From the
Recombinant methylmalonyl-coenzyme A (CoA) mutase from
Propionibacterium shermanii has been purified 20-fold to near
homogeneity in a highly active form. Neither the apoenzyme (the form in
which the enzyme is isolated) nor the holoenzyme (reconstituted with
the cofactor, adenosylcobalamin) has an electron paramagnetic resonance
(EPR) spectrum associated with it. However, the addition of either the
substrate, methylmalonyl-CoA, or the product, succinyl-CoA, results in
the appearance of a transient EPR signal. The signal has hyperfine
features that indicate coupling of the unpaired electron to the cobalt
nucleus. In the presence of
[CD
Methylmalonyl-CoA mutase catalyzes the reversible isomerization
of methylmalonyl-CoA to succinyl-CoA (see Fig. SI) and channels
catabolites of odd chain fatty acids, branched chain amino acids, and
cholesterol to the Krebs cycle
(1) . The enzyme is dependent
upon the cofactor, adenosylcobalamin(AdoCbl),
This mechanism predicts the existence of a pair of
paramagnetic species (one centered on cobalt and the other distributed
between deoxyadenosine, substrate, product, and perhaps the protein)
during the course of the reaction. In order to test this mechanism, we
have examined the EPR spectra associated with the enzyme in the
presence of both proteo- and deuterosubstrates and product. In
addition, we have characterized the EPR spectrum of enzyme-bound
cob(II)alamin generated under noncatalytic conditions. This study
demonstrates the transient formation of an EPR-active species during
catalysis that is distinct from the spectrum associated with bound
cob(II)alamin under noncatalytic conditions. This provides evidence for
the involvement of radicals in the rearrangement reaction catalyzed by
methylmalonyl-CoA mutase.
( R,S)-methylmalonyl-CoA and
( R,S)-[CD
On-line formulae not verified for accuracy
The total spin
intensity of the signal in the presence of protiated substrate
(Fig. 1, upper trace) is 0.4 spin/mol of enzyme. When
deuterated substrate was incubated with the enzyme (Fig. 1,
lower trace) the spectrum was similar to that obtained with
protiated substrate; however, the spin intensity decreased to 0.15
spin/mol of enzyme. In the presence of succinyl-CoA (Fig. 2), a
spectrum that is very similar to that shown in the upper trace of Fig. 1was obtained, and the spin intensity was 0.18
spin/mol of enzyme.
The deuterated sample exhibits a
power dependence that is similar to that of the protiated sample.
However, the two components are not well resolved, and a shift in the
crossover point from g = 2.11 to g = 2.14 with increasing
power is not seen (data not shown). At higher temperatures (>25 K)
none of the spectra show the change in crossover point that results
from predominance of Signal B (Fig. 3 B).
We have purified the mutase to near homogeneity.
Chromatography on phenyl-Sepharose was found to be essential for
separating the mutase from a tenacious contaminant with a molecular
mass of 44 kDa as judged by polyacrylamide gel electrophoresis under
denaturing conditions. The contaminant was found by amino acid sequence
analysis of a tryptic fragment to be the bacterial elongation factor,
EF-Tu (the N terminus of this protein is blocked
(13) ).
The
activity of the bacterial mutase has been routinely measured in the
reverse direction in a coupled system that requires three other
enzymes. Thus, in order to compare the activities that we have measured
in the radiolabeled assay with the values reported in the literature,
we have reexamined the data presented in Fig. 3by Kellermeyer
et al. (12) . The ratio of V
According to the mechanism depicted in Scheme I, the
mutase-catalyzed reaction involves different pairs of biradical
intermediates. This mechanism is postulated to be general for the
AdoCbl-dependent enzymes
(6, 15, 16) . When the
bacterial mutase is reacted with substrate, a two-component transient
EPR spectrum is obtained. Signal A is slow relaxing with a crossover g
value of 2.11, whereas Signal B is fast relaxing with a crossover g
value of 2.14. Hyperfine splittings resulting from coupling of the
unpaired electron with the cobalt nucleus ( I = ) are
evident in the high field region of the spectrum, where 5 out of the 8
lines are clearly resolved and centered at g = 2.00. The spacing
(50 G) is considerably smaller than that of the splittings observed
previously for enzyme-bound or -free cob(II)alamin
(17, 18, 19) , indicating less electron density
on the cobalt in this intermediate. The spacing is generally
Similar EPR spectra (showing only Signal A) have been
reported for the wild type bacterial mutase
(20, 21) and with the recombinant enzyme
(22) . However,
these EPR spectra were not characterized either with respect to spin
quantitation or power and temperature dependences. Thus, the
contribution of the two components to the signal was missed. In
addition, most of these spectra had poor resolution, and hyperfine
splittings resulting from coupling to the cobalt nucleus were not
observed
(20, 22) .
Other AdoCbl-dependent enzymes in
the presence of their respective substrates give rise to similar EPR
spectra. These include ethanolamine ammonia lyase
(23) ,
ribonucleotide reductase
(24) , glutamate mutase
(25) ,
and methyleneglutarate mutase
(26) . With both ribonucleotide
reductase and glutamate mutase, Signal A was generated under
freeze-quench conditions, and, with the former enzyme, the paramagnetic
intermediate was shown to be kinetically competent. A different type of
EPR spectrum has been reported for other AdoCbl-dependent enzymes in
which the organic substrate radical centered at g = 2.0 appears
as a doublet due to coupling to the unpaired electron on cobalamin
(27, 28, 29) . Based on simulations, it has been
proposed that the two radicals are
There are at least two possible
interpretations of the observed EPR signal. One is that it is due
only to cob(II)alamin in an unusual environment that results
in its EPR spectrum being very different from those observed previously
for the cofactor free in solution or bound to other enzymes
(19, 32, 33) . Alternatively, the signal could
represent an exchange-coupled system in which the two spins interact
strongly. Both possibilities have been raised previously
(34, 35) . The EPR spectrum observed with ribonucleotide
reductase was simulated as the sum of the line shapes corresponding to
two distinct hexacoordinate cob(II)alamin species
(34) .
However, the problem with this interpretation is that it did not
account for the ``other radical'' that must exist according
to a mechanism in which a diamagnetic parent generates paramagnetic
products. Coffman and co-workers
(35) have observed a similar
signal when partially dehydrated polycrystalline AdoCbl was photolyzed
by lasers. Their interpretation was that this signal represented an
exchange-coupled cob(II)alamin
In an attempt to distinguish between these possibilities, we
have examined the EPR spectrum of cob(II)alamin generated under
noncatalytic conditions. Regardless of whether the cob(II)alamin is
generated by electrochemical reduction or generated abortively in the
presence of a substrate analog, the resulting EPR spectrum is typical
of base-on cobalamin with g
The power dependence studies reveal
the presence of two components that are partially resolved at low
temperatures and high powers. It is not known whether they represent
two different radical species (for instance a
cob(II)alamin
What is the identity of the
second radical contributing to Signal A? Based on the prevalence of
Signal A (g = 2.11) in AdoCbl-dependent reactions
(20, 22, 23, 24, 25, 26) as well as its formation in photolysed AdoCbl crystals
(35) , it is tempting to speculate that it is the biradical
intermediate common to all the enzymes ( i.e. a
deoxyadenosyl radical
In conclusion, our EPR spectra
of cob(II)alamin generated under catalytic and noncatalytic conditions
provide strong support for the interpretation that Signal A observed
with this and other AdoCbl-dependent enzymes represents an
exchange-coupled biradical intermediate in which the two dissimilar
spins are strongly interacting. Whereas one of the two radicals is
clearly on cob(II)alamin, the identity of the second radical in these
EPR spectra, as well as the identity of the two components observed in
the mutase-catalyzed reaction, remain important and as yet unanswered
questions.
]methylmalonyl-CoA, an EPR signal is also seen
and is similar to that obtained in the presence of protiated substrate.
Power saturation studies reveal the presence of two components, a slow
relaxing species (with an apparent g value of 2.11) and a fast relaxing
species (with an apparent g value of 2.14) that can be partially
resolved at low temperature and high power. The EPR-active intermediate
is observed under catalytic conditions and is approximately midway in
its resonance position between a free radical and cob(II)alamin. It is
postulated to represent an exchange-coupled
cob(II)alamin
free radical pair. The signal bears
close resemblance to those observed with partially dehydrated
polycrystalline adenosylcobalamin following laser photolysis (Ghanekar,
V. D., Lin, R. J., Coffman, R. E., and Blakley, R. L. (1981)
Biochem. Biophys. Res. Commun. 101, 215-221) and with
the adenosylcobalamin-dependent ribonucleotide reductase under
freeze-quench conditions (Orme-Johnson, W. H., Beinert, H., and
Blakley, R. L. (1974) J. Biol. Chem. 249, 2338-2343).
When cob(II)alamin is generated under noncatalytic conditions ( i.e. in the presence of propionyl-CoA or by electrochemical reduction
of enzyme-bound hydroxocob(III)alamin), a different EPR signal is
observed with g
= 2.26 and g
=
2.00, typical of base-on cob(II)alamin.
(
)
a
derivative of vitamin B
, for activity. Dysfunction of this
enzyme leads to methylmalonic aciduria, an inborn error of organic acid
metabolism
(1) . In some bacteria, such as Propionibacterium
shermanii, the mutase is important in the reverse metabolic
direction, linking production of propionate from succinate, a pathway
elucidated by Wood and co-workers
(2) .
Figure SI:
Postulated reaction mechanism of
methylmalonyl-CoA mutase. X is either the deoxyadenosyl
radical or a secondarily generated protein radical (AdoCH+ XH
AdoCH
+
X).
The first step in the
mutase-catalyzed reaction is postulated to be homolytic fission of the
Co-C bond of the cofactor to generate a carbon-centered
deoxyadenosyl radical and a metal-centered cob(II)alamin radical (see
Fig. SI
, step i). Homolysis of the reactive Co-C
bond is accelerated by a factor of 10
by the enzyme
(3) . In the next step, the adenosyl radical, either directly or
via a protein radical, is believed to initiate the rearrangement by
abstracting a hydrogen atom from the methyl group of methylmalonyl-CoA
to generate a reactive primary radical on the substrate (see
Fig. SI
, step ii). The latter then rearranges to a more
stable secondary radical on the product (see Fig. SI, step
iii). The nature of the rearrangement step itself ( i.e. whether it occurs via a free radical, a carbonium ion, a
carbanion, or an organocobalt intermediate) is not known, and a large
number of studies have attempted to model this step
(4, 5, 6, 7) . Reabstraction of a
hydrogen atom (see Fig. SI, step iv) to give product and
recombination of the deoxyadenosyl and cobalamin radicals complete the
catalytic cycle.
Materials
The following were purchased from
Sigma: coenzyme A (sodium salt), coenzyme B,
hydroxocobalamin hydrochloride, benzyl viologen, bovine serum albumin,
lysozyme, phenylmethylsulfonyl flouride, EDTA,
-mercaptoethanol,
Q-Sepharose (fast flow), and dithiothreitol.
[CD
]Methylmalonic acid was purchased from MSD
Isotopes. Phenyl-Sepharose (fast flow) and Superose 12 were purchased
from Pharmacia Biotech Inc. Matrex Gel Blue A was purchased from
Amicon, and casamino acids were from Difco.
]methylmalonyl-CoA were
synthesized and characterized as described previously
(8) .
[
C]CH
-methylmalonyl-CoA (56.4
Ci/mol) was purchased from DuPont NEN.
Purification of Methylmalonyl-CoA Mutase
The
recombinant P. shermanii enzyme expressed in Escherichia
coli was purified by a modification of the procedure employed for
the human methylmalonyl-CoA mutase
(9) . The E. coli strain K38 harboring the plasmids pMEX2 (containing the mutase
gene behind the T7 RNA polymerase promoter) and pGP1-2 (carrying
the gene for the T7 RNA polymerase under control of a
temperature-sensitive repressor) were generous gifts from Ian Scott
(Texas A & M University). The cells were grown, and the mutase was
induced essentially as described by McKie et al. (14) .
E. coli cells (120 g, wet weight, obtained from 12 liters
of culture) were suspended in 400 ml of 0.1
M potassium
P
, pH 7.5, containing 0.1 m
M EDTA, 5 m
M
benzamidine HCl, 2 m
M
-mercaptoethanol, and 0.1
m
M phenylmethylsulfonyl fluoride. The cells were disrupted
with a Heat Systems Ultrasonic Processor XL operated at an output
setting of 7 for 6
1.5 min with 2-min breaks between the cycles
to prevent overheating. The suspension was centrifuged at
75,000
g for 1 h to remove cell debris and unbroken cells.
The supernatant fluid was immediately loaded on a Q-Sepharose (fast
flow) column (5
20 cm) equilibrated with 50 m
M
potassium P
, pH 7.5. The first three steps of the
purification were identical to those reported for the human mutase
(9) . The presence of the mutase was detected by Western
analysis with rabbit antimutase antibodies generously provided by Janos
Rétey (Universität Karlsruhe, Germany). The enzyme eluted
from the Q-Sepharose column at
350 m
M potassium
P
, from phenyl-Sepharose at 100 m
M ammonium
sulfate in 50 m
M potassium P
, and from the Matrex
Gel Blue column at 300 m
M potassium P
. The final
step involved chromatography on a fast protein liquid chromatography
Superose 12 (Pharmacia) gel filtration column. The protein was eluted
isocratically with 50 m
M potassium P
, pH 7.5,
containing 150 m
M KCl at a flow rate of 0.5 ml/min. Protein
concentration was determined by the Bradford method using bovine serum
albumin as a standard.
Kinetic Characterization of the Mutase
Activity of
the bacterial mutase was either determined in the radiolabel assay at
37 °C using
[C]CH
-methylmalonyl-CoA or in the
coupled spectrophotometric assay at 30 °C
(9) . 1 unit of
activity catalyzes the formation of 1 µmol of succinyl-CoA/min at
37 °C unless stated otherwise. Steady state kinetic data were
analyzed by nonlinear regression analysis with Sigma Plot (Jandel
Scientific, Corte Madera, CA) using the Michaelis-Menten equation.
EPR Spectroscopy
EPR spectra were recorded on a
Bruker ESP 300E spectrometer equipped with an Oxford ITC4 temperature
controller, a model 5340 automatic frequency counter from
Hewlett-Packard, and a gaussmeter. The specific conditions are provided
in the figure legends. Spin quantitation was performed by comparison of
the second integral of the sample spectra with that of a 1 m
M
cupric perchlorate standard. The samples were prepared as described in
the figure legends. The value of the power for half saturation
( P) was calculated by fitting the data to
Equation 1,
Electrochemical Generation of Enzyme-bound
Cob(II)alamin
The bacterial apoenzyme was reconstituted by
mixing apoenzyme with a 4-fold excess of AdoCbl on ice for 30 min. The
protein was then concentrated in a Centricon-30 microconcentrator
(Amicon) and washed extensively with 50 m
M potassium
P, pH 7.5, until the filtrate was colorless and had no
UV-visible absorption spectrum. The enzyme-bound cofactor (22
µ
M) was reduced electrochemically using a titration cell
described previously
(11) . Benzyl viologen (1 m
M) was
employed as a dye mediator, and the potential was poised at -350
mV versus the standard hydrogen electrode. Once a stable
current was observed, the sample was tipped in from the electrochemical
chamber into the EPR tube side arm and frozen in liquid nitrogen.
Purification and Characterization of the
Mutase
The recombinant enzyme from E. coli was purified
20-fold to near homogeneity. The summary of the purification is
presented in Table I. Steady state kinetic analysis of the recombinant
enzyme was performed using the radiolabel assay. The
Kfor
( R,S)-methylmalonyl-CoA is 168 ± 23 µ
M,
comparable with the K
of
( R,S)-methylmalonyl-CoA of 160 µ
M reported for
the wild type enzyme
(12) . The maximal specific activity is
38.5 ± 2 units/mg protein at 30 °C and 50 ± 2
units/mg protein at 37 °C.
EPR Spectra of Holoenzyme in the Presence of
Methylmalonyl-CoA or Succinyl-CoA
Neither the enzyme as isolated
nor the holoenzyme reconstituted with AdoCbl have EPR spectra
associated with them. When the holoenzyme was incubated with protiated
or deuterated substrate (Fig. 1) or with succinyl-CoA (Fig. 2) and
rapidly frozen in liquid nitrogen, an EPR spectrum was obtained. The
signal disappeared if the sample was thawed and refrozen within 5 min
(data not shown). Hyperfine splittings are well resolved in the high
field region and have a spacing of 50 G.
Figure 1:
X-band EPR spectra of holoenzyme under
catalytic conditions. Upper spectrum, mutase after addition of
protiated substrate. The sample was prepared by mixing a cold (5
°C) anaerobic solution of holoenzyme (final concentration, 160
µ
M) in 50 m
M potassium phosphate buffer, pH 7.5,
with ( R,S)-methylmalonyl-CoA (final concentration, 20
m
M) in the same anaerobic buffer and rapidly freezing it in
liquid nitrogen. All manipulations were performed under reduced
illumination due to the photolability of AdoCbl. Lower
spectrum, holoenzyme (200 µ
M) rapidly mixed with
[CD]methylmalonyl-CoA (20 m
M). The
spectra were recorded using the following conditions: sweep width, 2000
G; modulation frequency, 100 kHz; modulation amplitude, 10 G; power, 5
mW; and temperature, 25 K. The resonator frequency was 9.446 GHz. Both
spectra were obtained by addition of five scans. The line markers indicate the following g values: 1.90, 1.93, 1.95, 1.98, 2.02, and
2.11.
Figure 2:
EPR
spectrum of methylmalonyl-CoA mutase in the presence of succinyl-CoA.
An anaerobic and cold solution of holoenzyme (200 µ
M) was
rapidly mixed with succinyl-CoA (10 m
M) in the dark and then
frozen. The instrument conditions were the same as those reported in
the legend of Fig. 1 except that the resonator frequency was 9.64 GHz,
and the sweep width was 1000 G. The line marker is at g
= 2.1. Inset, power dependence of Signal A generated in
the presence of succinyl-CoA. The data were fit to Equation 1 with the
parameters reported in the text (the scaling factor (Equation 1,
A) was 30.9 ± 0.8). Power dependence at 10 () and
25 K (
) is shown.
Power and Temperature Dependence of the EPR
Spectrum
By varying the temperature and power, the signal shown
in Fig. 1( upper trace) can be clearly seen to be
composed of two features (Fig. 3 A). At low powers, Signal A
(with g values of 2.11 and 2.0) predominates and shows saturation at 1
mW. The intensity of this signal remains unchanged up to 20 mW. With
increasing microwave power, a fast relaxing species, Signal B, becomes
well resolved. At 100 mW, Signal B is the major component, whereas at
intermediate powers (5-20 mW) substantial amounts of both signals
lead to a double-humped absorption feature. The peak to trough widths
of Signals A and B are 155 and 120 G, respectively. Signal B shows
splittings with spacing similar to that of Signal A (50 G),
indicative of hyperfine coupling to the cobalt nucleus. The g values of
Signal B are difficult to assign, but the crossover point is at g
= 2.14. The power saturation data for Signal A could not be fit
at any temperature to Equation 1.
Figure 3:
Power dependence of the EPR signal in the
presence of protiated substrate (Fig. 1, upper trace) at two
different temperatures. A, power dependence at 10 K showing
progressive resolution of two species. The arrow points to the
spectrum at 100 mW; successive spectra were recorded at 80, 40, 20, 10,
5, 1, and 0.5 mW of power, respectively. The line markers represent g values of 2.11 and 2.14, respectively. B,
power saturation at 25 K. The arrow points to the spectrum at
100 mW; successive spectra were recorded at 80, 40, 20, 10, 5, 1, 0.5,
0.1 and 0.05 mW of power, respectively. The instrument settings were
the same as in Fig. 1.
The power
dependence of Signal A observed in the presence of succinyl-CoA is less
complex, and a typical dependence of signal intensity on the square
root of power is observed. The power for half-saturation at 10 K is
0.99 ± 0.2 mW and at 25 K is 5.7 ± 0.8 mW (Fig. 2,
inset) with an inhomogeneity parameter (Equation 1,
b) of 1.00, indicating inhomogeneous broadening. At higher
temperatures (100 K), saturation is not evident at a microwave power of
100 mW.
EPR Spectrum of Enzyme-bound Cob(II)alamin under
Noncatalytic Conditions
In order to better understand the
origins of Signals A and B, enzyme-bound cob(II)alamin was generated in
one of two ways. In the first case, holoenzyme was exposed to
propionyl-CoA, a substrate analog that slowly catalyzes the
irreversible homolysis of the carbon-cobalt bond. Alternatively,
enzyme-bound hydroxocobalamin was electrochemically reduced to
cob(II)alamin. In both instances, the resulting EPR spectrum is typical
of base-on cobalamin with g= 2.26 and g
= 2.0 (Fig. 4). Superhyperfine structure is observed in
the high field lines that are split into triplets due to coupling to a
nucleus with I = 1 at the lower axial position.
/ V
for the mutase from
P. shermanii is
2.25. The specific activity (determined
under V
conditions) of the bacterial mutase
assayed in the reverse direction is reported to be 14.4 µmol of
methylmalonyl-CoA formed per min/mg of protein at 30 °C
(12) . This would correspond to a maximal specific activity in
the forward direction of
32 µmol of succinyl-CoA formed per
min/mg of protein. Our enzyme (maximal specific activity of 38.5
µmol of succinyl-CoA formed per min/mg of protein at 30 °C) is
thus comparable to the wild type enzyme and is
6-fold more active
than the recombinant P. shermanii mutase isolated from the
same E. coli strain by Leadlay and co-workers
(14) .
105 G
for base-on cobalamin and 140-160 G for base-off cobalamins and
cobinamides.
10 Å apart in these
systems
(30, 31) .
deoxyadenosyl radical
pair.
= 2.26 and g
= 2.0 (Fig. 4). In the presence of propionyl-CoA, a
very poor substrate analog
(36) , the unstable deoxyadenosyl
radical is presumably quenched to deoxyadenosine. The observed EPR
spectra rule out the possibility that Signal A is only due to
cob(II)alamin in an unusual environment. Thus, the interpretation that
we favor is that Signal A represents an intermediate in which the
metal- and carbon-centered radicals interact strongly (Scheme II).
When, as in this case, the spins are dissimilar in nature, there is a
high rate of mutual spin ``flipping,'' which is rapid
compared with the difference in the precession frequencies in the
external field. The field therefore sees only an average of the two
radicals
(37) , and the external magnetic field spectral line is
observed at
(g
+ g
)
H
= g
H
instead of at or near the
individual frequencies (g = 2.26 for cobalt and g = 2.00
for a free radical). A recent study on the magnetic field dependence of
and magnetic isotope effects on the reaction catalyzed by ethanolamine
ammonia-lyase has provided evidence for the existence of a radical pair
intermediate on this enzyme
(38) .
Figure 4:
Spectrum of enzyme-bound cob(II)alamin
under noncatalytic conditions. Upper spectrum, anaerobic
holoenzyme (80 µ
M) in the presence of 2 m
M
propionyl-CoA. The sample was incubated at ambient temperature under
reduced illumination for 20 min before freezing. Lower
spectrum, electrochemically reduced enzyme-bound hydroxocobalamin
(22 µ
M) generated as described under ``Experimental
Procedures.'' The instrument settings were the same as described
in Fig. 1 with the following exceptions: temperature, 100 K; modulation
amplitude, 5 G; and microwave power, 40 mW. The lower spectrum was obtained by the addition of four scans. The g = 2
signal seen in the lower trace is due to reduced
benzylviologen used as a dye mediator. The line markers indicate the following g values: 2.26 and 2.0,
respectively.
Because the substrate is
one of the possible residences for the migrating radical
(Fig. SI), we investigated whether the unpaired electron
experiences magnetic effects due to the nuclear spins of adjacent
protons on the substrate. The spectrum of the enzyme treated with
[CD]methylmalonyl-CoA is similar to that obtained
with protiated substrate. The simplest explanation for the apparent
absence of an isotope effect on the EPR spectrum is that the methyl
protons of the substrate do not contribute to the observed hyperfine
structure of the signal. However, this is not an unambiguous conclusion
because the signal to noise ratio is low and weak hyperfine couplings
due to protons could be missed.
deoxyadenosyl radical pair and a
cob(II)alamin
substrate radical pair) or whether
they are two different states of the same biradical intermediate. The
signals generated in the presence of deuterated substrate but not
succinyl-CoA show the presence of both Signals A and B. This suggests
that the two Signals A and B arise from two intermediates with their
relative proportions varying in the different samples. Furthermore,
with these two substrate-containing samples, we have been unable to
obtain reasonable fits to the power saturation data using Equation 1
that is used to estimate the power for half-saturation of EPR signals
due to a single species
(10) . This also supports the conclusion
that the EPR signals observed in the presence of substrate are
comprised of more than one species.
cob(II)alamin pair).
Alternatively, it could represent a
cob(II)alamin
protein radical pair, assuming that a
structurally similar protein radical is involved in all the enzymes in
which this signal has been observed.
Table: Purification of recombinant P. shermanii
methylmalonyl-CoA mutase
(Dolphin, D., ed) pp. 431-462, Wiley-Interscience, New York
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.