in Luria-Bertani medium containing 1% glucose and 40 mM sodium fumarate as electron acceptor. Hydrogen gas
evolved during growth was allowed to escape through a 22-gauge syringe needle fitted to one of two ports constructed in the stopper used to
seal the culture vessels. A second port provided the means to remove
aliquots for monitoring the growth and for required additions. Cultures
were inoculated at 1:100 using an anaerobic starter culture grown
overnight. About 3 h later, after reaching an OD600 nm of
0.7-0.9, the cells were induced by addition of 0.4 mM isopropyl-
-D-thiogalactopyranoside
(Research Organics) and immediately supplemented with iron,
sulfide, and nickel, 100 µM
Fe(NH4)2(SO4)2, 200 µM Na2S, and 5 µM
NiCl2 added from sterile, anaerobic stock solutions. The
cultures were further supplemented after 2 and 4 h following
induction. These subsequent additions were needed to provide sufficient
iron, as was evident from small amounts of black iron sulfide that
remained in the final cell pellets under these conditions, but not when
either lower levels or fewer additions of iron and sulfide were
employed. Cells were harvested by centrifugation under anaerobic
conditions 5 h after induction (at an OD600 nm of around
2-2.5), and the cell paste was frozen in liquid nitrogen.
Crude buffer-soluble extracts were prepared by French press cell lysis
at 20,000 pounds/square inch at 4 °C of 6.5 g of cell paste
resuspended in 30 ml of 50 mM Tris·SO4, 25 mM Na2SO4, 10% glycerol, pH 7.5, and the lysate was centrifuged at 34,000 × g for 20 min at 4 °C. These steps as well as all others subsequently used for
purification of various forms of the ACDS subunit were performed
under anaerobic conditions. The supernatant obtained following
centrifugation was applied at ~1.6 ml/min to a 12 × 2.5 cm
diameter column of Q-Sepharose Fast Flow (Amersham Biosciences) equilibrated in buffer A (50 mM Tris·SO4, 25 mM Na2SO4, pH 7.5) at room
temperature. The column was then washed with ~270 ml of buffer A and
eluted with 400 ml of linear gradient of 0-0.4 M Na2SO4 in buffer A. Fractions (8 ml) were
collected and analyzed by SDS-PAGE and for UV-visible absorbance.
Fractions with the maximum absorbance ratio
A400 nm/A280 nm and
displaying the highest purity on SDS gels were pooled, concentrated by
ultrafiltration using an Amicon stirred cell, and diafiltered to remove
salt and low molecular mass contaminants. Samples were adjusted to
contain 10% glycerol prior to storage by freezing in liquid nitrogen. Protein was determined on the basis of absorbance at 280 nm using an
absorptivity coefficient for CdhC of 72,300 M
1 cm
1 (66,600 M
1 cm
1 for CdhC*) obtained from
the expression 1.03 × (5550
Trp + 1340
Tyr + 150
Cysnon-cluster) + 4500
CysFe/S-cluster,
modified from the original method of Perkins (15) to include the number of cysteine residues involved in Fe/S clusters in order to account for
absorbance contributed by Fe/S clusters. Comparable results were
obtained from protein measurements performed by the method of Bradford
(16) using bovine serum albumin as a standard. Metal analyses were
carried out by ICP-atomic emission spectroscopy using a Thermo
Jarrell-Ash 965 AtomComp plasma emission spectrometer on samples
submitted to the University of Georgia, Research Services, Chemical
Analysis Laboratory.
Nickel Reconstitution-- Purified recombinant CdhC at a concentration of 7-35 µM that contained iron but lacked nickel was reconstituted with Ni2+ by incubation under anaerobic conditions in the presence of 100 µM NiCl2 in 30 mM HEPES, pH 7.2, at room temperature. Aliquots were removed at different times, diluted with sufficient water to give 1.4 µM CdhC, and immediately assayed by transferring 10 µl of the diluted sample to 110 µl of acetyltransferase assay solution, and the reactions were completed as described below. Large scale reconstitution of the enzyme employed 60-80 µM CdhC* and 180 µM NiCl2 in reaction volumes of up to 12 ml. Sufficient time was allowed for metal incorporation (as determined in separate trials), and the reaction mixtures were thereafter concentrated to 1.3 ml by ultrafiltration on an Amicon YM30 ultrafiltration membrane, 44.5 mm diameter. Excess Ni2+ was removed by applying the concentrated enzyme to a 1.5-cm diameter, 10-ml bed volume column of Sephadex G-25 equilibrated in 40 mM HEPES, 10 mM Na2SO4, pH 7.2, and 1.0-ml fractions were collected. The protein was recovered in three brown fractions, which were combined and reconcentrated to yield a preparation of holoenzyme at a final concentration equal to that of the starting apoenzyme.
Enzymatic Assays of Acetyltransferase and Acetyl-CoA Synthesis
Catalyzed by the ACDS Subunit--
The ability of recombinant CdhC
to catalyze redox-dependent transfer of the acetyl group
from acetyl-CoA (acetyltransferase) was measured by a modified method
similar to that described previously (3). The reaction mixtures
contained 100 µM acetyl-CoA, 100 µM
3'-dephospho-CoA, 50 µM aquocobalamin, 4.0 mM
Ti3+-EDTA, 50 mM MOPS buffer, pH 7.2. Reactions
were carried out in 12 × 75-mm glass tubes at 25 °C and were
initiated by addition of a 10-µl aliquot of sufficiently diluted
protein to 110 µl of an assay solution that contained all the above
components at concentrations needed to give the final values indicated
in a total reaction volume of 120 µl. A series of reactions were
carried out for each enzymatic rate determination and stopped at
different time points (from 0 to 8 min) by addition of 120 µl of 2 mM TiCl3 in 0.5 M sodium citrate,
pH 4.0, and the mixtures were frozen in liquid nitrogen prior to
analysis by HPLC for the products CoA and
S-acetyl-3'-dephospho-CoA. HPLC analyses and calculation of
reaction rates were performed as described previously (3).
Net synthesis of acetyl-CoA by the recombinant ACDS subunit from
carbon monoxide, CoA, and methylcobalamin was measured in reaction
mixtures containing 0.65-3.9 µM nickel-reconstituted CdhC, 500 µM methylcobalamin, 100 µM CoA, 1 mM Ti3+-citrate in 50 mM MOPS
buffer, pH 7.2, under an atmosphere of 100% CO at room temperature.
Reactions were initiated by addition of CdhC and were followed over
time by removal of aliquots that were mixed with an equal volume of 2 mM TiCl3 in 0.5 M sodium citrate,
pH 4.0, frozen in liquid nitrogen, and subsequently analyzed by HPLC
for the formation of acetyl-CoA.
Titration of Nickel-deficient ACDS Subunit with Different
Metal Ions--
Metal ion incorporation into nickel-deficient CdhC* in
titration experiments was monitored spectrophotometrically on the basis of increase in absorbance around 336-340 nm observed due to
ligand-to-metal charge transfer band formation when the
nickel-deficient protein was exposed to different divalent metal ions
in the late 3d transition series. Titration of 16.4 nmol of CdhC*
contained in 590 µl of 50 mM HEPES, pH 8.0, was carried
out in a semi-microspectrophotometer cuvette by addition of 5.0-µl
aliquots of 1.20 mM stock solutions of divalent metal ions
(e.g. MnCl2,
Fe(NH4)2(SO4)2,
CoCl2, NiCl2, CuSO4). UV-visible
scans were recorded on a Hewlett-Packard 8452A spectrophotometer set up
inside the anaerobic chamber. After addition of each aliquot of
divalent metal solution, scans were recorded over time until no further
changes were observed in the spectrum.
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RESULTS |
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Overexpression and Purification of the ACDS Subunit--
The
gene encoding the
subunit of the ACDS complex from M. thermophila was overexpressed in two separate forms in E. coli. One of these was the full-length 472-amino acid protein,
CdhC, and the other was a 397-amino acid form truncated at the C
terminus, CdhC*. CdhC* includes all major regions of conservation among
subunit homologs and is about 30 amino acids smaller than the estimated size of the native protein isolated in truncated form following disruption of the ACDS complex by partial proteolysis (2, 3).
SDS-gel electrophoresis showed that similar amounts of CdhC and CdhC*
were produced over time after induction of E. coli cultures
grown under anaerobic conditions, as described under "Materials and
Methods." However, CdhC was considerably less soluble than CdhC* as
found by analysis of cell extracts and fractions from subsequent
purification procedures. Yields of the purified proteins were around 3 mg per g of cell paste for CdhC versus 20-22 mg per g of
cells for CdhC*. As shown in Fig. 1,
Q-Sepharose anion exchange chromatography of the supernatant obtained
from an extract of E. coli expressing CdhC* resulted in
elution of a major peak of protein with absorbance at 280 and at 400 nm
(due to Fe/S clusters), with fractions containing highly purified
CdhC*. The final preparation consisting of the pooled and concentrated peak fractions was ~90-92% pure as judged by densitometric analysis of SDS gels (Fig. 1, inset). Samples analyzed for metal
content by plasma emission spectroscopy contained 2.7 to 3.0 g
atom of iron per mol of CdhC*, but nickel was not detected, even though 5 µM NiCl2 had been added to the growth
medium.
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Activation of a Nickel-deficient Form of the Enzyme by
Reconstitution with Nickel--
Samples of purified CdhC and CdhC*
were assayed for acetyltransferase activity to determine their ability
to react with acetyl-CoA under low redox potential conditions
generating an acetyl-enzyme intermediate. As isolated, both recombinant
proteins showed very low levels of acetyltransferase activity, less
than 1% of the specific activity of the native subunit. However,
high activity was found for both proteins, CdhC and CdhC*, after
incubation with Ni2+. A progressive increase in activity
was observed over time, reaching a maximum after several hours of
incubation of CdhC, 7.1 µM, with NiCl2, 100 µM, as shown in Fig. 2.
Activation of the enzyme under these conditions in which
[Ni2+]
[apoCdhC] followed pseudo-first order
kinetics with an apparent half-time for activation of about 32 min.
Activity in the assay showed absolute dependence on low redox
potential, as expected, because it is a characteristic property of the
subunit isolated from the ACDS complex (3). Turnover rates for acetyl
transfer were up to 1250 min
1 for CdhC and 4500 min
1 for CdhC*, as compared with the value of 3100 min
1 (3) for the native
subunit isolated from the
ACDS complex.
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Plasma emission spectroscopic analyses on samples of the subunit
isolated from the ACDS complex and on samples from large scale nickel
reconstitution of CdhC* showed significant levels of iron and nickel
but only trace amounts of other metals. In particular, levels of copper
were extremely low, as shown in Fig. 3.
Notably, the measured iron/nickel ratio was 1.9 for
nickel-reconstituted CdhC*, and a similar value of 2.2 was observed for
the isolated
subunit. These results indicate that binding of nickel
is required for activation of the enzyme and that the enzyme contains
iron and nickel in a ratio of ~2:1. Therefore, direct
spectrophotometric methods were developed to examine further the
interaction of nickel with the
subunit.
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Binding of Nickel to the Subunit Results in Characteristic
UV-visible Changes and Proceeds with Defined Stoichiometry and
Kinetics--
Preparations of the brown, iron-containing,
nickel-deficient
subunit (CdhC and CdhC*) exhibited absorbance at
around 400 nm typical of a simple Fe/S protein with minimum values for
the ratio
A280 nm/A400 nm of
around 5.1. Upon addition of NiCl2 a marked,
time-dependent change was observed in the UV-visible spectrum of the recombinant protein resulting in a final spectrum closely resembling that of the subunit isolated from the ACDS complex.
Difference spectra obtained by subtracting the spectrum of the
apoenzyme from that of the nickel-reconstituted protein showed a
sharp peak of absorbance increase centered around 332-336 nm and a
broader peak at around 550 nm, as shown in Fig.
4A (middle panel).
A third peak with the highest intensity was found at 262 nm (not
shown). These features are attributed to ligand-to-metal charge
transfer absorption formed when nickel binds to the enzyme, with
multiple S ligation indicated by the high values of molar absorptivity. In addition, similar features were observed for the
enzyme after incubation with other divalent first row transition metals
including Co2+ and Cu2+, Fig. 4A
(top and bottom panels), with lower intensity
d-d transitions at 680 and 720 nm found in the spectrum of
the Co2+-substituted protein.
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Titration of the subunit apoprotein (CdhC*) was carried out with
various different metal ions using the changes in absorbance to monitor
the extent of binding. The results, shown in Fig. 4B, indicated that Co2+, Ni2+, and Cu2+
bind to the
subunit with end points all significantly greater than
1 eq. The values for Ni2+ and Cu2+ were close
to 2, indicating a stoichiometry of 2 g atom of nickel per mol of
CdhC*. Evidence for binding of Mn2+ and Fe2+
was also obtained; however, much higher levels were required to
saturate the enzyme, suggesting a weaker affinity for these metals.
Acetyltransferase assays were performed on samples removed after the
titrations and showed expected high levels of activity in the presence
of nickel; however, no activity could be detected with any of the other
metals (including manganese, iron, cobalt, and copper). These results
indicate that the enzyme is capable of binding other divalent metal
ions at the nickel sites, involving multiple thiolate ligation, but
that only nickel is able to generate the active enzyme.
The kinetics of nickel binding to the subunit were monitored
spectrophotometrically by following the increase in absorbance over
time after a single addition of excess Ni2+ to CdhC*. Under
conditions in which the concentration of CdhC* was significant relative
to [Ni2+], the reaction followed second order kinetics in
which the rate was dependent upon the concentration of both apoenzyme
and [Ni2+], with an apparent second order rate constant
of ~3 × 10
4 µM
1
min
1, as shown in Fig. 5.
These results agree with the time course for activation of the enzyme
in Fig. 2 in which [Ni2+]
[apoenzyme],
corresponding to pseudo-first order conditions, indicating that
activation is limited by the rate of nickel binding. Nickel
reconstitution at pH 7.2 (Fig. 5) was much faster than at pH 6.5 but
markedly slower than the reaction at pH 8.0. The magnitude of these
differences was consistent with deprotonation of more than one cysteine
thiol group in metal ion coordination and is in accordance with results
described later from site-directed mutagenesis.
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The ACDS Subunit Catalyzes Nickel-dependent Acetyl
C-C Bond Activation in the Absence of All Other Protein
Subunits--
Methods were developed to assay for net synthesis of
acetyl-CoA from one-carbon precursors using the purified recombinant
subunit as the only protein component. Reactions contained
nickel-reconstituted
subunit protein and CoA under an atmosphere of
CO, serving as the precursor of the carbonyl group of acetyl-CoA, in
the presence of methylcobalamin as a methyl group donor and
Ti3+-citrate as a reducing agent. The results of HPLC
analyses of samples removed over time showed that acetyl-CoA was indeed
formed in the absence of all other ACDS subunit proteins (Fig.
6). Besides methylcobalamin, methyl
iodide was also tested, but no evidence was found for activity of
methyl iodide as a methyl group donor. The rate of acetyl-CoA synthesis
(Fig. 6) was only about 1/3000th of the rate catalyzed by the native
ACDS complex. However, because the
subunit protein was expressed in
E. coli, which lacks ACDS and other CO dehydrogenase
proteins, these levels of activity cannot be due to contamination from
other ACDS proteins. Thus, the results reliably demonstrate that the
active site for acetyl-CoA cleavage and C-C bond activation resides on
the
subunit. Furthermore, as shown in Fig. 6, formation of
acetyl-CoA was observed only with nickel-reconstituted samples of the
protein and was undetectable with samples that had not been
preincubated with Ni2+, which demonstrates that nickel
is essential for net acetyl-CoA synthesis activity.
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Formation of a Characteristic A-cluster EPR Spectrum by Reaction of
the ACDS Subunit with CO Requires Ni2+ and Occurs in
the Absence of Reducing Agents--
Preparations of the isolated
subunit were tested both in the absence of nickel and after nickel
reconstitution for the formation of an EPR-detectable adduct in the
presence of CO. As shown in Fig. 7,
reaction of the nickel-reconstituted
subunit with CO generated a
strong signal with g values and power saturation
characteristics of an S = 1/2 system similar to those found for
the A-cluster NiFeC species observed in the native methanogen ACDS
complex (17, 18) and clostridial CO dehydrogenase/acetyl-CoA synthase
(19, 20). The signal was observed only in samples of the
nickel-reconstituted protein and was undetectable in preparations of
the nickel-deficient enzyme. Notably, exposure of the nickel-deficient
enzyme to CO resulted in rapid bleaching of Fe/S center absorbance,
which was not found for the holoenzyme. This finding indicates that CO
reacts with the Fe/S center in the apoenzyme causing its destruction in
the absence of nickel and suggests that nickel plays a structural role
in addition to its catalytic function, modulating the properties of the
Fe/S center and preventing permanent alteration by strong ligands such
as CO.
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In contrast to previous studies (11, 12), these results show that addition of a reducing agent was not required to form the NiFeC species. Samples identical to those employed in Fig. 7 were also prepared except that 1 mM Ti3+-citrate was included as a reducing agent during the reaction with CO. Under these conditions the NiFeC signal intensity was equivalent to that observed in the absence of reducing agent. These results indicate that reduction of the enzyme is not required for reaction with CO to generate the A center NiFeC signal and that the paramagnetic enzyme-CO adduct contains nickel in the Ni2+ oxidation state.
Site-directed Mutagenesis Indicates That Amino Acid Residues
Cys-278 and Cys-280 Function as Ligands to Nickel, and Cys-189 Is
Required for Fe/S Cluster Formation--
A comparison of available DNA
sequence data indicated that there are 6 cysteine, 3 histidine, and 2 tryptophan residues conserved in the subunit, with a number of
conserved proline residues found at positions in close proximity, as
shown in Scheme 3. To obtain information
on which of the histidine or cysteine residues may function as ligands
to nickel and/or iron, several single site-directed mutants of CdhC*
were prepared, including C189S, C278S, C280S, and H394N, and
overexpressed and purified as described for the wild type CdhC*. As
shown in Fig. 8, substitution of
asparagine for histidine at position 394, an invariant residue near the
end of the last region of conservation among all
subunit homologs, had little or no effect on the UV-visible spectrum of the protein or on
the process of nickel binding as judged from the difference spectrum
obtained following incubation with Ni2+. In contrast,
replacement of cysteine residues at either position 278 or 280 with
serine resulted in a pronounced alteration of the difference spectrum
with a complete lack of the peak at 550 nm and marked attenuation of
the absorbance around 330-340 nm (Fig. 8, right). This
demonstrates that both Cys-278 and Cys-280 are essential for proper
binding of nickel and indicates that coordination of nickel involves
thiolate ligation from both cysteine residues. Regardless of the major
effects on the formation of the nickel site, the UV-visible spectra of
the C278S and C280S mutants in the absence of nickel were virtually
identical to that of the nickel-deficient wild type protein (Fig. 8,
left), indicating in both mutants that an Fe/S center was
present with little or no structural variation. However, mutation of
Cys-189 resulted in a colorless protein (soluble and purified in high
yield) lacking an Fe/S center, as shown by the absence of absorbance
around 400 nm (Fig. 8, left). These results suggest that
Cys-189 is required for proper coordination of iron in the assembly of
the Fe/S cluster. Difference spectra recorded after addition of
Ni2+ were unique, showing that nickel was bound to the
C189S mutant with substantial thiolate ligation but in an altered form
compared with the wild type protein. This demonstrates that the
presence of the Fe/S center has a significant influence on the
coordination environment of the nickel-binding site.
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Acetyltransferase activity of the mutants was measured after incubation with Ni2+ and corresponded to the results from UV-visible spectroscopic analyses. No significant level of activity was found with the mutants C278S or C280S, both lacking a thiol group needed for coordination to nickel (Table I). The mutant C189S in which the Fe/S center is absent was also inactive. However, high levels of activity were found with the mutant H394N (Table I) which exhibited native Fe/S and nickel binding characteristics (Fig. 8), despite the fact that His-394 is highly conserved.
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DISCUSSION |
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To investigate the role of the ACDS subunit in activation of
the acetyl C-C bond, two forms of the protein were overexpressed in
E. coli under anaerobic growth conditions and purified and characterized. One of these was the 472-amino acid full-length ACDS
subunit CdhC, and the other was a truncated form of the protein lacking
75 amino acid residues at the C terminus designated CdhC*, designed to
mimic the form of the
subunit isolated previously by partial
proteolytic digestion of the ACDS complex (2, 3). CdhC* was obtained in
higher yields than CdhC and was considerably more soluble as indicated
by the finding that significant amounts of CdhC but not CdhC* were
found in the pellet obtained after centrifugation of E. coli
extracts and were present in cloudy fractions that did not bind to the
Q-Sepharose column used for purification. The increase in solubility
resulting from truncation of the C-terminal region is consistent with
previous findings that proteolytic removal of the C terminus takes
place at an early stage during dissociation of the ACDS complex by
limited proteolytic digestion (2). Loss of the C terminus also
correlates closely with a marked decline in the ability of the complex
to catalyze net synthesis of acetyl-CoA (see Fig. 4 in Ref. 2). Thus,
it is suggested that the C-terminal region may function as a docking domain in the ACDS complex needed for protein-protein interactions and
proper communication between subunits (while promoting the non-physiological aggregation of the full-length protein when expressed
unaccompanied by the other ACDS subunits).
All three proteins CdhC, CdhC*, and the native subunit isolated
from the ACDS complex had similar Fe/S cluster contents, as judged by
comparisons of their UV-visible spectra. Metal analyses showed that
CdhC and CdhC* lacked nickel under the conditions used for their
expression and purification, and both proteins were inactive in
catalysis of acetyl group transfer at low redox potential.
Nevertheless, high enzymatic activity was observed following incubation
with Ni2+ (Fig. 2) demonstrating that the
nickel-reconstituted recombinant enzyme is able to form an
acetyl-enzyme intermediate in reaction with acetyl-CoA and possesses
redox properties comparable with those of the native protein. Although
NiCl2 was added during expression, one explanation for why
nickel was not incorporated into the protein in E. coli
might be that levels of the metal ion were limited due to competition
for insertion into other nickel-containing proteins such as
hydrogenases needed for anaerobic growth. However, such competition
should be overcome at least in part by the large amounts of
subunit
protein formed (Fig. 1), and the finding of less than 1% active
protein with an undetectably low level of nickel suggests that other
factors, lacking in E. coli, are needed for specific
incorporation of nickel into the methanogen
subunit. Indeed, the
strong dependence on Ni2+ concentration in the apparent
second order kinetics (Fig. 5) indicates that the rate of nickel
insertion would be too low to support formation of the active enzyme
under physiological conditions of pH and free Ni2+
concentration. Studies are now in progress to determine whether nickel
incorporation in vivo may involve the putative nickel
insertion accessory protein encoded by the ACDS operon.
Acetyl C-C Bond Activation--
Direct evidence that the ACDS subunit contains the site for activation of the acetyl C-C bond was
obtained from the finding that significant levels of acetyl-CoA were
formed in reactions of the
subunit with CoA, CO, and
methylcobalamin in the absence of all other ACDS protein components. As
shown in Fig. 6, at low redox potential acetyl-CoA was generated over
time from CoA and the one-carbon precursors CO and methylcobalamin.
Previously, it was shown unequivocally that the ACDS
subunit is
responsible for activation of the C-S bond of acetyl-CoA (2, 3);
however, only indirect evidence for activation of the C-C bond was
available (2, 4). The results presented here demonstrate for the first time that the
subunit active site indeed has the intrinsic chemical properties needed for C-C bond activation. These results are in agreement with recent findings on the homologous subunit from the
clostridial CODH/ACS enzyme (12) and further show that the
subunit
is capable of interacting with the free methyl-B12
cofactor, not bound to a corrinoid protein. This indicates that the
subunit forms a complex with the corrinoid cofactor, to a significant degree, even in the absence of corrinoid protein. Binding of methylated corrinoid would thereby increase the effective concentration of methyl
groups at the active site; thus, the failure to form acetyl-CoA in
reactions using methyl iodide as methyl donor may result from an
inability of methyl iodide to bind to the enzyme. If methylation of the
enzyme is rate-limiting to begin with, then inefficient binding of a
methyl donor would be especially detrimental.
Absolute Requirement for Nickel--
Binding of nickel to the subunit was a conditio sine qua non for each of the four
major characteristics investigated as follows: acetyltransferase
activity involving formation of an acetyl-enzyme intermediate (Fig. 2);
the ability to carry out C-C bond activation as indicated by net
synthesis of acetyl-CoA (Fig. 6); formation of an EPR-detectable
enzyme-CO adduct (Fig. 7); and the ability to generate a UV-visible
spectrum characteristic of the native enzyme (Fig. 4A and
Fig. 8). The binding of nickel occurs with a stoichiometry of 2 eq/mol
protein, as shown by values of the iron/nickel ratio close to 2:1 by
inductively coupled plasma-atomic emission spectroscopy metal analyses
of the native and recombinant proteins (Fig. 3) and by direct titration
of the apoenzyme (Fig. 4B). Divalent metal ions other than
Ni2+ exhibited comparable stoichiometries in titrations and
generated UV-visible spectral changes related to those produced by
Ni2+. However, none of the other metal ions tested
(Mn2+, Fe2+, Co2+, and
Cu2+) was able to substitute for nickel in formation of a
catalytically active enzyme. This all-or-none quality indicates that
remarkably specific electronic structure and reactivity are demanded
for the metal ion to participate successfully in the catalytic process, properties possessed only by nickel.
Ni-Fe/S Center--
The recombinant subunit
expressed in E. coli contained all of the ligands needed
for nickel binding to generate a functionally active Ni-Fe/S site,
because no additions other than NiCl2 were required to
reconstitute activity. Multiple thiolate ligation to each nickel was
indicated by the high values of molar absorptivity of the charge
transfer bands formed upon nickel reconstitution. Characteristic
UV-visible difference spectra obtained for binding of three different
metals (Co2+, Ni2+, and Cu2+) also
implied multiple thiolate ligation, consistent with findings from
extended x-ray absorption fine structure (EXAFS) studies on the
related clostridial CODH/ACS
metallosubunit of nickel coordination
by more than one sulfur ligand (21, 22). The strong effect of pH on the
rate of nickel incorporation was also consistent with deprotonation of
more than one thiol group in the process of nickel binding, and
site-directed mutagenesis (Fig. 8 and Table I) identified Cys-278 and
Cys-280 as ligands potentially involved in direct coordination to
nickel. In addition, the results may be viewed in relation to the
recently published crystallographic structure of the A center of
clostridial CODH/ACS (13), in which an Fe4S4
cluster is bridged to a binuclear metal site with two cysteine residues
in shared coordination to both metal ions, corresponding to Cys-278 and
Cys-280 in the methanogen
subunit. The binuclear center of CODH/ACS
contained nickel, but the metal ion proximal to the Fe/S center was
suggested to be copper. In contrast, our results indicate that copper
is not a component of the Ni-Fe/S center in the
subunit, because
only trace levels of copper are detectable (Fig. 3). Rather, the data
presented here are consistent with a structure for the Ni-Fe/S center
similar to that of the clostridial CODH/ACS, but containing a binuclear
metal site with nickel residing at both positions in an
Ni2[Fe4S4] arrangement.
In some respects the Fe4S4 and nickel-nickel subsites appear to act relatively independently, e.g. altered nickel binding in the C278S and C280S mutants had little effect on the spectrum of the Fe/S center itself (Fig. 8). However, a notable exception was revealed in reactions with CO, which provided evidence for interaction of the nickel and Fe/S sites. We found that CO caused a rapid bleaching of the nickel-deficient enzyme, not observed in reactions with the nickel-reconstituted protein, suggesting decomposition of the Fe/S center in the absence of nickel by reaction of the strong ligand CO with iron in the Fe/S center. This provides the first evidence to date that nickel modulates the reactivity of the Fe/S center and would be consistent with bridging of the Fe/S center to nickel at the proximal site, by analogy to the A center structure in CODH/ACS (13).
Site-directed mutagenesis experiments also provided information on groups involved at the Fe/S center. The complete absence of an Fe/S center in the C189S mutant (Fig. 8) implied that Cys-189 is directly coordinated to one of the iron atoms in the Fe/S center. Although mutations that strongly affected the binding of nickel had marginal effects on the UV-visible spectrum of the Fe/S center, the absence of an Fe/S center in C189S strongly influenced the nickel-binding characteristics as observed by the pronounced alteration of the difference spectrum formed upon addition of Ni2+ (Fig. 8). These changes may result from local conformational rearrangements and displacement of groups normally involved in coordination of nickel. Alternatively, the altered nickel binding properties in the C189S mutant may reflect the loss of interaction between the Fe/S cluster and the nickel-nickel site needed for proper coordination of the binuclear nickel center and formation of its characteristic UV-visible spectrum. The close proximity of Cys-189 to Cys-192 indicates that Cys-192 also may be coordinated to the Fe/S cluster, and these residues correspond to two of the four cysteines that are coordinated to the Fe/S center in the structure of the A center in CODH/ACS (13). Because an Fe/S cluster is not formed even partially in the C189S mutant, Cys-189 along with another group such as Cys-192 may provide the site of nucleation for the initial step in Fe/S cluster insertion.
Mechanism of C-C Bond Activation--
The mechanism of acetyl
C-C bond activation by the subunit can be interpreted on the basis
of the present results together with previous findings on the
subunit isolated from the methanogen ACDS complex and information
available from studies on the homologous subunit from clostridial
CODH/ACS. It has been established that activation of the enzyme by
one-electron reduction is required for acetyltransferase activity,
involving reaction with acetyl-CoA to form an acetyl intermediate. The
enzyme nucleophile needed to generate this intermediate is depicted as
Ni(I) in Scheme 4. The Ni(I) species
would be formed by reduction of one of the nickel ions at the binuclear
site in the as-isolated, all Ni(II)-enzyme. Formation of the
acetyl-enzyme intermediate would occur by attack of Ni(I) on the
carbonyl group of acetyl-CoA. In the case of the copper-substituted
enzyme, reduction to the Cu(I) level with its d10 closed shell configuration may be readily
accomplished; however, subsequent action of Cu(I) as a nucleophile
would be highly unfavorable. Therefore, it is not surprising that the
copper-enzyme is completely inactive. Reduction of the
Co(II)-substituted enzyme would be substantially more difficult, but if
a Co(I) species was formed, then the ensuing nucleophilic attack would
yield an acetyl-Co(III)-enzyme potentially too stable to undergo
rapidly the reverse reaction, i.e. attack by CoA (or homolog
3'-dephospho-CoA) as required for the overall exchange. Thus, Ni(I)
provides sufficient nucleophilicity without generating unreactive
products, and the indicated Ni(III) intermediates should be relatively
high energy species. Accordingly, we propose that the role of the Fe/S
center is to stabilize the nascent Ni(III) adducts formed in reactions
a and b (Scheme 4) by converting them to the more
stable Ni(II) forms. This would also serve to maintain electron density
on nickel, as required for interaction with the
-acid acceptor CO in
the step of carbonylation/decarbonylation involving migration of the methyl group, reaction c. In addition to migratory steps that may occur within the coordination sphere of a single nickel, the
presence of a binuclear nickel-nickel center opens the possibility for
migration of species between the two nickel ions distinguished by
different coordination environments. The overall cleavage of acetyl-CoA
would require the reverse of reaction a, i.e. transfer of the methyl group from the
subunit to the corrinoid protein by nucleophilic attack of the Co(I) corrinoid on the bound methyl group. Similarly, the reverse of reaction b is needed for acetyltransferase activity through nucleophilic attack of CoA on
the
subunit acetyl group. The Fe/S center in its oxidized form
would facilitate both processes, generating the corresponding reactive
Ni(III) species by accepting an electron in the equilibrium internal
electron transfer steps indicated in Scheme 4.
|
The findings from EPR analyses of reactions of the subunit with CO
are relevant for understanding the role of the paramagnetic NiFeC
species in the mechanism of C-C bond activation. In Scheme 4, reaction
d, CO is indicated to react with the enzyme at the Ni(II)
level, forming an EPR active species not directly involved in the
catalytic cycle. This is based on the finding that the active enzyme
obtained following reconstitution with NiCl2 contains
nickel in the Ni(II) form and that addition of a reducing agent was not
needed to generate the NiFeC EPR signal (Fig. 7). This finding
contrasts with the apparent requirement for reducing agent reported for
NiFeC signal formation in the isolated
metallosubunit from
clostridial CODH/ACS (11, 12). The fact that reduction is not required
to form the EPR signal with the ACDS
subunit raises questions about
the relevance of proposed species such as Ared-CO and
Ni+-CO. Theoretically, one-electron reduction of the
paramagnetic enzyme-CO adduct could convert it directly to an EPR
silent form involved in the catalytic cycle. However, we found that the
EPR signal intensity was unchanged by the presence of a strong
reductant (1 mM Ti3+-citrate). This indicates
that disappearance of the NiFeC signal upon reaction with an
appropriate methyl donor or during catalytic turnover (8, 18, 23) takes
place by the reversal of reaction d, with the shift in
equilibrium becoming favorable only in the presence of substrates.
In conclusion, the present work highlights the importance of the subunit in the overall process of acetyl-CoA cleavage by the methanogen
ACDS complex. The focus on characterization of the spectroscopic and
catalytic properties of nickel as part of an unusual Ni-Fe/S cluster at
the active center of the
subunit emphasizes its role in activation
of the acetyl C-C bond.
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FOOTNOTES |
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* This work was supported by National Science Foundation Grant MCB-0215160, the United States Department of Energy Grant DE-FG02-00ER15108, and the Uniformed Services University of the Health Sciences Grant R071GM.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Uniformed Services University of the Health
Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814-4799. Tel.:
301-295-3555; Fax: 301-295-3512; E-mail: dgrahame@usuhs.mil.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M210484200
2 M. barkeri Fusaro Genome Project, the United States Department of Energy Joint Genome Institute (www.jgi.doe.gov).
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ABBREVIATIONS |
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The abbreviations used are: ACDS, acetyl-CoA decarbonylase/synthase; H4SPt, tetrahydrosarcinapterin; MOPS, 3-(N-morpholino)propanesulfonic acid; HPLC, high pressure liquid chromatography; CODH/ACS, CO dehydrogenase/acetyl-CoA synthase.
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