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Originally published In Press as doi:10.1074/jbc.M411842200 on January 12, 2005

J. Biol. Chem., Vol. 280, Issue 11, 10073-10082, March 18, 2005
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Nitric Oxide Inhibits Mammalian Methylmalonyl-CoA Mutase*

Amanpreet Kambo{ddagger}, Vijay S. Sharma, Darren E. Casteel, Virgil L. Woods, Jr., Renate B. Pilz, and Gerry R. Boss§

From the Department of Medicine, and Cancer Center, University of California, La Jolla, California 92093-0652

Received for publication, October 18, 2004 , and in revised form, January 7, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Methylmalonyl-CoA mutase is a key enzyme in intermediary metabolism, and children deficient in enzyme activity have severe metabolic acidosis. We found that nitric oxide (NO) inhibits methylmalonyl-CoA mutase activity in rodent cell extracts. The inhibition of enzyme activity occurred within minutes and was not prevented by thiols, suggesting that enzyme inhibition was not occurring via NO reaction with cysteine residues to form nitrosothiol groups. Enzyme inhibition was dependent on the presence of substrate, implying that NO was reacting with cobalamin(II) (Cbl(II)) and/or the deoxyadenosyl radical (·CH2-Ado), both of which are generated from the co-factor of the enzyme, 5'-deoxyadenosyl-cobalamin (AdoCbl), on substrate binding. Consistent with this hypothesis was the finding that high micromolar concentrations (≥600 µM) of oxygen also inhibited enzyme activity. To study the mechanism of NO reaction with AdoCbl, we simulated the enzymatic reaction by photolyzing AdoCbl, and found that even at low NO concentrations, NO reacted with both the generated Cbl(II) and ·CH2-Ado indicating that NO could effectively compete with the back formation of AdoCbl. Thus, NO inhibition of methylmalonyl-CoA mutase appeared to be from the reaction of NO with both AdoCbl intermediates (Cbl(II) and ·CH2-Ado) generated during the enzymatic reaction. The inhibition of methylmalonyl-CoA mutase by NO was likely of physiological relevance because a NO donor inhibited enzyme activity in intact cells, and scavenging NO from cells or inhibiting cellular NO synthesis increased methylmalonyl-CoA mutase activity when measured subsequently in cell extracts.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammalian cells only two enzymes are known to require cobalamin (vitamin B12) as a cofactor: methionine synthase, which uses methylcobalamin, and methylmalonyl-coenzyme A (CoA) mutase, which uses 5'-deoxyadenosyl-cobalamin (AdoCbl).1 During the reaction catalyzed by methionine synthase, methylcobalamin is cleaved heterolytically generating cobalamin I (Cbl(I)) and a methyl carbocation, whereas during the reaction catalyzed by methylmalonyl-CoA mutase, AdoCbl is cleaved homolytically generating cobalamin II (Cbl(II)) and a deoxyadenosyl radical (·CH2-Ado) (1, 2). The latter serves to abstract a hydrogen atom from the substrate methylmalonyl-CoA, and after intramolecular migration of a thioester moiety, the hydrogen atom is transferred back to generate the product succinyl-CoA. In the final step of the reaction, the deoxyadenosyl radical recombines with Cbl(II) to regenerate AdoCbl (3). It should be noted that the homolytic cleavage of AdoCbl occurs essentially only on substrate binding to the enzyme (3).

Nitric oxide (NO) is produced by a variety of mammalian cell types, and has a diverse array of cellular and physiological functions including regulation of cell growth, differentiation, and apoptosis, and modulation of blood pressure, platelet aggregation, and synaptic plasticity (4–6). At pharmacological concentrations, NO reacts with several different chemical groups, but at physiological concentrations, one of its major targets appears to be the iron atom in heme groups, and in particular the iron in heme-containing guanylate cyclase (7, 8).

Heme and cobalamin are structurally similar, and although there have been conflicting data in the literature, it is now generally agreed that NO binds to Cbl(II) (9–14), and we showed that NO rapidly oxidizes Cbl(I) to Cbl(II) (15). NO inhibits methionine synthase activity in vitro (9, 16, 17), and we showed that it inhibits the enzyme in vivo, thereby disrupting carbon flow through the folate pathway (15). We and others have shown that NO does not react with methylcobalamin (9, 15), and thus the mechanism of methionine synthase inhibition is likely through NO oxidation of the Cbl(I) intermediate to Cbl(II), and formation of nitrosylcobalamin. The effect of NO on methylmalonyl-CoA mutase has not been studied, and similar to methylcobalamin, NO does not react with AdoCbl (9). Analogous to the inhibition of NO for methionine synthase, it seemed possible that NO could inhibit mutase activity by binding to the Cbl(II) intermediate, or even to the free deoxyadenosyl radical intermediate because NO reacts readily with free radicals, but the two intermediates are generated only transiently, and their recombination rate back to AdoCbl is very high (18, 19). Moreover, the Cbl(II) and deoxyadenosyl radical are generated at the active site, which at least in the case of the bacterial enzyme, is buried at the end of a ({beta}{alpha})8 TIM barrel (20, 21). AdoCbl is light-sensitive being cleaved into Cbl(II) and the deoxyadenosyl radical, and when AdoCbl is subjected to complete photolysis in the presence of high NO concentrations, the NO reacts with the generated Cbl(II) (9), but it does not necessarily follow that at low physiological conditions NO will be able to compete with the back reaction of the deoxyadenosyl radical with Cbl(II). In addition, NO may not be able to penetrate to the active site of the enzyme.

We, therefore, sought to determine the effects of NO on methylmalonyl-CoA mutase activity, and found that the activity of the mammalian enzyme was inhibited by NO. The mechanism of inhibition appeared to be from NO reacting with Cbl(II) and the deoxyadenosyl radical, rather than with a sulfhydryl group(s). Based on in vivo work with cultured cells, it appeared that NO may be a physiological regulator of methylmalonyl-CoA mutase activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Arginine-deficient Eagle's minimal essential media (MEM) was either from ICN Pharmaceuticals or was made as described previously (22). High performance liquid chromatography (HPLC) was performed as previously described (23), with C18 reversed-phase columns from Whatman. [1-14C]Propionic acid (53 mCi/mmol) and [1,4-14C]succinic acid (54 mCi/mmol) were from Moravek Biochemicals, and [methyl-14C]methylmalonic acid (50 mCi/mmol) and L-[4,5-3H]leucine (40 Ci/mmol) were from American Radiolabeled Chemicals. DEA-NONOate (2-(N,N-diethylamino)-diazenolate-2-oxide), DETA-NONOate ((Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate), and L-NAME (NG-nitro-L-arginine methyl ester) were from Alexis Biochemicals. Hemoglobin was made from freshly lysed red blood cells (24). NO gas, >99% pure from Matheson Gas Products, was passed through 5 M NaOH immediately prior to use to remove contaminating nitrogen oxide species. AdoCbl and hydroxycobalamin (OH-Cbl) were from Sigma with the AdoCbl protected from light except as indicated.

Cell Culture Conditions
Baby hamster kidney (BHK) fibroblast-like cells were obtained from the American Tissue Culture Collection, and cultured as described previously (25).

Measurement of Methylmalonyl-CoA Mutase Activity in Vitro
BHK cells were incubated for 16 h in Dulbecco's modified Eagle's (DME) medium supplemented with 10% fetal bovine serum (FBS) in the absence or presence of the indicated concentrations of OH-Cbl. Parallel cultures were incubated in MEM containing 100 µM arginine and 10% dialyzed FBS. As indicated, cells received 10 nM OH-Cbl with or without 1 mM L-NAME. The standard assay for measuring methylmalonyl-CoA mutase activity was performed essentially as described by Kikuchi et al. (26), and Riedel et al. (27). All steps were performed in subdued light, and when possible in complete darkness. Briefly, ~1 x 106 cells were extracted in 0.1 ml of ice-cold 1 mM Tris-HCl, pH 7.4, by three 5-s bursts of sonication. Tris-HCl was increased to 100 mM, the extracts centrifuged at 10,000 x g for 5 min, and the supernatants were preincubated for 10 min at 37 °C in the absence and presence of 5 µM AdoCbl (to measure the amount of enzyme in the extracts in the holoenzyme form, and to convert all enzyme to the holoenzyme form, respectively). The extracts were returned to 4 °C, and transferred to either polypropylene microcentrifuge tubes or gas tight glass syringes, both of which were sealed with a tight-fitting rubber septum. The extracts were deoxygenated by passing argon gas over the liquid surface for 15 min with frequent gently shaking of the tubes, and all subsequent additions to the extracts were performed using gas tight syringes. To some of the samples, 1 mM deoxygenated DEA-NONOate was added, and in the standard assay, the reaction was started immediately thereafter by adding deoxygenated methylmalonyl-CoA to a final concentration of 400 µM. Variations on the standard assay included: (i) adding 25 mM deoxygenated {beta}-mercaptoethanol, dithiothreitol, or reduced glutathione simultaneous with the DEA-NONOate; (ii) preincubating extracts with DEA-NONOate for 10 min at 37 °C prior to adding AdoCbl or substrate, with half of the samples receiving 1 mM hemoglobin at the time of substrate addition; and (iii) adding 100 mM Tris buffer saturated with pure NO gas or oxygen with the final NO and oxygen concentrations calculated using the solubility of the two gases in aqueous solutions. After adding the methylmalonyl-CoA, samples were incubated for the indicated times at 37 °C, with the reaction stopped by adding ice-cold perchloric acid (final concentration, 0.4 N). Acid-insoluble material was removed by centrifugation, and the supernatants were neutralized using 2.2 M KHCO3. The neutralized samples were injected onto a C18 reversed-phase HPLC column with methylmalonyl-CoA resolved isocratically from succinyl-CoA using 100 mM sodium phosphate, pH 4.0, with 12% methanol (26, 27). The amounts of methylmalonyl-CoA and succinyl-CoA were determined by comparing the areas of their respective peaks at 254 nm to those of standards. The assay was linear with time from 2 to 15 min, and with protein concentration from 50 to 500 µg. Enzyme activity is expressed as nanomole/min/mg of protein with protein was measured by the method of Bradford (28).

Reactions of NO with AdoCbl
Qualitative Rate Studies—To 3 ml of 0.1 M sodium phosphate, pH 7.4, in a spectrophotometer cuvette was added 85 µl of 3.2 mM AdoCbl yielding a final AdoCbl concentration of 88 µM. The cuvette was sealed with two rubber septums, and after passing ultrahigh purity argon gas through the solution for 30 min, the cuvette was transferred to the closed cell compartment of a Uvikon 860 spectrophotometer (Kontron Instruments) with the lamp light covered. After a 2-h dwell time to allow the solution in the cuvette to equilibrate fully with the temperature in the spectrophotometer compartment, the lamp light was uncovered and allowed to shine on the cell. Absorbance changes were followed at 544 nm for 2 h, at which time 0.1 ml of NO-saturated phosphate buffer was added to the solution under subdued light. The solution was thoroughly mixed and absorbance changes were followed for an additional 2 h. By considering the total volume of the cuvette, the volume of the solution, and the solubility of NO in aqueous systems (1.946 mM at 24 °C), the calculated NO concentration in the solution was ~6 µM, or ~1/15th of the AdoCbl concentration.

Spectroscopic Studies—Solutions were prepared as described above for the rate studies, but instead of observing absorbance changes as a function of time at a single wavelength, the solutions were scanned over the wavelengths indicated (using the Uvikon 860 spectrophotometer). Complete photolysis of AdoCbl was achieved by exposing cuvettes containing AdoCbl solutions to a 500 watt tungsten lamp for 3 min. In some experiments, photolysis of AdoCbl was conducted in the presence of the indicated concentrations of NO.

Mass Spectral Analyses—AdoCbl (750 µM in water) was subjected to photolysis in the absence or presence of NO as described above. The samples were diluted 1:10 (v/v) into 50% water, 40.75% methanol, 0.25% formic acid, and then infused at a rate of 20 µl/min into a Finnegan LCQ mass spectrometer (San Jose, CA) equipped with an ESI assembly, operating in positive-ion mode (5.0 kV spray voltage) with a 200 °C capillary temperature. Data were acquired in profile mode from m/z 100–2000 with spectra averaged over 1.5 min of continuous acquisition. Results are reported as the m/z of monoisotopic peaks, with all ions of interest present as the +1 charge state.

Measurement of Methylmalonyl-CoA Mutase Activity in Vivo
A standard method for measuring methylmalonyl-CoA mutase activity in vivo is to incubate cells with [1-14C]propionic acid, and follow incorporation of radioactivity into acid-precipitable material (29–31). The resulting propionyl-CoA is converted to methylmalonyl-CoA and then to succinyl-CoA by methylmalonyl-CoA mutase. As an intermediate in the tricarboxylic acid cycle, succinyl-CoA is a precursor to other molecules including amino acids, purines, and pyrimidines. We showed by removing DNA and RNA from the precipitates by a 30-min heating at 80 °C that >90% of the radioactivity in the precipitates was in protein (32). Because the assay includes multiple steps after methylmalonyl-CoA conversion to succinyl-CoA, we simultaneously measured [1,4-14C]succinic acid incorporation into acid-precipitable material. Because succinic acid is a completely symmetrical molecule, the radioactive labels from [1,4-14C]succinic acid will be incorporated into amino acids similarly to the radioactive label from [1-14C]propionic acid, allowing the former compound to control for effects of the experimental conditions on steps distal to the methylmalonyl-CoA mutase reaction. To control for potential changes by the experimental conditions on propionyl-CoA conversion to methylmalonyl-CoA, we performed some experiments in which we incubated cells with [methyl-14C]methylmalonic acid. Although the radioactive label will be in carbon 2 of succinic acid rather than in carbons 1 or 4, this should not affect incorporation into amino acids because the full 4-carbon skeleton of succinic acid is converted to aspartate, the major amino acid derived from succinate. Briefly, –1 x 106 BHK cells were plated in a 6-well cluster dish, and after an overnight incubation, the growth medium was replaced by 1 ml of experimental medium. As indicated in the legend to Fig. 5A, the experimental medium was either DME supplemented with 10% FBS, or MEM containing 10% dialyzed FBS and 100 µM arginine. To half of the cultures, 1 mM DETA-NONOate was added, and immediately thereafter, 0.5 µCi of [1-14C]propionic acid, [1,4-14C]succinic acid, or [methyl-14C]methylmalonic acid were added to the cultures. The cells were incubated for 12 h, washed once in ice-cold phosphate-buffered saline, and extracted in 10% trichloroacetic acid. After 20 min on ice, acid-precipitated material was collected on glass microfiber filters, and radioactivity on the filters was measured by liquid scintillation counting as described previously (22). Incorporation of radioactive labels was linear with time for at least 16 h and cell number from 1 x 106 to 2 x 106. By comparing the effects of DETA-NONOate on the incorporation of propionic acid to the incorporation of succinic acid, one can focus on the effect of DETA-NONOate on methylmalonyl-CoA mutase activity.



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  		FIG. 5.
NO inhibits methylmalonyl-CoA mutase activity in vivo. A, NO inhibits enzyme activity. Approximately 1 x 106 logarithmically growing BHK cells were cultured in 6-well dishes in DME supplemented with 10% FBS, with half of the cultures receiving 1 mM DETA-NONOate. In vivo methylmalonyl-CoA mutase activity was measured as described under "Experimental Procedures" by following for 12 h the incorporation of [1-14C]propionic acid and [1,4-14C]succinic acid into the acid-precipitable material. The amount of propionic acid and succinic acid incorporation in the absence of DETA-NONOate was considered to be 100%, with the amount reduction induced by DETA-NONOate plotted. DETA-NONOate inhibited propionic acid incorporation significantly more than succinic acid incorporation (p < 0.05). B, decreased cellular NO availability increases methylmalonyl-CoA mutase activity. BHK cells were incubated for 16 h in DME containing 10% FBS and a limiting amount of OH-Cbl (10 nM), with or without 10 µM hemoglobin (Hb). Parallel cultures were incubated in MEM containing 100 µM arginine, 10% dialyzed FBS, and 10 nM OH-Cbl, with or without 1 mM L-NAME. The cells were processed as described in the legend to Fig. 1, with methylmalonyl-CoA mutase activity measured in cell extracts. In the absence of hemoglobin and L-NAME, enzyme activity was the same whether the cells were incubated in full DME medium or the low arginine MEM. The samples with hemoglobin and L-NAME were significantly different from the untreated sample (p < 0.05).

 
Data Presentation and Statistical Analyses
In bar graphs, the data are the mean ± S.D. of at least three independent experiments performed in duplicate. Spectrophotometric data were performed at least three times with highly reproducible results. Statistical analyses were performed using the Student's t test, with p < 0.05 considered to indicate statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of NO on Methylmalonyl-CoA Mutase Activity in Vitro
Many standard tissue culture media including DME, MEM, and Medium 199 do not contain vitamin B12, and cells derive the vitamin from serum supplements. When BHK cells were grown in DME supplemented with 10% FBS, but lacking added OH-Cbl, methylmalonyl-CoA mutase activity in cell extracts was 0.52 ± 0.21 nmol/min/mg of protein (Fig. 1, open bar on left). When the cell extracts were preincubated with 5 µM AdoCbl, enzyme activity increased ~4-fold to 2.01 ± 0.19 nmol/min/mg of protein (Fig. 1, diagonally striped bar on left). Similar amounts of total holoenzyme activity have been found in human fibroblasts and glioma cells (26, 27). Thus, under conditions of relatively limited cobalamin availability, about three-fourths of cellular methylmalonyl-CoA mutase was present as apoenzyme, and it could be converted to holoenzyme in vitro. Other workers have also found that the enzyme in vivo is largely in the apoenzyme form, including human skin and lung fibroblasts, human glioma cells, and human, mouse, and rat liver (26, 27, 33–35). When cells were incubated for 16 h in DME medium containing 10% FBS supplemented with 1 µM OH-Cbl, enzyme activity in the cell extracts increased to the same level as observed when extracts were incubated with AdoCbl (Fig. 1, open bar on right), and adding AdoCbl to the extract had only a marginal effect (Fig. 1, diagonally striped bar on right).



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  		FIG. 1.
The NO donor DEA-NONOate inhibits methylmalonyl-CoA mutase activity in vitro. Logarithmically growing BHK cells were incubated for 16 h in DME medium supplemented with 10% FBS in the absence or presence of 1 µM OH-Cbl as indicated. Under subdued light, cells were harvested, sonicated in hypotonic buffer, and the resulting extracts were clarified by centrifugation. The extracts were deoxygenated in tightly sealed microcentrifuge tubes, and methylmalonyl-CoA mutase activity was measured for 10 min at 37 °C in the presence of 400 µM L-methylmalonyl-CoA, with substrate and product separated and quantified by HPLC as described under "Experimental Procedures." As indicated, some of the extracts were preincubated for 10 min with 5 µM AdoCbl, and to some of the samples 1 mM DEA-NONOate was added during measurement of enzyme activity.

 
To study the effect of NO on methylmalonyl-CoA mutase activity, we added the NO donor DEA-NONOate to cell extracts, and found that 1 mM DEA-NONOate inhibited methylmalonyl-CoA mutase activity by ~50% (Fig. 1, solid bars). Inhibition was observed when holoenzyme was generated either in vitro by adding AdoCbl to the extracts, or in vivo by adding OH-Cbl to the culture medium (Fig. 1). DEA-NONOate also inhibited the small amount of holoenzyme activity present in cell extracts not preincubated with AdoCbl. Although the initial DEA-NONOate concentration was 1 mM and DEA-NONOate liberates 1.5 mol of NO per mol with a half-life of 2 min at 37 °C, pH 7.4 (36), the actual NO concentration was likely to be <50 µM during the 10-min incubation period because of partitioning of NO between the liquid (50 µl) and gaseous (1.5 ml) phases, with preferential partitioning into the gaseous phase (37).

Studies on NO Inhibition of Methylmalonyl-CoA Mutase
NO could inhibit methylmalonyl-CoA mutase activity by one or more of at least three possible mechanisms: by reacting with cysteine residues to form nitrosothiol groups, by reacting with the Cbl(II) generated during the enzyme reaction, or by reacting with the deoxyadenosyl radical generated during the enzyme reaction.

NO reacts slowly with cysteine in aqueous solutions at neutral pH with a reaction time of 21/2 h (38). Because we found that NO inhibited mutase activity during a 10-min incubation (Fig. 1), enzyme inhibition by nitrosothiol formation seemed unlikely. However, to address this issue further, we performed two additional experiments. First, we studied the kinetics of enzyme inhibition by NO, and found that even after 2 min (the earliest time point at which we could reproducibly measure enzyme activity), NO significantly inhibited mutase activity (Fig. 2A). Second, we found that NO inhibited enzyme activity even in the presence of a 25-fold excess of {beta}-mercaptoethanol, which should prevent nitrosothiol formation or reduce any nitrosothiols formed back to –SH groups (Fig. 2B). Similar inhibitory effects of 1 mM DEA-NONOate were found in the presence of 25 mM glutathione and dithiothreitol. These results suggest that nitrosothiol formation is an unlikely cause of enzyme inhibition.



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  		FIG. 2.
NO inhibition of methylmalonyl-CoA mutase activity: kinetics, effect of {beta}-mercaptoethanol, and substrate dependence. Logarithmically growing BHK cells were extracted and methylmalonyl-CoA mutase activity was measured in the cell extracts as described in the legend to Fig. 1. A, kinetics. Enzyme activity was measured at 2, 5, and 10 min in the absence (closed circles) and presence (open circles) of 1 mM DEA-NONOate. B, effect of {beta}-mercaptoethanol. To some extracts either 25 mM {beta}-mercaptoethanol alone or 25 mM {beta}-mercaptoethanol plus 1 mM DEA-NONOate were added immediately prior to adding substrate. C, substrate dependence. Cell extracts were preincubated for 10 min at 37 °C either in extract buffer alone (open bar) or extract buffer containing 1 mM DEA-NONOate (cross-hatched and filled bars). At the time of substrate addition, 1 mM hemoglobin (Hb) was added either to non-treated extracts (diagonally stripped bars) or extracts that had been treated with 1 mM DEA-NONOate (filled bars).

 
Thus, it appeared that NO was inhibiting enzyme activity by reacting with Cbl(II) and/or the deoxyadenosyl radical. If this hypothesis is correct, then the inhibition would occur only in the presence of substrate. To study this possibility, we modified the experimental protocol described in the legend to Fig. 1, and incubated the cell extracts with DEA-NONOate for 10 min prior to adding methylmalonyl-CoA, and then added substrate either alone or with sufficient hemoglobin to trap all NO released by DEA-NONOate. We found that in the absence of added hemoglobin, DEA-NONOate inhibited enzyme activity, but that adding hemoglobin at the time of substrate completely prevented the inhibition by DEA-NONOate (Fig. 2C). Similar results were obtained when the DEA-NONOate was added prior to incubating extracts with AdoCbl, i.e. inhibition by DEA-NONOate was eliminated by adding hemoglobin at the time of substrate, both in extracts incubated with AdoCbl and extracts not incubated with AdoCbl. These observations indicate that the inhibition of NO by enzyme activity required the presence of substrate, and thus that NO was likely reacting with Cbl(II) or the deoxyadenosyl radical. Although it is possible that substrate-induced conformational changes could expose an otherwise concealed cysteine residue that could then react with NO, this seems rather unlikely as there are no cysteines in the TIM barrel or at the active site (20, 21, 39).

Effect of Oxygen on Methylmalonyl-CoA Mutase Activity in Vitro
Because the data suggest that NO inhibits methylmalonyl-CoA mutase activity by reacting with Cbl(II) or the deoxyadenosyl radical, oxygen should also inhibit enzyme activity because it could potentially also react with these two cofactor intermediates. Previous workers studying the Propionibacterium shermanii enzyme have shown that at ambient oxygen concentrations (~200 µM), oxygen reacts with the Cbl(II) generated during the enzymatic reaction, albeit at a slow rate (40). Other workers found no inhibition of the P. shermanii enzyme under ambient conditions, but enzyme activity was measured over only a 3-min interval (41). We found that at 200 µM, oxygen had a small but statistically non-significant effect on the activity of mammalian methylmalonyl-CoA mutase during a 10-min incubation, but at 600 µM oxygen significantly inhibited enzyme activity (Fig. 3, p < 0.05 for the difference between anerobic conditions and 600 µM oxygen). On an equimolar basis, NO was a more potent inhibitor of methylmalonyl-CoA mutase activity than oxygen (Fig. 3), and possible reasons for this difference will be considered under "Discussion." The inhibition of methylmalonyl-CoA mutase activity by pure NO gas suggests that the NO released by DEA-NONOate is responsible for its enzyme inhibition (Figs. 1 and 2).



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  		FIG. 3.
Oxygen and NO gas inhibit methylmalonyl-CoA mutase activity in vitro. Methylmalonyl-CoA mutase activity was measured in deoxygenated cell extracts derived from logarithmically growing BHK cells as described in the legend to Fig. 1, except the experiments were performed in gas tight syringes (open bar). Oxygen-saturated Tris buffer (100 mM Tris HCl, pH 7.4) was added to the cell extracts to achieve a final oxygen concentration of either 200 µM (diagonally striped bars) or 600 µM (cross-hatched bars). Fully deoxygenated Tris buffer was saturated with pure NO gas and added to cell extracts to achieve an NO concentration of 600 µM (filled bars). The difference between the conditions of 600 µM oxygen and no additions is statistically significant at p < 0.05, as is the difference between 600 µM oxygen and 600 µM NO.

 
Reactions of NO with AdoCbl
Because NO appeared to be inhibiting methylmalonyl-CoA mutase activity by reacting with Cbl(II) or the deoxyadenosyl radical, we decided to simulate the enzymatic reaction by subjecting AdoCbl to photolysis, and study the reaction of NO with the photolytic products. As mentioned previously, NO at high non-physiological concentrations has been shown to react with Cbl(II) during the full photolysis of AdoCbl (9), but the details of this reaction and whether it occurs under limited AdoCbl photolysis and physiological, i.e. low micromolar, concentrations of NO are unknown. To address these questions, we performed the following series of experiments.

Photoinduced Reactions of NO with AdoCbl: Qualitative Rate Studies—First, we asked whether low NO concentrations can compete with reformation of AdoCbl from steady state concentrations of Cbl(II) and the deoxyadenosyl radical generated from limited AdoCbl photolysis. Deoxygenated AdoCbl (88 µM) was placed into a light-tight spectrophotometer compartment with the only light shining on the sample coming from the spectrophotometer beam. By measuring the decrease in absorption at 544 nm, a {lambda}max for AdoCbl, we followed the dissociation of AdoCbl into Cbl(II) and the deoxyadenosyl radical. Because of the back reaction of some of the Cbl(II) and the deoxyadenosyl radical to re-form AdoCbl, the data provide a measurement of the net dissociation of AdoCbl. Full photolysis of the AdoCbl would have yielded a decrease of 0.18 absorbance units, but after 2 h a decrease of only 2.8 x 10–4 absorbance units occurred, indicating a net dissociation of 0.0.1% of the AdoCbl (Fig. 4A, curve 1). Under subdued light, NO-saturated buffer was added to the sample to yield a final NO concentration of 6 µM. The dissociation reaction was once again followed, and was now found to be increased ~10-fold compared with incubation in the absence of NO (Fig. 4A, curve 2 shows a decrease of 2.7 x 10–3 absorbance units over 2 h). The small decrease in absorption at zero time likely reflects a 3.3% dilution of the AdoCbl solution on adding the NO-saturated buffer, which would, if anything, slow the reaction rate of NO with the newly formed species, and thereby retard the rate of decrease in absorption. These experiments indicate that even under conditions of limited AdoCbl photolysis (into Cbl(II) and the deoxyadenosyl radical), and at concentrations of NO that occur physiologically, NO can effectively compete with the back formation of AdoCbl from Cbl(II) and the deoxyadenosyl radical. These experiments do not distinguish between whether NO is reacting with Cbl(II), the deoxyadenosyl radical, or both species.



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  		FIG. 4.
Photoinduced reactions of NO with AdoCbl. A, qualitative rate studies. An 88 µM AdoCbl solution in a stoppered cuvette was deoxygenated with argon gas and placed into a light-tight spectrophotometer compartment with the light beam covered. After a 2-h equilibration time, the beam was uncovered and the absorbance at 544 nm was followed for 2 h (curve 1). Under subdued light, NO saturated buffer was added to the sample to yield a final NO concentration of 6 µM, the sample was mixed, and returned to the spectrophotometer. The absorbance at 544 nm was followed for an additional 2 h (curve 2). B, NO reaction with Cbl(II). Deoxygenated AdoCbl (105 µM) fully photolyzed in the presence of 1 eq of NO was scanned between 300 and 700 nm. Inset, curve 1 is the spectrum of AdoCbl, curve 2 is the spectrum of fully photolyzed AdoCbl, and curve 3 is the spectrum of AdoCbl fully photolyzed in the presence of 1.96 mM NO. C, NO reaction with deoxyadenosyl radical. AdoCbl (105 µM) was scanned between 220 and 300 nm. Curve 1 is the initial scan, curve 2 is the scan after full photolysis of the AdoCbl, and curve 3 is the scan after full photolysis of the AdoCbl in the presence of 1.96 mM NO.

 
Photoinduced Reactions of NO with AdoCbl: Reaction with Cbl(II)—Brouwer et al. (9) showed previously that when NO is present in great excess it reacts with Cbl(II) generated from AdoCbl (9). To determine whether low concentrations of NO react with Cbl(II) generated from AdoCbl, we photolyzed AdoCbl in the presence of 1 eq of NO. The resulting spectrum is shown in Fig. 4B. By comparing it to the spectra in the inset that shows AdoCbl (curve 1), and photolyzed AdoCbl in the absence (curve 2) and presence (curve 3) of excess NO, it is clear that even in the presence of only 1 eq of NO, some NO-Cbl(II) was formed. By performing calculations at 310 nm (Fig. 4B), and taking into account the {Delta}A for the formation of Cbl(II) and NO-Cbl(II) at [NO] = 1 eq of AdoCbl, we found that about 50% of the NO was combining with Cbl(II). This suggested that the remaining 50% of the NO was reacting with some other species generated during the photolysis of AdoCbl, i.e. the deoxyadenosyl radical.

Photoinduced Reactions of NO with AdoCbl: Reaction with Deoxyadenosyl Radical—There are no previous reports of NO reacting with the deoxyadenosyl radical generated during AdoCbl photolysis. However, while studying thermolysis of AdoCbl, which like photolysis yields Cbl(II) and the deoxyadenosyl radical, Finke and Hay (42, 43) showed that adding the free radical trap 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO) to the reaction at pH 7 yielded a new product, 5'-deoxy-5'-(TEMPO)adenosine. In the absence of TEMPO, the cyclic compound 8,5'-anhydro-5'-deoxyadenosine was the dominant species formed from the unstable deoxyadenosyl radical (42, 43). Thus, the deoxyadenosyl radical can react with the nitroxide group of TEMPO, or presumably with other radical traps. To determine whether the deoxyadenosyl radical reacts with NO, we subjected AdoCbl to full photolysis in the absence or presence of NO, and assessed spectral changes between 220 and 300 nm, which is where the adenosyl moiety absorbs maximally (Fig. 4C). Curve 1 of Fig. 4C shows the spectrum of AdoCbl prior to photolysis with a {lambda}max of 260 nm. Curve 2 of Fig. 4C shows that photolysis of AdoCbl in the absence of NO caused the {lambda}max to increase to 262 nm, and the absorbance to decrease markedly. These changes were likely because of the formation of 8,5'-anhydro-5'-deoxyadenosine, the main product of AdoCbl photolysis in anerobic solutions at neutral pH (42, 43), and for which a {lambda}max of 262 nm has been reported (44). Photolysis of AdoCbl in the presence of excess NO decreased the {lambda}max by 5–257 nm (Fig. 4C, curve 3), which is the same as for 5'-deoxyadenosine, and suggests that the species 5'-deoxy-5'-(NO)adenosine was formed. Further evidence for this conclusion came from mass spectral analyses that showed that photolysis of AdoCbl in the presence of NO generated a monoisotopic peak at m/z 281.0, the calculated mass of 5'-deoxy-5'-(NO)adenosine. This peak was not present when AdoCbl was photolyzed in the absence of NO. An unidentified peak with m/z of 282.0 was also observed in the presence of NO.

Effect of NO on Methylmalonyl-CoA Mutase Activity in Vivo
To determine whether NO inhibited methylmalonyl-CoA mutase activity in vivo, we treated BHK cells with DETA-NONOate, which releases NO with a half-life of about 20 h at 37 °C, pH 7.4 (36). As discussed under "Experimental Procedures," the standard method for measuring methylmalonyl-CoA mutase activity in vivo is to follow [1-14C]propionic acid incorporation into acid-precipitable material (29–31, 33). This assay assesses the activity of enzymatic reactions in addition to the mutase reaction, and we, therefore, simultaneously measured [1,4-14C]succinic acid incorporation into acid-precipitable material in replicate cultures. By comparing results between propionic and succinic acid incorporation, we could concentrate on changes in mutase activity. We found that 1 mM DETA-NONOate inhibited both propionic acid and succinic acid incorporation into acid-precipitable material, but that inhibition of propionic acid incorporation was significantly more than that of succinic acid incorporation (Fig. 5A, DETA-NONOate reduced propionic acid incorporation by 53 ± 4% and succinic acid incorporation by 39 ± 5%). DETA-NONOate inhibited [methyl-14C]methylmalonic acid incorporation into acid-precipitable material to a similar extent as it inhibited [1-14C]propionic acid incorporation, indicating that it appeared to have no effect on propionyl-CoA conversion to methylmalonyl-CoA. As mentioned in the previous section, the actual NO concentration in the tissue culture medium was likely to be considerably less than 1 mM (even though DETA-NONOate releases 2 mol of NO per mol) because of preferential partitioning of NO into the gaseous phase, which was in free equilibrium with ambient air. Other workers have found that 1 mM DETA-NONOate is required to yield NO levels similar to those produced by cytokine-treated cells (45).

Effect of Decreased Availability of Endogenous NO on Methylmalonyl-CoA Mutase Activity
Because NO inhibited methylmalonyl-CoA mutase activity both in vitro and in vivo, it seemed possible that endogenously produced NO could inhibit methylmalonyl-CoA mutase activity. To evaluate this possibility, we quenched endogenously produced NO by treating cells with the NO scavenger hemoglobin or decreased endogenous NO production using the NO synthase inhibitor L-NAME, and then measured enzyme activity in vitro. We found that treating BHK cells with either 10 µM hemoglobin or 1 mM L-NAME for 16 h prior to cell extraction significantly increased methylmalonyl-CoA mutase activity (Fig. 5B, compare diagonally striped bars and solid bars to open bars). Because L-NAME is a competitive inhibitor of NO synthase with respect to arginine, the experiments with this agent were performed at a reduced arginine concentration of 100 µM. These data could potentially be explained by endogenous NO inhibiting protein synthesis. However, we found no significant effect of hemoglobin or L-NAME on rates of protein synthesis as measured by L-[4,5-3H]leucine incorporation into acid-precipitable material. Thus, scavenging NO from cells or inhibiting cellular NO synthesis increased methylmalonyl-CoA mutase activity, suggesting that endogenous NO inhibits enzyme activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Methylmalonyl-CoA mutase occupies a key position in the pathway converting propionyl-CoA to succinyl-CoA, with the catabolism of isoleucine, methionine, threonine, and valine, as well as of cholesterol, odd chain fatty acids, thymine, and uracil leading to propionyl-CoA production. The enzyme is, therefore, part of a gluconeogenic pathway for converting amino acids, lipids, and pyrimidines to carbohydrates (46). Genetic deficiency of methylmalonyl-CoA mutase activity or of AdoCbl synthesis leads to the disease state of methylmalonic acidemia. These patients present early in life with metabolic ketoacidosis, developmental retardation, and in severe cases die as neonates (46).

We found that NO inhibited methylmalonyl-CoA mutase, both in vitro in cell extracts and in vivo in intact rodent cells. Evidence that endogenous NO may serve to regulate methylmalonyl-CoA mutase activity is suggested by the experiments in which scavenging NO by hemoglobin and inhibiting NO synthesis by L-NAME increased methylmalonyl-CoA mutase activity. The increase in enzyme activity could be from decreased inhibition of newly synthesized enzyme. Methylmalonyl-CoA mutase is a mitochondrial matrix enzyme that could be exposed to NO by passive diffusion of the gas from the cytoplasm into the mitochondria. However, in addition to NO production by cytoplasmic and plasma membrane-bound NO synthases, NO is also produced by a recently described mitochondrial NO synthase. The latter form of NO synthase accounts for 50% of cellular NO production in rat liver, and low micromolar concentrations of NO have been found in rat heart mitochondria (47, 48). Thus, methylmalonyl-CoA mutase may be exposed to sufficiently high NO concentrations for NO to serve as an endogenous regulator of enzyme activity.

NO reacts with cysteine residues of several proteins including hemoglobin and the small GTPase Ras, thereby yielding nitrosothiols groups (49, 50). Our data suggest that this mechanism is unlikely the cause of NO inhibiting mammalian methylmalonyl-CoA mutase because of the slow rate of the NO reaction with cysteines and the lack of protection of enzyme activity by thiol reagents. Moreover, in a recent study of S-nitrosylation of mitochondrial proteins, Foster and Stamler (51) identified five major proteins that underwent S-nitrosylation after treating rat liver mitochondria with DEA-NONOate or S-nitrosoglutathione, but these did not include methylmalonyl-CoA mutase. Thus, it appears that NO inhibits methylmalonyl-CoA mutase activity by reacting with Cbl(II) or the deoxyadenosyl radical, or more likely with both. These two intermediates are generated transiently at the active site, which from crystallographic studies of the P. shermanii enzyme has been shown to be buried at the base of a narrow ({alpha}{beta})8 TIM barrel (20, 21). After the substrate enters the barrel, the barrel closes in around the substrate, implying that the active site should be inaccessible to other molecules (21). However, this may not apply to non-polar diatomic molecules like oxygen or NO, as oxygen has been shown to react with Cbl(II) generated by the P. shermanii enzyme, albeit at a slow rate (40). Mutation of the P. shermanii mutase at histidine 244 markedly increases the sensitivity of the enzyme to oxygen, indicating that this residue is critical for protecting the Cbl(II) and deoxyadenosyl radical intermediates (40, 41). Modeling the possible structure of the homodimeric human enzyme against the known structure of the heterodimeric P. shermanii enzyme using the Swiss Model program suggests that the active sites of the two enzymes are similar, but such modeling can overlook subtle structural differences that could lead to increased sensitivity to NO and oxygen (52).

At equimolar concentrations, NO was a more potent inhibitor of mutase activity than oxygen, and there are at least two possible reasons for this observation. First, the reactivity of NO with Cbl(II) is much greater than the reactivity of oxygen with Cbl(II), with removal of NO from cobalamin requiring the use of an NO trap, whereas oxygen can be removed from cobalamin simply by passing argon over the complex (14, 53). In this respect, Cbl(II) is similar to heme that has a much higher affinity for NO than for oxygen (54). Second, in the case of ethanolamine deaminase, another AdoCbl requiring enzyme, the protein cage around the cobalamin maintains it in the Cbl(II) state, which would favor the formation of Cbl(II)-NO and inhibit the formation of Cbl(III)-, the latter being the product of oxygen reaction with Cbl(II) (55). A similar conclusion for methylmalonyl-CoA mutase can be drawn from recent work by Vlasie and Banerjee (56) who found that the protein exercises control over internal electron transfer in the prosthetic group (56). Thus, one would anticipate that NO would be a more effective inhibitor of methylmalonyl-CoA mutase activity than oxygen.

The finding that NO can react with Cbl(II) and the deoxyadenosyl radical derived from AdoCbl was not apparent a priori because in the case of methylcobalamin, the second order rate constant for recombination of the methyl radical with Cbl(II) is ~2 x 109 M–1 s–1, and in the protein cage provided by the enzyme, geminate recombination also becomes important (kgeminate recombination 1 x 109 s–1 (18)). Therefore, at low concentrations (i.e. <10 µM), NO might not be able to compete with such a high reaction rate because its rate of reaction with Cbl(II) is somewhat lower at 0.5 x 108 M–1 s–1 (14). Similarly, for NO to react with the deoxyadenosyl radical, it has to compete with cyclization of the radical to 8,5'-anhydro-5-deoxyadenosine, the predominant species formed in the absence of NO (42, 43). We found that approximately half of the NO was reacting with Cbl(II), the other half reacting with the deoxyadenosyl radical. The latter data suggest that the rate constant for the reaction of NO with the deoxyadenosyl radical is also around 108–109 M–1 s–1, similar to the rate constant for the reaction of TEMPO with the deoxyadenosyl radical (3–5 x 108 M–1 s–1 (42, 43)).

Nitrous oxide, another oxide of nitrogen commonly used as an anesthetic, inhibits methylmalonyl-CoA mutase activity both in cultured cells and whole animals, but prolonged exposure (2–16 days) to extremely high concentrations (gaseous mixtures containing 50% nitrous oxide) is required to significantly reduce enzyme activity (57, 58). Thus, both NO and nitrous oxide inhibit methylmalonyl-CoA mutase activity, but NO appears to be a much more potent inhibitor.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant CA90932 (to G. R. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by National Institutes of Health Training Grant CA81211. Back

§ To whom correspondence should be addressed: Dept. of Medicine, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0652. Tel.: 858-534-8805; Fax: 858-534-1421; E-mail: gboss{at}ucsd.edu.

1 The abbreviations used are: AdoCbl, adenosylcobalamin; BHK, baby hamster kidney cells; Cbl(I) and Cbl(II), cobalamin in the +1 or +2 valency state, respectively; DEA-NONOate, 2-(N,N-diethylamino)-diazenolate-2-oxide; DETA-NONOate, (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; DME medium, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; HPLC, high performance liquid chromatography; L-NAME, NG-nitro-L-arginine methyl ester; MEM medium, Eagle's minimal essential medium; NO, nitric oxide; OH-Cbl, hydroxycobalamin; TEMPO, 2,2,6,6-tetramethylpiperidinyl-1-oxy. Back



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