From the Department of Biochemistry, Beadle Center, University of Nebraska, Lincoln, Nebraska 68588-0664
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ABSTRACT |
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This paper focuses on how a methyl group is
transferred from a methyl-cobalt(III) species on one protein (the
corrinoid iron-sulfur protein (CFeSP)) to a nickel iron-sulfur cluster
on another protein (carbon monoxide dehydrogenase/acetyl-CoA synthase).
This is an essential step in the Wood-Ljungdahl pathway of anaerobic CO
and CO2 fixation. The results described here strongly
indicate that transfer of methyl group to carbon monoxide
dehydrogenase/acetyl-CoA synthase occurs by an SN2 pathway.
They also provide convincing evidence that oxidative inactivation of
Co(I) competes with methylation. Under the conditions of our anaerobic
assay, Co(I) escapes from the catalytic cycle one in every 100 turnover
cycles. Reductive activation of the CFeSP is required to regenerate
Co(I) and recruit the protein back into the catalytic cycle. Our
results strongly indicate that the [4Fe-4S] cluster of the CFeSP is
required for reductive activation. They support the hypothesis that the
[4Fe-4S] cluster of the CFeSP does not participate directly in the
methyl transfer step but provides a conduit for electron flow from
physiological reductants to the cobalt center.
The Wood-Ljungdahl pathway allows anaerobic microbes to grow with
CO or CO2 as their sole carbon source (1-4). This pathway, which has been most extensively studied in the acetogenic bacterium, Clostridium thermoaceticum, allows microbes to generate
three molecules of acetyl-CoA from a single molecule of glucose. A
cobamide-dependent methyl carrier protein, the CFeSP, is
involved in two methyl transfer steps in this pathway (Fig. 1). It
accepts the N5 methyl group of methyl-tetrahydrofolate
(CH3-H4folate)1
in a reaction catalyzed by a CH3-H4folate/CFeSP
methyltransferase (MeTr) to form methyl-cob(III)amide (5, 6). Co(I) is
the nucleophilic species that attacks the methyl group of
CH3-H4folate. Then the methyl group is
transferred from the CFeSP to CODH/ACS, where acetyl-CoA is assembled
from the methyl group, CO, and CoA (7, 8). The catalytic cycles
involving the CFeSP (Fig. 1) are thought to involve shuttling between
the methyl-Co(III) and Co(I) states.
Two hypotheses guide the experiments described in this paper. The first
hypothesis is that methylation of the CFeSP competes with oxidative
inactivation of Co(I) to the Co(II) state. There is substantial
evidence that oxidation of Co(I) to Co(II) competes with catalytic
turnover and leads to inactivated enzyme (Fig. 1). The Co(II/I) midpoint potential
(Em) is below
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
500 mV (9). Thus, when the CFeSP is
purified, it is in the inactive Co(II) state (9, 10). CODH and CO,
pyruvate ferredoxin oxidoreductase and pyruvate, and reduced ferredoxin
can activate the CFeSP and convert it to the Co(I) state (10). The
reductive activation hypothesis is consistent with the observations
that (i) the rate of Co(I) decay equals the rate of methyl-Co(III)
formation during MeTr-catalyzed methylation of the CFeSP by
CH3-H4folate and (ii) the rate of Co(I)
formation equals the rate of methyl-Co(III) decay in the reverse
reaction (methylation of H4folate) (6). The reductive
activation reaction is thought to be crucial for survival of the cells.
This is because the Em of the Co(II/I) couple is low
enough that, even under the anaerobic conditions required for growth of
acetogens and methanogens, the CFeSP is expected to undergo oxidative
inactivation sporadically. There is strong evidence that the
cobalamin-dependent methionine synthase also requires the Co(I)
state for catalytic turnover (11, 12). This protein does not contain an
Fe-S cluster but uses an
S-adenosyl-L-methionine-dependent reactivation mechanism (13-15).
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Fig. 1.
Methylation reactions involving the
CFeSP. The CFeSP is methylated by
CH3-H4folate and demethylated by CODH/ACS.
Methylation of the Co(I) center of the CFeSP competes with oxidative
inactivation. The G0 is from Ref. 31. Fd indicates
ferredoxin.
The second hypothesis is that reductive reactivation of the Co(II)
center requires the FeS cluster of the CFeSP to communicate with
physiological reductants. The large 55-kDa subunit of the CFeSP
contains a [4Fe-4S] cluster (10). This was the first
"B12" protein identified that also contains an
iron-sulfur cluster. Subsequently, a similar CFeSP was purified from
Methanosarcina thermophila, where it is involved in the
conversion of acetyl-CoA to methane (16). Analogs have been identified
in the genome sequences of Methanobacterium
thermoautotrophicum (17), Methanococcus janaschii (18),
and Archaeoglobus fulgidus (19). What is the role of the
cluster? Several years ago, we proposed that the [4Fe-4S] cluster
facilitates the conversion of Co(II) to Co(I) (9, 10). This proposal is
supported by recent studies of a C20A variant of the CFeSP in which the
4Fe cluster (Em = 523 mV) is converted to a 3Fe
cluster (Em =
31 mV) (20). This 500-mV increase in
the midpoint potential severely cripples the CFeSP's ability to be
activated by physiological electron donors like CODH/ACS or reduced
ferredoxin. These studies led to the hypothesis that the low potential
4Fe cluster relays electrons from the CODH subunit of CODH/ACS to the
cob(II)amide CFeSP, thereby facilitating its reactivation to Co(I).
Optimal conditions for studying acetyl-CoA synthesis from CO,
CH3-H4folate, and CoA have been established
(21). The reaction requires MeTr, the CFeSP, and CODH/ACS. Ferredoxin
stimulates the reaction 4-fold. Under these conditions, the
Co(II)-CFeSP must be reduced to Co(I) before it can enter the catalytic
cycle. In the studies described here, we monitored the synthesis of
acetyl-CoA from CO, CH3-H4folate, CoA, and the
methylated form of either the wild type or C20A variant CFeSP. If the
two hypotheses described above are correct, then, when the C20A variant
of the methylated CFeSP is used, the rate of acetyl-CoA synthesis will
slowly decrease at the rate of oxidative inactivation of the cobalt
center. Furthermore, we can measure the UV-visible spectra of the C20A
variant during and after the reaction to directly determine if Co(I)
forms during the linear phase of the reaction and if Co(II) forms when
the reaction has undergone inactivation. These results can be compared with those for the wild type protein. According to our hypotheses, when
the wild type protein is used, this inactivation will not be observed
because CODH and CO2 will
rapidly reduce the Co(II) protein, recruiting the inactive protein back
into the catalytic cycle.
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EXPERIMENTAL PROCEDURES |
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Materials-- N2 (99.98%) and CO (99.99%) were obtained from Linweld (Lincoln, NE). N2 was deoxygenated by passing through a heated column containing BASF catalyst. Reagents were of the highest purity available. 14CH3-H4folate was purchased from Amersham Pharmacia Biotech.
Organism and Enzyme Purification-- Construction of the C20A CFeSP variant, cloning the gene into Escherichia coli, and reconstitution of the protein with B12 and the FeS cluster was described earlier (20). The wild type (10) and variant (20) CFeSPs were purified under strictly anaerobic conditions as described. CODH/ACS (22), ferredoxin II (23), and MeTr (6) were purified as described under strictly anaerobic conditions at 17 °C in a Vacuum Atmospheres chamber maintained below 1 ppm oxygen. Protein concentrations were determined by the Rose Bengal method (24).
Enzyme Assays-- The wild type and variant CFeSPs were methylated with methyl iodide essentially as described earlier (8).3 The as-isolated protein was first reduced by reacting with 10 mM titanium(III) citrate. The solution was then incubated for 15-30 min at 13 °C with 20-fold excess 14CH3I and centrifuged through a Sephadex G-50 column (26) to remove the unreacted methyl iodide and the titanium citrate. Therefore, methyl iodide, which inhibits CODH/ACS, was absent from reactions involving the methylated CFeSP.
The reaction of CH3-H4folate, CO, and CoA to
form acetyl-CoA was performed as described previously (21). Under these
conditions, the concentration of CODH/ACS is rate-limiting. The
reaction was performed in the dark in a glass V-shaped reaction vial
capped with a red rubber serum stopper. Details of the reaction mixture are given in the Fig. 2 legend. The reaction was quenched at various times by removing 5-µl aliquots into 5 µl of 2.2 N
perchloric acid. The amount of acetyl-CoA formed was measured by Dowex
50W-H+ chromatography as described (27). UV-visible spectra
were obtained on an OLIS-modified Cary 14 spectrometer. The data were
then analyzed using the program KINSIM (28, 29) and modified by Gary
Xin Hua and Dr. Bryce Plapp of the University of Iowa. The input rate constants are given under "Results." The simulated data were
plotted using SigmaPlot (Jandel Scientific, San Rafael, CA).
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RESULTS |
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Synthesis of Acetyl-CoA from the Methylated CFeSP,
CH3-H4folate, CO, and CoA--
We measured the
synthesis of acetyl-CoA from CH3-H4folate, CO,
and CoA using the methylated CFeSP as the methyl carrier
protein. If the variant CFeSP is used, this reaction is very slow,
since the protein is in the Co(II) state and cannot be activated by physiological reductants (20). In the reaction studied here, the first
turnover generates cob(I)amide, which undergoes remethylation by
CH3-H4folate or oxidation to cob(II)amide. When
the wild type methylated CFeSP is used, acetyl-CoA synthesis continues
linearly with time until the limiting reagent is depleted. The
concentration of CO (1 mM) is limiting in these reactions.
With the C20A variant, the rate of acetyl-CoA synthesis initially is
the same as with the wild type protein and then slowly decreases and
becomes negligible after ~100 turnovers (Figs.
2 and
3A, inset). This
reaction was performed using three different concentrations of
methylated CFeSP. The data with the variant proteins were fit to single
exponential equations to estimate the inactivation rate constants (Fig.
2, solid lines; Table
I). These rate constants were inversely
proportional to the CFeSP concentration. The rate constant for
reactivation of the mutant protein (kact = 0.0015 s1) has been measured earlier (20). It is
30-90-fold lower than these rates of inactivation. The UV-visible
spectrum of the variant CFeSP after 40 min, when the reaction is fully
inhibited and acetyl-CoA is no longer being formed, is characteristic
of the cob(II)amide state (Fig. 3A).
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These results demonstrate that reductive activation is required to
regenerate the active form of the CFeSP. After it converts to the
Co(II) state, the C20A variant cannot be reactivated at significant
rates by its physiological electron donors (CODH and CO), because its
high potential 3Fe-4S cluster cannot provide the driving force to
reduce Co(II) to Co(I). The wild type protein undergoes reactivation
over 4000-fold faster (kact = 0.88 s1) than the mutant, so the reaction continues unabated
until the substrates are depleted. Therefore, a low potential Fe-S
cluster in the CFeSP is required for reductive activation of the CFeSP by physiological electron donors.
Single Turnover Kinetics of the Variant Methylated CFeSP--
When
the wild type methylated CFeSP is reacted with CODH/ACS, a low
potential metal center (cluster A) on ACS undergoes methylation as the
CFeSP is converted to the Co(I) state (25). It remained a possibility
that there is some degree of radical chemistry in this reaction (see
below for discussion). We considered that disabling the electron
transfer pathway to the cobalt center might uncover or shift the
predominantly SN2 mechanism to a radical pathway. If so,
the initial cobamide product of the methyl transfer reaction would be
cob(II)amide, instead of cob(I)amide. We mixed the methylated C20A
variant CFeSP and CODH/ACS in the presence of CoA and CO and followed
the spectrum of the reaction mixture. The broad absorption band from
methyl-Co(III) at 450 nm decreases as the intensity of the 390-nm peak
from cob(I)amide increases (Fig. 3B). The reaction yields
clean isosbestic points, clearly demonstrating that demethylation of
the variant methyl-CFeSP (like the wild type protein) by CODH/ACS is
accompanied by conversion of CH3-Co(III) to Co(I). The
final absorption changes at 390 nm yield a difference extinction
coefficient () of 17 mM
1
cm
1, which indicates complete conversion of
methyl-Co(III) to Co(I). The rate of Co(I) formation is 0.21 µM min
1, which yields a
kcat for CODH/ACS under these conditions of 1 min
1. This is similar to values measured earlier under
these conditions of pH and ionic strength (21). As described above
(Fig. 2B), Co(I) undergoes oxidation to Co(II) over a longer time.
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DISCUSSION |
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The Wood-Ljungdahl pathway of acetyl-CoA synthesis is unusual in
that the so-called "Western half" (1) of the pathway occurs through
enzyme-bound intermediates. The first of these is methylcobamide, formed by transfer of the methyl group from
CH3-H4folate to the CFeSP. Studies of the
C. thermoaceticum MeTr and an analogous reaction catalyzed
by the E. coli methionine synthase strongly indicate that
this reaction occurs by an SN2 mechanism (6, 20, 30). The
results described here clearly show that methylation of Co(I) competes
with oxidative inactivation. The rate constant for Co(I) oxidation,
calculated by fitting the data shown in Fig. 2 to an exponential, is
0.05 min1. The competition between oxidative inactivation
and methyl transfer can also be measured from these data. As shown in
Fig. 3A, there is one inactivation event per 100 turnovers,
where one turnover consists of 1 mol of acetyl-CoA formed. These two
values are internally consistent, since the inactivation rate is
100-fold lower than the turnover number for the acetyl-CoA synthase
reaction (60 min
1 at pH 5.8). A variety of conditions can
affect this rate constant. For example, if the protein is exposed to
oxygen, the inactivation rate constant increases dramatically.
Oxidation of Co(I)-CFeSP with an air-saturated buffer occurs with a
rate constant of >300 s
1.4 However,
acetogenic bacteria growing in their natural habitats would rarely
experience such high concentrations of oxygen. If the conditions of our
assay approximate physiological conditions, the need to extract one
electron equivalent from reduced CODH or ferredoxin per 100 mol of
acetyl-CoA formed would not be overly demanding.
We observe a striking decay in the rate of acetyl-CoA synthesis when
the C20A variant CFeSP is used as the methyl carrier protein (Fig. 2).
We can estimate the inactivation rate constant by fitting the data to
an exponential equation. To analyze the data more quantitatively, we
simulated the progress curve. This procedure requires inputting the
relevant rate constants and concentrations of substrates and enzymes
for each reaction in this multistep process. We have determined all of
the elementary reaction rates and the steady-state kinetic parameters
for the MeTr5 and the CODH
(32) reactions. We also have determined the rate of formation of Co(I)
from Co(II) for the wild type and C20A CFeSP (20). However, the full
set of rate constants for acetyl-CoA synthesis from the methylated
CFeSP, CO, and CoA have not yet been measured. A full simulation that
included all of these steps would contain about 40 rate constants. We
made several simplifying assumptions to wrap all of these reactions
into three steps (Scheme 1). The first
step is the MeTr-catalyzed methylation of the CFeSP (B + E C + E). The CFeSP and
methyl-CFeSP concentrations are below their Km
values, and the CH3-H4folate concentration is
much higher than its Km value. Therefore, the rate constants for the methylation the CFeSP and demethylation of the methyl-CFeSP by MeTr can be substituted by the
kcat/Km values for the CFeSP and the
methylated CFeSP, respectively. Since the
CH3-H4folate concentration (2.5 mM)
is much higher than its Km (10 µM)
value in these reactions, we also omitted this term from the
simulation. In addition, we simplified the acetyl-CoA synthesis steps
by summarizing them into a single reaction (C + F + G
B + F + H). Since
the CoA and CO concentrations are significantly above their
Km values, the rate of regeneration of Co(I) by
CODH/ACS is governed by the
kcat/Km for the methylated
CFeSP. The kcat for acetyl-CoA synthesis is
approximately 1 s
1. This was an adjustable parameter. Our
first estimates used a Km value of 10 µM for the methylated CFeSP, which is similar to that for
MeTr. Furthermore, acetyl-CoA synthesis is irreversible at these high
concentrations of CO and CoA, so the reverse rate constant is set to 0. For the third step (B
B'), the rate of reactivation of Co(II) to
Co(I) is 0.88 s
1 for the wild type and 0.0015 s
1 for the C20A variant (20) CFeSP. These simplifying
assumptions allowed us to focus attention on the oxidative inactivation
and reductive activation reactions and quantitatively test the
reductive activation hypothesis.
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After supplying the above rate constants, we input rate constants for
inactivation of Co(I) (beginning with those derived from the
exponential fits as a starting point) into the KINSIM program. The
dashed lines in Fig. 3 are the simulated progress curves for the reactions performed at three concentrations of the
methylated CFeSP. The simulated progress curves fit the data well,
indicating that our simplifying assumptions are reasonable. To better
understand the factors that control whether the CFeSP will undergo
oxidative inactivation, reductive reactivation, or methylation, we used
KINSIM to output the concentrations of the three states of the CFeSP,
Co(I), Co(II), and methyl-Co(III) for the mutant and wild type
proteins. Fig. 4 shows these species for
the reaction shown in Fig. 2A with 8 µM CFeSP.
The pre-steady state reaction is identical for the mutant and the wild
type proteins. With the wild type protein, the concentration of Co(II)
quickly reaches a value that is about 2% of the total CFeSP present
and slowly decays to about 0.4%. The predominant species at the end of
the reaction is methyl-Co(III). However, with the C20A mutant, Co(II)
increases in an exponential fashion as Co(I) decays. At the end of the
reaction, there is a mixture of methyl-Co(III) (32% of the total
CFeSP) and Co(II) (58% of the total). In both reactions, there is a
significant amount of methyl-Co(III) at the end of the reaction,
because the CH3-H4folate concentration is
higher than that of CO. The essential parameters are the relative rates
of inactivation relative to the rates of reactivation and Co(I)
methylation. With an inactivation rate constant of about 0.1 min1, the reaction profile begins to deviate from
linearity even when the reactivation rate is as high as the
inactivation rate constant. Interestingly, increasing the reactivation
rate constant above the measured value has minimal effect on the
progress curve. For example, increasing the reactivation rate 10-fold
(to 8.8 min
1) increases the slope of the reaction's
progress curve by only 3%. This indicates that the environment and
location that nature has selected for the FeS cluster in the CFeSP is
nearly optimal for its role in reductive activation.
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The redox potential for the [4Fe-4S]2+/1+ couple is about 20 mV more negative than that of the Co(II)/(I) couple and is nearly equal to that of the CO2/CO couple. The design of a low potential cluster is clearly important; however, based on a purely electrochemical argument, one would expect CO to reduce the Co(II) state about as easily as the cluster 2+ state. However, the requirement for the cluster in reductive activation of Co(II)-CFeSP by physiological electron donors is nearly absolute. The cluster is located in a separate subunit from the cobamide. Solving the crystal structures of the CFeSP, MeTr, and CODH/ACS would greatly enhance our understanding of how these proteins interact in electron and methyl transfer reactions.
The studies described here also provide insight into the mechanism of methyl group transfer from the methylated CFeSP to CODH/ACS. There are three possible mechanisms (33). The first is heterolytic cleavage of the Co-C bond by an SN2-type mechanism involving attack by a nucleophilic center of ACS (Mechanism I). This nucleophilic center has not been unambiguously identified. Evidence from our laboratory, summarized in recent reviews (1, 2, 34, 35), and from Lindahl's laboratory (7) indicates that the nucleophile is the nickel site of cluster A. We favor a mechanism involving a Ni(I) nucleophile; however, Barondeau and Lindahl (7) favor a Ni(II) nucleophile. A second possibility (Mechanism II) is a homolytic mechanism in which electron transfer from the reduced Fe-S cluster of the CFeSP would form a methyl-Co(II) species that would disproportionate to form Co(I) and methyl-nickel.6 A third possibility (Mechanism III), that the CH3-Co(III) species is cleaved homolytically to form a CH3-Ni species and Co(II), is inconsistent with stopped flow studies of the wild type CFeSP. Co(I) forms at the same rate that methyl-Co(III) decays, apparently ruling out a Co(II) intermediate (36). Mechanisms II and III invoke transfer of a methyl radical to CODH/ACS.
Methylation of CODH/ACS has been modeled using compounds such as CH3-Co(dmgBF2)py (where dmgBF2 represents (difluoroboryl)dimethylglyoximato and py represents pyridine) as the methyl group donor complex and Ni(tmc)+ (where tmc represents 1,4,8,11-tetramethyl-1,4,8,1-tetraazacyclotetradecane) as the methyl acceptor (37, 38). Two equivalents of the Ni(I) complex were required, one to reduce methyl-Co (III) to methyl-Co(II) and the other to capture the methyl radical generated upon cleavage of the methyl-Co(II) species. Thus, studies of the inorganic model system support Mechanism II.
On the other hand, several criteria favor Mechanism I for the analogous
enzymatic methyl transfer. Martin and Finke (39) noted the
thermodynamic problem with mechanism II: reduction of CH3-Co(III) requires redox potentials that are too low for
physiological electron donors. In the case of model compounds, Co-C
bond homolysis is possible, since the Ni(tmc)(II/I) couple, which has a
reduction potential of 1.18 V (versus SHE), is capable of
reducing CH3-Co(III), with an Em of
1.2 V. However, neither CO nor any of the redox centers present in
CODH/ACS from C. thermoaceticum have Em
values below
550 mV (8, 40-43). Thus, CODH is too weak as a
reductant to reduce methyl-cob(III)amide. We were unable to detect any
reduction or cleavage of the methyl-Co(III) state of the CFeSP after
several hours of incubation in the presence of CO and CODH/ACS (9).
Mechanism I is also supported by studies of acetyl-CoA synthesis using
chiral CH3-H4folate, which is converted to
acetyl-CoA with retention of configuration (44). The most straightforward interpretation of these studies is that transfer of the
methyl group to CODH occurs with inversion of configuration, as
expected for an SN2 type displacement. Although reactions
exist in which the radical is transferred before it has the chance to rotate (and thus randomize), if the methyl group is transferred as a
radical species, racemization would be the most likely outcome.
Further support for Mechanism I comes from recent studies using the
variant C20A CFeSP (20). The methylated C20A variant, which cannot
accept electrons from CODH and, therefore, cannot reduce methyl-Co(III)
to methyl-Co(II), generates acetyl-CoA as rapidly as the wild type
protein. This result appears to be inconsistent with Mechanism II.
However, this study did not evaluate the redox state of the CFeSP
during the reaction. The finding that electron transfer to the cobalt
center is crippled in the variant offers the possibility of uncovering
reaction intermediates that would be masked by the rapid reduction by
CODH of the wild type protein. Furthermore, it is possible that a
radical pathway (such as Mechanism III), which may be a minor component
of the reaction with the wild type protein, could become the dominant
pathway used by the variant form of the CFeSP. However, just as with
the wild type protein, demethylation of methyl-Co(III) is accompanied
by formation of Co(I), which is in redox equilibrium with Co(II). We
conclude that methylation of CODH/ACS, like the
CH3-H4folate-dependent methylation of the
CFeSP, proceeds through an SN2 displacement mechanism and
does not involve radical chemistry. An important question remains. What
conditions determine whether a methyl transfer reaction will occur
through a homolytic or heterolytic mechanism?
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM-39451 (to S. W. R.).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.
Present address: Genetics Institute, 87 Cambridge Park Dr.,
Cambridge, MA 02140.
§ To whom correspondence should be addressed: Dept. of Biochemistry, Beadle Center, University of Nebraska, Lincoln, NE 68588-0664. Tel.: 402-472-2943; Fax: 402-472-8912; E-mail: sragsdal{at}unlinfo.unl.edu.
2 CO and CODH are required for reductive activation, and CO and ACS are required for acetyl-CoA synthesis.
3 In these studies, we methylated the CFeSP with methyl iodide. This procedure was criticized by others who erroneously stated that our samples contained methyl iodide at concentrations sufficient to inhibit CO/acetyl-CoA exchange and CO oxidation activities (see Footnote 27 of Ref. 7). However, in our studies (25), we followed a protocol established after extensive studies to assure that the methyl-CODH is "viable" (8). After reacting the CFeSP with methyl iodide, the reaction mixture is chromatographed to remove methyl iodide and isolate the methylated CFeSP. Therefore, methyl iodide is absent from all enzymatic reactions in which the methylated CFeSP is used as a methyl donor. The catalytic competence of the methyl-CODH intermediate was demonstrated in three different reactions. First, in an exchange reaction between methylated CFeSP and methyl-CODH, 40% of the methyl groups underwent exchange. Second, in an exchange reaction between methyl-CODH and the methyl group of acetyl-CoA, 90% underwent exchange. Third, by measuring the conversion of methyl-CODH, CO, and CoA to acetyl-CoA, 80% of the methyl groups were converted to acetate or acetyl-CoA. Therefore, the methylation protocol is sound, and the published criticisms relating to this methodology are unjustified.
4 S. W. Ragsdale, unpublished data.
5 Seravilli, J., Zhao, S. Y., and Ragsdale, S. W. (1999) Biochemistry 38, in press.
6 It would be methyl-Ni(II) if Ni(I) is the nucleophile and methyl-Ni(III) if it is Ni(II).
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ABBREVIATIONS |
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The abbreviations used are: H4folate, tetrahydrofolate; MeTr, methyltransferase; CFeSP, corrinoid iron-sulfur protein; CODH, carbon monoxide dehydrogenase; ACS, acetyl-CoA synthase.
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REFERENCES |
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