NEW ASPECTS OF THE STRUCTURE OF CORRINOID COENZYMES Fritz Wagner and Konrad Bernhauer Lehrstuhl fur Biochemie der Technischen Hochschule, Stuttgart, Germany Recently the structure of the cobalamin-coenzyme has been elucidated by X-ray analysis1 and has been confirmed by partial s y n t h e ~ i s . ~ - ~ ~ With both methods, however, it was not possible to estimate the precise struc- ture of the chromophore in the corrin nucleus. We have been able to show that the reactivity of the corrin nucleus in the corrinoid coenzymes is greatly different from that in the cyano- or hydroxo-forms. Investigations by Todd and coworkers* have shown that cyano- cobalamin is converted to cyano-dehydrocobalamin by oxidation with air in alkaline solution (FIGURE 1). Under the same experimental conditions 60 per cent of the cobalamin-coenzyme is unchanged and 40 per cent is converted in the first step to hydroxo-cobalamin which is rapidly oxidized to hydroxo-dehydrocobalamin. The reaction mixture was separated by paper electrophoresis in the presence of 0.2 per cent NaHS03 in acetic acid solution of pH 2.5. The hydroxo-dehydrocobalamin was identified and the coenzyme fraction was decomposed with cyanide. This fraction showed the same activity as cyano-cobalamin by the E. coli mutant tube assay and is also identical with cyano-cobalamin by paper chromatography in four different solvents. With [ Co] -methyl-cobalamin or cobalamin-sulf~nate,~ however, no reaction takes place. In both cases, 95 per cent remain unchanged and only traces of deaminated products of [ Co] -methyl-cobalamin or cobala- min-sulfonate are produced. Both unchanged compounds showed after treatment with cyanide by the E. coli mutant tube assay the same activity as cyano-cobalamin and are identical with it by paper chromatog- raphy and ultraviolet and visible absorption. It is noteworthy that the carboxamide groups in cobalamin-coenzyme, [ Co] -methyl-cobalamin and cobalamin-sulfonate are greatly resistant to hydrolysis ( FIGURE 2). Treatment of cyano-cobalamin with an equimolar amount of chloramine T or bromine water at pH 4 yielded cobalamin-lactone which contains no halogen.* Further treatment with chloramine T led to chlorine containing cobalamin-lactone, but this substance was not fully characterised ( FIGURE 1). After the reaction of cyano-dehydrocobalamin with 1 mol of chlora- mine T it was possible to isolate a crystalline compound which contains one atom of chlorine and the structure was postulated as 10-chloro- dehydr~cobalamin.~ In contrast to these results the halogenations of cobalamin-coenzyme, [Co]-methyl-cobalamin and cobalamin-sulfonate give quite different prod- ucts. When [ Col-methyl-cobalamin is treated with an equimolar amount of chloramine T in 0.0025 M HC1 it is possible to isolate a crystalline compound (95 per cent) containing the [Col-methyl bond intact and one 580
Transcript
NEW ASPECTS OF THE STRUCTURE OF CORRINOID COENZYMES
Fritz Wagner and Konrad Bernhauer Lehrstuhl fur Biochemie der Technischen Hochschule, Stuttgart, Germany
Recently the structure of the cobalamin-coenzyme has been elucidated by X-ray analysis1 and has been confirmed by partial s y n t h e ~ i s . ~ - ~ ~ With both methods, however, it was not possible to estimate the precise struc- ture of the chromophore in the corrin nucleus. We have been able to show that the reactivity of the corrin nucleus in the corrinoid coenzymes is greatly different from that in the cyano- or hydroxo-forms.
Investigations by Todd and coworkers* have shown that cyano- cobalamin is converted to cyano-dehydrocobalamin by oxidation with air in alkaline solution (FIGURE 1). Under the same experimental conditions 60 per cent of the cobalamin-coenzyme is unchanged and 40 per cent is converted in the first step to hydroxo-cobalamin which is rapidly oxidized to hydroxo-dehydrocobalamin. The reaction mixture was separated by paper electrophoresis in the presence of 0.2 per cent NaHS03 in acetic acid solution of pH 2.5. The hydroxo-dehydrocobalamin was identified and the coenzyme fraction was decomposed with cyanide. This fraction showed the same activity as cyano-cobalamin by the E . coli mutant tube assay and is also identical with cyano-cobalamin by paper chromatography in four different solvents.
With [ Co] -methyl-cobalamin or cobalamin-sulf~nate,~ however, no reaction takes place. In both cases, 95 per cent remain unchanged and only traces of deaminated products of [ Co] -methyl-cobalamin or cobala- min-sulfonate are produced. Both unchanged compounds showed after treatment with cyanide by the E. coli mutant tube assay the same activity as cyano-cobalamin and are identical with it by paper chromatog- raphy and ultraviolet and visible absorption. It is noteworthy that the carboxamide groups in cobalamin-coenzyme, [ Co] -methyl-cobalamin and cobalamin-sulfonate are greatly resistant to hydrolysis ( FIGURE 2) .
Treatment of cyano-cobalamin with an equimolar amount of chloramine T or bromine water at pH 4 yielded cobalamin-lactone which contains no halogen.* Further treatment with chloramine T led to chlorine containing cobalamin-lactone, but this substance was not fully characterised ( FIGURE 1). After the reaction of cyano-dehydrocobalamin with 1 mol of chlora- mine T it was possible to isolate a crystalline compound which contains one atom of chlorine and the structure was postulated as 10-chloro- dehydr~cobalamin.~
In contrast to these results the halogenations of cobalamin-coenzyme, [Co] -methyl-cobalamin and cobalamin-sulfonate give quite different prod- ucts. When [ Col-methyl-cobalamin is treated with an equimolar amount of chloramine T in 0.0025 M HC1 it is possible to isolate a crystalline compound (95 per cent) containing the [Col-methyl bond intact and one
FIGURE 1. Oxidation of cyano- or hydroxo-cobalamin.
0.1 N NaOH IOO'C 10min. + Air 95% Unchanged *
R = C H j >5% Deamidrted products of
HjC,. ,CHz-CONHz [Go] -Methyl -CObJlJmin a N' \N -CHz-CONHz Same R = Conditiom SO3H
I
95% Unchanged
>5% Dermidrted products of Cobalamin-sulfonic acid
60% Unchanged 40% Hydroao-cobalrmin, which reacts to Hydroro- dahydro- cobdamin
R = C; - Adenosyl
I I Nucleotide
FIGURE 2. Behaviour of [Col-methyl-cobalamin, cobalamin-sulfonate and cobala- min-coenzyme against alkali in air.
582 Annals New York Academy of Sciences atom of chlorine. Only a small amount ( 5 per cent) of an amorphous chlorine-containing lactone is produced. The chlorinated [ CO] -methyl- cobalamin has a marked bathochromic shift in the visible spectrum (TABLE 1 ) . This product is converted on light with cyanide to a crystal-
TABLE 1 THE INFLUENCE OF CHLORINE SUBSTITUTION IN THE CHROMOPHORE
line compound. The elemental analysis indicates that the chlorination product is a monocyano-monochloro-cobalamin. Its spectral characteristics are very similar to those of 10-chloro-dehydrocobalamin (TABLE 1 ) . The electrophoretical, chromatographical and spectral behaviour suggest that the chlorine attack on the chromophore of [ Co]-methyl-cobalamin led to [ Co] -methyl-10-chloro-cobalamin and after treatment with cyanide on light to cyano-10-chloro-cobalamin.
By the similar chlorination with an equimolar amount of chloramine T both cobalamin-coenzyme and cobalamin-sulfonate yield the correspond- ing 10-monochloro-derivatives. Cyanide converts these products to the same crystalline cyano-10-chloro-cobalamin which was obtained from [ Co] -methyl-l0-chloro-cobalamin ( FIGURE 3 ) .
[ Co] -methyl-cobalamin or cobalamin-coenzyme when treated with 3 equivalents of chloramine T yielded in both cases 6&70 per cent of the corresponding 10-chloro compound and 30-40 per cent of chloro-lactones which were not further characterised.
It is remarkable that the 10-chloro-cobalamin-coenzyme and the [ Co]- methyl-10-chloro-cobalamin have the same great difference in spectra be- tween the red and yellow forms as cobalamin-coenzyme and [ Col-methyl- cobalamin respectively (TABLE 1). Williams and coworkers6 postulated
FIGURE 3. Behaviour of [ Co] -methyl-cobalamin, cobalamin-coenzyme and co- balamin-sulfonate against chloramine T.
that this reversible change may be due to a protonation of a methene bridge Cn, Clo or CIS. The spectra of the 10-chloro-corrinoid-coenzymes show that a protonation at Clo is not possible.
The above experiments about the aerial oxidations and halogenations indicate that the chromophore in the corrinoid-coenzymes and in the cobalamin-sulfonate has a very different reactivity compared with the chromophore of cyano- or hydroxocorrinoids. These differences cannot result only from the ligands, because for exampIe the cyano- and sulfonate group are both electron attracting and the methyl or C-Y-deoxyadenosyl-
584 Annals New York Academy of Sciences group are electron repelling, but these ligands behave similarly to cobalamin-sulfonate. In the chromophore of the corrinoid-coenzymes or cabalamin-sulfonate the Clo-position seemed to be activated against halo- genations and not the C8-position as in the cyano- or hydroxo-corrinoids. In view of these results it seems possible that the six double bonds in the coenzyme-forms are de-conjugated or one of the double bonds is hydro- genated.
It has been reported' that if hydroxo-cobalamin is mixed in the dark with 7-picoline the spectrum of the resultant solution is similar to those of corrinoid-coenzymes. The authors concluded that the conjugated double bonds in the corrinoid-coenzymes are broken to form B (FIGURE 4). How-
FIGURE 4. Forms of corrinnucleus.
ever, if form B already exists in the coenzymes it should not be possible to obtain the coenzyme of dehydrocobalamin. But it has been possible to prepare the coenzyme forms of dehydrocobalamin, 10-chloro-dehydro- cobalamin and also the [ Co] -methyl-dehydrocobalamin and [ Co] -methyl- 10-chloro-dehydrocobalamin in the usual chemical ~ a y . ~ - ~ ~ By the reaction of these coenzyme forms with cyanide we have obtained in all cases the corresponding cyano-dehydrocobalamin or cyano-10-chloro-dehydro- cobalamin. The coenzymes were also obtained by fermentation of hydroxo- dehydrocobalamin and hydroxo-10-chloro-dehydrocobalamin with grow- ing cultures of P . shermanii in 10 to 20 per cent yield. These results strongly suggest that there is no double bond between C8 and C9 in the corrinoid-coenzymes.
In agreement with Pratt and Williams7 we have found that hydroxo- cobalamin gives the described spectral shift on reaction with y-picoline. However, hydroxo-dehydrocobalamin and hydroxo-10-chloro-dehydro- cobalamin do not give this spectral shift under the same conditions. It is possible that only the hydroxo-corrinoids can exist in the discussed two forms A and B (see FIGURE 4).
Wagner & Bernhauer : Corrinoid Coenzymes 585 All derivatives shown in FIGURE 5 are inactive in the E.co1i mutant tube
assay, only [ Co] -5’-deobyandenosyl-dehydrocobalamin shows a non-com- petitive inhibition.
All the above results could be explained by postulating that the double bond in the chromophore between NZl-Cg is hydrogenated in the cor- rinoid-coenzymes. Such a structure could explain that the H-atom on Cs has lost the “activation” caused by carbon-nitrogen double bond. We hoped to decide this question by studies about the preparation of [Col- methylcobalamin in tritiated water.
R z Methyl or C; - deoxyadenosyl
I : X = N H ; Y = H
II : X=NH; Y = C I
FIGURE 5. Coenzyme like forms of dehydrocobalamin.
When cyano-cobalamin was reduced with Zn/acetic acid in the presence of TOH and then alkylated with dimethylsulfate a tritium-containing [ Co]-methyl-cobalamin was obtained in good yield. This [ Co] -methyl- cobalamin which has been washed to constant activity loses the whole incorporated radioactivity when exposed to light in the presence of air. Basing on this result and by the behaviour on alkaline oxidation and halogenation we suggested in a preliminary notes that in the corrinoid- coenzymes the N21-Cg double bond was hydrogenated. This view now requires modification.
Further investigations of the aerobic photolysis of [ Co] -methyl- cobalamin have shown that the Co-methyl bond is split by light in a limited amount of air to yield hydroxocobalamin and formaldehyde. The latter substance was estimated with dimedone. The resulting dimedone- formaldehyde condensation product obtained in nearly quantitative yield was identical with an authentic sampIe. A re-examination of this reaction with tritium-labeled [ Co]-methyl-cobalamin has shown that the photolysis in the presence of air and dimedone gives inactive hydroxo- cobalamin and nearly all the radioactivity there is surprisingly in the dimedone-formaldehyde condensation product. These results suggest that the tritium content in the [Col-methylcobalamin is in the methyl group
586 Annals New York Academy of Sciences
(FIGURE 6) and probably no doubIe bond is hydrogenated in the chromo- phore of [ Co] -methylcobalamin. On the other hand the results indicate that the H-atom of the methyl group bonded to the cobalt are more active than those of methyl groups in the corrin nucleus.
It should be briefly mentioned that in order to obtain more details about the reactivity of the corrinoids we looked at reactions with Grignard reagents and mercaptans.
OH H(T) Zn/CH&OOH
TOH AO]@ -
Nucleotide'
Hydroiocobalamin Hydridocobalamin
I I Nucleotide'
[Col- Methyl- Cobalamin ~ o o ~ c / ~ . ~ x I o - ~ ~ o I
h u + 02 + Dimedone I
Dimedone-Formaldehyde Adduct 180pC/7.5 x 10-5moI
Formaldehyde Hydroxocobalamin
FIGURE 6. Synthesis of [Col-methyl-cobalamin in presence of TOH and photolysis of it.
Cobalamin, cobinamide or cobyrinic acid itself is converted into cobyrinic acid-ethylester by heating in ethanol in the presence of strong acids. The structure of this ester was determined by elemental analysis and by alkaline hydrolysis to the free cobyrinic acid. The ultraviolet and visible spectra of this ester were like those of cobinamide with and with- out cyanide, suggesting the chromophoric system was still unchanged. The infrared spectrum in KBr shows a new band at 1725 cm-l which is consistent with the presence of ester groups.
The dicyano-cobyrinic acid heptaethylester prepared as above was then reduced with Zn/acetic acid and alkylated with methyl iodide to yield a [ Co] -methyl-cobyrinic acid heptaethylester. This compound has an almost identical absorption spectrum to that of [ CO] -methyl-cobinamide and behaves in its reaction to light and cyanide typically coenzymic. When treated with excess methyl magnesium iodide in ether/tetrahydrofuran it was converted into the correspondent hepta-tert.alcoho1. This is iden- tical in physical and chemical properties with a compound obtained directly from the dicyano-cobyrinic acid heptaethylester and methyl magnesium iodide (FIGURE 7) . A similar direct alkylation of the cobalt
Wagner & Bernhauer : Corrinoid Coenzymes 587
H3C‘ H3C‘
FIGURE 7 . Two routes of the synthesis of [Col-methyl-hydroxo-heptatert. alcohol from cobyrinic acid heptaethylester.
588 Annals New York Academy of Sciences has been found to be possible using lithium-alkyls. This seems to indicate that tervalent cobalt is capable of being alkylated by an alkyl anion unlike the previous which has required electrophilic reagents. The reaction also suggests strongly that in the coenzyme-forms the cobalt atom is in the +I11 state.
The [ Co] -methyl-hepta-tert.alcoho1 obtained by the two routes men- tioned above was then investigated by chromatography, electrophoresis, and infrared spectrum. As a result its properties were found to be con- sistent with the structure shown in FIGURE 7 . On photolysis in water or chloroform it gave a product resembling diaquo-cobinamide but in ethanol or tetrahydrofuran photolysis in the presence of air resulted in a stable yellow compound with an absorption spectrum like that of cobina- mide R (the analogue of BIxr).
OH2 I
NuclebtideQ
pH 1-10 GSH I
r[ t o I" Nucleot idee
FIGURE 8. Synthesis of [Co]-methyl-cobalamin complex.
+SG 1 Nucleotide r[ F I:]
over the glutathione-cobalamin
Wagner & Bernhauer : Corrinoid Coenzymes 589
Another interesting synthesis of corrinoid coenzymes has been found in the reaction of hydroxo-corrinoids with mercaptans. When hydroxo- cobalamin is treated with an excess of glutathione a purple compound is formed which reacted in good yields with alkylating agents to give co- enzyme analogues with the alkyl group directly linked to cobalt (FIGURE 8 ) . The purple compound was chromatographically and electrophoreti- cally homogeneous and relatively stable in light and air. The main absorp- tion maxima are at 287,337, 374, 434, and 536 mp. This spectrum does not change with pH between 1-10. The electrophoretical behaviour indicates a 1:l molar complex of glutathione and cobalamin. The complex is neutral at pH 2.5 and shows one negative charge from pH 4-7 and two negative charges at pH 11. It is possible that this reaction may be a model for the first step in the biological pathway of the corrinoid-coenzymes. The work is being continued.
Acknowledgments
This work was done together with P. Renz and P. Rietz to both of whom we wish to express our thanks.
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5. K. BERNHAUER & 0. WAGNER. 1963. Biochem. Z. 337: 366. 6. J. A. HILL, J. M. PRATT & R. J. P. WILLIAMS. 1962. J. Theoretical Biol. 3: 423. 7. J. M. PRATT & R. J. P. WILLIAMS. 1961. Biochim. Biophys. Acta 46: 191. 8. F. WAGNER & P. RENZ. 1963. Tetrahedron letters: 259.
