©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence from Electron Paramagnetic Resonance Spectroscopy of the Participation of Radical Intermediates in the Reaction Catalyzed by Methylmalonyl-coenzyme A Mutase (*)

Rugmini Padmakumar , Ruma Banerjee (§)

From the (1) Biochemistry Department, University of Nebraska, Lincoln, Nebraska 68583-0718

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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, 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)alaminfree 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.


INTRODUCTION

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),() 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 10by 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.

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.


EXPERIMENTAL PROCEDURES

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.

( R,S)-methylmalonyl-CoA and ( R,S)-[CD]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,

 

On-line formulae not verified for accuracy

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.


RESULTS

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 Kof ( 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.

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.


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.

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).


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.


DISCUSSION

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/ Vfor the mutase from P. shermanii is 2.25. The specific activity (determined under Vconditions) 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) .

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 105 G for base-on cobalamin and 140-160 G for base-off cobalamins and cobinamides.

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 10 Å apart in these systems (30, 31) .

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)alamindeoxyadenosyl radical pair.

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= 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 = gHinstead 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.

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)alamindeoxyadenosyl radical pair and a cob(II)alaminsubstrate 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.

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 radicalcob(II)alamin pair). Alternatively, it could represent a cob(II)alaminprotein radical pair, assuming that a structurally similar protein radical is involved in all the enzymes in which this signal has been observed.

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.

  
Table: Purification of recombinant P. shermanii methylmalonyl-CoA mutase



FOOTNOTES

*
This research was supported by Grant DK45776 from the National Institutes of Health (to R. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Biochemistry Dept., University of Nebraska, Lincoln, NE 68583-0718. Tel.: 402-472-2941; Fax: 402-472-7842; E-mail: RBANERJEE@crcvms.unl.edu.

The abbreviations used are: AdoCbl, adenosylcobalamin; mW, milliwatt(s).


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