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An electron-spin resonance study of coenzyme B12
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
An Electron-Spin
100,
Resonance
Study
H. P. C. HOGENKAMP2 From
the Department
of Biochemistry,
(1963)
353-359
AND
University
of Coenzyme
B,,
H. A. BARKER of California,
Uerkeley,
California
,4ND
H. S. MASOK From
the Depmtment
of Biochemistry, Received
Z’niversity
of Oregon
November
Medicul
School,
Portland,
Oregon
16, 1962
The absence of electron-spin resonance spectra from three different samples of coeneyme BU either in the solid crystalline state or in aqueous solution suggests t,hat coenayme Brz is diamagnetic. These results agree with several magnetic susceptibility measurements and with the chemical degradation studies; they are, however, in disagreement with other magnetic susceptibility measurements which indicate t,hat coenzyme Biz is paramagnetic. In contrast, electron-spin resonance spectra of coenzyme Biz photolyzed under anaerobic condition indicate that the photolysis product is paramagnetic. Since this spectrum is identical to that obtained for vitamin Bigr, it has been concluded that the anaerobic photolysis product contains bivalent cobalt. Furthermore, exposure of the anaerobic photolysis product or vitamin Bizr to oxygen completely abolishes the electron-spin resonance spectra. This observation is in accord with the magnetic susceptibility studies of aquocobalamin, known to be the product of this oxidation reaction. These results are interpreted to mean that coenzyme Bi2 does not, contain bivalent cobalt. Consequently, the photolytic decomposition of coenzyme Bie must involve an intramolecular oxidation reduction or a homolytic cleavage of the carbon-cobalt linkage. The electron-spin resonance spectra do not establish the valence state of cobalt in coenzyme RI?; however, the absence of a signal suggests that coenzyme B,? is diamagnetic and consequently contains trivalent cobalt.
Vitamin HI2 (cyanocohalamin) is diamagnetic (l-3). From this property and x-ray diffraction studies (4), it has been concluded that the vitamin is a cobaltic complex with octahedral d2sp3bonding (3). The valence of the cobalt, atom has been confirmed by x-ray 1 This investigation was supported in part by research grants from the Sat,ional Institutes of Health (E-653, E-563 and A-971), U. S. Public Health Service, the Xat,ional Science Foundation (g-7599), and from the American Cancer Society, and by a research cont)ract with the U. S. Atomic Energy Commission. Station, 2 Present address : Technological Fisheries Research Board of Canada, 6610 S. W. Marine Drive, Vancouver, British Columbia. 353
spectroscopic measurements by Boehm et al. (5). On the other hand, the valence of cobalt in coenzyme B12is still unknown; its magnetic susceptibility has been the subject of conflicting reports. Bernhauer et al. (6) found that coenzyme B,, is diamagnetic in the solid crystalline state and paramagnetic in solution, the magnetic moment being 1.63 Bohr magnetons. Nowicki and Pawelkiewicz (7) made similar observations on aqueous solutions of the coenzyme form of cobinamide (Factor B) and reported a magnetic moment of 1.3 Bohr magnetons. B. B. Cunningham (unpublished observation) examined three highly purified crystalline coenzyme RI2 preparations from different
354
HOGENKAMP,
BARKER,
sources by the Faraday method and found them to be diamagnetic. In contrast, Johnson and Shaw (8) stated that a solid crystalline preparation obtained from E. R. Squibb and Sons was paramagnetic (1.8 Bohr magnetons). The same preparation was found to be paramagnetic by Kratochvil and Diehl (personal communication) using the Gouy method. Bernhauer et al. (6) suggested that the solid form of coenayme B12 exists as a dimer, in order to explain apparent differences in magnetism reported for the solid and dissolved states, and proposed that coenzyme B,z contains bivalent cobalt e-8). Studies on the degradation of coenzyme B12 by acid and cyanide suggest that coenzyme B,, contains trivalent cobalt. Alkaline cyanide treatment of coenzyme Blz yields dicyanocobalamin, adenine, and the cyanohydrins of D-erythro-2,3-dihydroxypenten-4-al, indicating a heterolytic cleavage of the carbon-cobalt bond (9). The same products are formed when the reaction is carried out in the absence of oxygen.3 Thus the cyanide ion takes the position of the 5-deoxyadenosyl moiety; the 5-deoxyadenosyl anion, the primary product of this reaction, is unstable and decomposes to adenine and the unsaturated sugar. No change in the valence of cobalt appears to be required for these reactions; this suggests that both coenzyme B,z and vitamin Blz contain trivalent cobalt. In view of the contradictory evidence concerning cobalt valence in coenzyme B12, we have undertaken an electron-spin resonance study of the coenzyme and the vitamin in the crystalline and dissolved states. We have also examined the changes in electron-spin resonance spectra which accompany the inactivation of the coenzyme by light. EXPERIMENTAL
PROCEDURE
MATERIALS Three crystalline preparations of coenzyme Bit were used. Preparation DVP2 was isolated from Propionibacterium shermanii (10) ; preparation PFl, obtained from D. R. Walters and D. Perlman, 3 H. P. unpublished
C. Hogenkamp results.
and
H.
A.
Barker,
AND
MASON
Squibb Institute for Medical Research, was isolated from P. freudenreichii; and preparation ML2, obtained from K. Folkers, Merck, Sharp and Dohme Research Laboratories, was isolated from an unidentified microorganism. Both the Squibb and Merck preparations were rechromatographed on a Dowex 50 column and recrystallized by previously described methods (10) before use. The three coenzyme preparations were initially free of colored or ultraviolet light-absorbing impurities as judged by the uniformity of their absorption spectra and their homogeneity in paper chromatographic and ionophoretic tests (10, 11). All t.he preparations had been stored for several months to several years in the dark at -10”. Vitamin B,,, was prepared from vitamin Bls (Nutritional Biochemicals) by catalytic hydrogenation (12).
METHODS Electron-spin resonance spectra were obtained with a Varian V4500 x-band spectrometer using loo-kc. field modulation, and modulation amplitudes of 0.2-16.0 gauss. Klystron frequencies were determined wit)h a Hewlett-Packard K532B frequency meter, and field strengths were monitored through a I’arian flux meter with a HewlettPackard 524C electronic frequency counter. The concentrations of unpaired electrons were determined with 5 rnM CuSOa-EDTA in water as a standard with all conditions fixed, including sample geometry and temperature. Spectra were recorded as t,he derivative of absorption with respect to field st’rength. RESULTS
ELECTRON-SPIN RESONAWE SPECTRA OF THREE CRYSTALLINE COENZYME Blz PI~EPARATIONY
Figure 1 depicts the electron-spin resonance spectra of three crystalline coenzyme B,, samples. The signals were observed with samples at - 165”. Even at high gain (500X) sample M gave no signal. The other two samples showed similar complex signals 130 gauss wide; one component appeared to be a narrow line, peak-to-peak width 90 gauss, with a g value at about 2.04. The concentrations of unpaired electrons in these samples were calculated by comparing the double integrals of the signals with that of cupric ethylenediamine tetraacetate observed under identical conditions, and were found to be, PFl, 3.9 %, and DVPs, 1.2 % of the concentration of cobalt present. This indicates the
ESR STUDY
OF COEWYME
B,z
355
FIG. 1. Electrtrn-spin resonance spectra of crystalline coenzyme B,, preparations ML2, PFl, and IIVP~. Field modulation amplitude: IF gauss. This and subsequent spectra are recorded as derivatives of the actual absorption with respect to field strengt.11.
absence of unpaired electrons in the coenzyme HI2 component of the samples, which appear to contain small amounts of unidentified tram&ion elementSs or an organic free radical. IPITOTOLYYIS
in
OF COENZYME soLuTIorvs
ES,1
-1. solution of coenzyme HI., (PFl), 7.1 i-d4 deionized and deoxygcnatcd water,
prepared in the dark, was placed in an electron-spin resonance quartz sample tube and cooled to - 190” in liquid nitrogen. The frozen sample, kept in liquid nitrogen in a narrow unsilvered Dewar vessel, was exposed to illumination from a 300-w. General Electric Co. Refl&or Spotlight at a distance of 30 cm.; a barely det,ectable signal having a width of 2500 gauss centered around 9 = 2.a appeared after 3 min. exposure (Fig,
356
HOGENKAMP,
BARKER,
AND MASON
FIG. 2. Changes occurring during the photolysis of coenzyme Blz. A solution of coenzyme Br2 (PFl, 7.1 m&f in deoxygenated, deionized water, 0.2 ml.) contained in a quartz sample tube, 2 mm. i.d., was illuminated (A) at -195” by a 300-w. lamp at 30 cm., for 3 min., gain 100 X; (B) at 0” with the same apparatus at 135 cm. for 1 min., gain 109 X; (C) at 0” for 5 min., gain 100 X; (D) at 0” for 46 min., gain 25 X. The field marker pips are 100 gauss apart..
ESR STUDY
OF
COENZYME B12
357
Fro. 3. Identity of the electron-spin resonance spectra of vitamin B12r and photolyzed coenzyme B~z. (A) 4.5 mM,BIPr in deoxygenated deionized water, gain 3.2 X ; (B) coenzyme Bls, 7.1 mM, photolyzed at 0” for 30 min. at a distance of 25 cm. from a 300-w. G. E. lamp under anaerobic conditions, gain 3.2 X ; (C) solution (A) after 5 min. bubbling with 02, gain 100 X ; (D) solution (B) after bubbling with 02 for 15 min., gain 32 X. The double integrals of (A) and (R) were119sq. cm. and 122sq. cm. per 5 mM CO+~, respectively.
211). If, however, the sample was illuminated in the liquid state at O”, and at a distance of 135 cm. from the lamp, a signal with g = 2.015 with a peak-to-peak width of 100 gauss appeared within a few seconds of illumination (sample retooled to -165” for electron-spin resonance observation (Fig. 2B). Upon further illumination under these conditions at O”, the signal was partially submerged in a much more intense signal about 1000 gauss wide, consisting of a major line at g,,, = 2.2, and several lines 100 gauss apart and centered at approximately g = 1.96 (Figs. 2C and 2D). After 46 min. of illumination, no further change in the electron-spin resonance spectrum was observed (Fig. 20). Evidence that the signal at g,,, 2.2 was in fact causedby a Co+2-containing molecule closely related to vitamin Ble was obtained by comparison with the electron-spin resonance spectrum of authentic vitamin B12r, which was identical to that of the
photolysis product of coenzyme B,, (Fig. 3). After photolysis was complete, the concentration of unpaired electrons was equivalent to 9.4 PM Co+*. On the assumption that the paramagnetic species responsible for the absorption contained only one unpaired electron, this is equivalent to 132 % of the coenzyme RI2 originally present. The concentration of unpaired electrons of vitamin BIZr was 4.0 PM, equivalent to 89% of the vitamin Bn reduced. When the photolysis product or vitamin B1?, was exposed to oxygen for a few minutes, the characteristic spectrum vanished. PHOTOLYSIS
OF COENZYME CRYSTALS
B,?
A number of attempts were made to stabilize and detect organic free radicals formed from coenzyme Blz during photolysis, but these were unsuccessful, except during photolysis of coenzyme B,, crystals at 0”. When the crpst’als, in a quartz electron-
358
HOGENKAMP,
BARKER,
AND MASON
FIG. 4. The electron-spin resonance spectrum of illuminated coenzyme B~z crystals. Sample PFl illuminated for 27 min. under the conditions described in the text,.
On the other hand, the final state of cobalt in coenzyme B,, photolyzed under anaerobic conditions is identical with that of . . . cobalt m vltamm BIZr (Fig. 3), which has, been established as a cobaltous state (14). Accordingly, the photolytic decomposition of coenzyme Blz must involve an intraDISCWSSION molecular oxidation-reduction, or a homoThree crystalline samples of coenzyme lytic splitting of a carbon-cobalt covalency. B 12, prepared from different sources in The electron-spin resonance signal of photodifferent laboratories, displayed no electron- lyzed coenzyme BIz or of vitamin B,z, should contain S-line hyperfine splittings spin resonance spectra commensurate with the amount of cobalt in the samples. These corresponding to the nuclear spin of 76 of results suggest that coenzyme B,, is dia- cobalt-59; what appears to be at least a magnetic; this presumption is in agreement six-line hyperfine splitting is centered at with the chemical degradation studies and g = 1.96, but because the samples were someof the magnetic susceptibility measure- amorphous or polycrystalline, the interpretaments. However, it should be pointed out tion of these spectra in terms of g values that our failure to detect a signal does not must be ambiguous; in any case the spectra prove the diamagnetism of coenzyme B12, resemble that already reported for cobaltous since several structural features (spin-spin phthalocyanine (15, 16). The signal is a low-spin or spin-lattice interactions) may so broaden therefore consistent with cobaltous state, but the experiments cannot an electron-spin resonance spectrum that it cannot be distinguished from electronic give unambiguous evidence concerning t’he reaction. noise in the detection system (13). The mechanism of the photolytic small absorptions observed with two of the During the initial stages of photolysis of three samples (Fig. 1) suggest that a small either solutions of coenzyme B,Z (Fig. 2) amount of paramagnetic impurities, possibly or crystals of coenzyme Blz (Fig. 4), there is both free radical and transition element, is clear evidence of the formation of an organic free radical having an electron-spin present in these samples.
spin resonance sample tube, are illuminated with a 300-w. lamp at 15 cm., a narrow signal at g = 2.00, peak-to-peak width about 40 gauss, develops simultaneously with the broad signal of the photolysis product. This is shown in Fig. 4.
ESR
resonance spectrum centered at this radical may be involved in tion of the two nucleosides which identified as products of the cleavage (17).
STUDY
OF
g = 2.0; the formahave been photolytic
REFERENCES 1. ~)IEHL, H., VANDER HAAR, R. W., AND SEA I.OCK, R. R., J. Am. Chem. Sot. 72, 5312 (1950). 2. GRUN, F. AND MENASSE, R., Experientia 6, 263 (1950). 3. WOLLMANN, J. C., CUNNINGHAM, B. B., AND CALVIN, M., Science 113, 55 (1951). 1. HODGKIN, 1). C., KAMPER, J., LINDSEY, J., MACKAY, M., PICKWORTH, J., ROBERTSON, J. H., SHOEMAKER, C. B., WHITE, J. G., PROSEX, R. J., AND TRUEBLOOD, K. N., Proc. Roy. Sot. (London) Ser. A 242, 228 (1957). 5. BOEHM, G., FAESSLER, A., AND RITTMAYER, G., Zeit. A’aturforsch. 9b, 509 (1954). 6. BERNHAGER, Ii., GAISER, P., MULLER, O., MULLER, E., AND GUNTER, F., Riochem. 2. 333, 560 (1961).
COENZYME
B12
359
7. NOWICKI, L., AND PAWELKIEWICZ, J., Bull. Acad. Polonaise Sci., Cl II 8, 433 (1960). 8. JOHNSON, A. W., AND SHAW, N., Proc. Chem. Sot. 1960, 420 (1960). 9. JOHNSON, A. W., AND SHAW, N., Proc. Chem. Sot. 1961, 447 (1961). 10. BARKER, H. A., SME-TH, R. D., WEISSBACH, H., TOOHEY, J. I., LADD, J. N., AND VOLCANI, B. E., J. Biol. Chem. 236, 480 (1960). 11. LADD, J. N., HOGENKAMP, H. P. C., AND BARKER, H. A., J. Biol. Chem. 236, 2114 (1961). 12. BRADY, R. O., AND BARKER, H. A., Biochem. Biophys. Res. Commun. 4, 464 (1961). 13. INGRAM, D. J. E., “Free Radicals as Studied by Electron Spin Resonance,” p. 102. Butterworth, London, 1958. 14. DIEHL, H., AND MURIE, R., Zowa State Coil. J. Sci. 23, 4555 (1952). 15. GIBSON, J. F., INGRAM, D. J. E., AND SCHONLAND, D., Discussions Faraday Sot. Ko. 26, 72 (1958). 16. GRIFFITH, J. 8., Discussions Faraday Sot. No. 26, 81 (1958). 17. HOGENKAMP, H. P. C., LAUD, J. N., AND BARKER, H. A., J. Biol. Chew 237, 1950 (1962).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
An Electron-Spin
100,
Resonance
Study
H. P. C. HOGENKAMP2 From
the Department
of Biochemistry,
(1963)
353-359
AND
University
of Coenzyme
B,,
H. A. BARKER of California,
Uerkeley,
California
,4ND
H. S. MASOK From
the Depmtment
of Biochemistry, Received
Z’niversity
of Oregon
November
Medicul
School,
Portland,
Oregon
16, 1962
The absence of electron-spin resonance spectra from three different samples of coeneyme BU either in the solid crystalline state or in aqueous solution suggests t,hat coenayme Brz is diamagnetic. These results agree with several magnetic susceptibility measurements and with the chemical degradation studies; they are, however, in disagreement with other magnetic susceptibility measurements which indicate t,hat coenzyme Biz is paramagnetic. In contrast, electron-spin resonance spectra of coenzyme Biz photolyzed under anaerobic condition indicate that the photolysis product is paramagnetic. Since this spectrum is identical to that obtained for vitamin Bigr, it has been concluded that the anaerobic photolysis product contains bivalent cobalt. Furthermore, exposure of the anaerobic photolysis product or vitamin Bizr to oxygen completely abolishes the electron-spin resonance spectra. This observation is in accord with the magnetic susceptibility studies of aquocobalamin, known to be the product of this oxidation reaction. These results are interpreted to mean that coenzyme Bi2 does not, contain bivalent cobalt. Consequently, the photolytic decomposition of coenzyme Bie must involve an intramolecular oxidation reduction or a homolytic cleavage of the carbon-cobalt linkage. The electron-spin resonance spectra do not establish the valence state of cobalt in coenzyme RI?; however, the absence of a signal suggests that coenzyme B,? is diamagnetic and consequently contains trivalent cobalt.
Vitamin HI2 (cyanocohalamin) is diamagnetic (l-3). From this property and x-ray diffraction studies (4), it has been concluded that the vitamin is a cobaltic complex with octahedral d2sp3bonding (3). The valence of the cobalt, atom has been confirmed by x-ray 1 This investigation was supported in part by research grants from the Sat,ional Institutes of Health (E-653, E-563 and A-971), U. S. Public Health Service, the Xat,ional Science Foundation (g-7599), and from the American Cancer Society, and by a research cont)ract with the U. S. Atomic Energy Commission. Station, 2 Present address : Technological Fisheries Research Board of Canada, 6610 S. W. Marine Drive, Vancouver, British Columbia. 353
spectroscopic measurements by Boehm et al. (5). On the other hand, the valence of cobalt in coenzyme B12is still unknown; its magnetic susceptibility has been the subject of conflicting reports. Bernhauer et al. (6) found that coenzyme B,, is diamagnetic in the solid crystalline state and paramagnetic in solution, the magnetic moment being 1.63 Bohr magnetons. Nowicki and Pawelkiewicz (7) made similar observations on aqueous solutions of the coenzyme form of cobinamide (Factor B) and reported a magnetic moment of 1.3 Bohr magnetons. B. B. Cunningham (unpublished observation) examined three highly purified crystalline coenzyme RI2 preparations from different
354
HOGENKAMP,
BARKER,
sources by the Faraday method and found them to be diamagnetic. In contrast, Johnson and Shaw (8) stated that a solid crystalline preparation obtained from E. R. Squibb and Sons was paramagnetic (1.8 Bohr magnetons). The same preparation was found to be paramagnetic by Kratochvil and Diehl (personal communication) using the Gouy method. Bernhauer et al. (6) suggested that the solid form of coenayme B12 exists as a dimer, in order to explain apparent differences in magnetism reported for the solid and dissolved states, and proposed that coenzyme B,z contains bivalent cobalt e-8). Studies on the degradation of coenzyme B12 by acid and cyanide suggest that coenzyme B,, contains trivalent cobalt. Alkaline cyanide treatment of coenzyme Blz yields dicyanocobalamin, adenine, and the cyanohydrins of D-erythro-2,3-dihydroxypenten-4-al, indicating a heterolytic cleavage of the carbon-cobalt bond (9). The same products are formed when the reaction is carried out in the absence of oxygen.3 Thus the cyanide ion takes the position of the 5-deoxyadenosyl moiety; the 5-deoxyadenosyl anion, the primary product of this reaction, is unstable and decomposes to adenine and the unsaturated sugar. No change in the valence of cobalt appears to be required for these reactions; this suggests that both coenzyme B,z and vitamin Blz contain trivalent cobalt. In view of the contradictory evidence concerning cobalt valence in coenzyme B12, we have undertaken an electron-spin resonance study of the coenzyme and the vitamin in the crystalline and dissolved states. We have also examined the changes in electron-spin resonance spectra which accompany the inactivation of the coenzyme by light. EXPERIMENTAL
PROCEDURE
MATERIALS Three crystalline preparations of coenzyme Bit were used. Preparation DVP2 was isolated from Propionibacterium shermanii (10) ; preparation PFl, obtained from D. R. Walters and D. Perlman, 3 H. P. unpublished
C. Hogenkamp results.
and
H.
A.
Barker,
AND
MASON
Squibb Institute for Medical Research, was isolated from P. freudenreichii; and preparation ML2, obtained from K. Folkers, Merck, Sharp and Dohme Research Laboratories, was isolated from an unidentified microorganism. Both the Squibb and Merck preparations were rechromatographed on a Dowex 50 column and recrystallized by previously described methods (10) before use. The three coenzyme preparations were initially free of colored or ultraviolet light-absorbing impurities as judged by the uniformity of their absorption spectra and their homogeneity in paper chromatographic and ionophoretic tests (10, 11). All t.he preparations had been stored for several months to several years in the dark at -10”. Vitamin B,,, was prepared from vitamin Bls (Nutritional Biochemicals) by catalytic hydrogenation (12).
METHODS Electron-spin resonance spectra were obtained with a Varian V4500 x-band spectrometer using loo-kc. field modulation, and modulation amplitudes of 0.2-16.0 gauss. Klystron frequencies were determined wit)h a Hewlett-Packard K532B frequency meter, and field strengths were monitored through a I’arian flux meter with a HewlettPackard 524C electronic frequency counter. The concentrations of unpaired electrons were determined with 5 rnM CuSOa-EDTA in water as a standard with all conditions fixed, including sample geometry and temperature. Spectra were recorded as t,he derivative of absorption with respect to field st’rength. RESULTS
ELECTRON-SPIN RESONAWE SPECTRA OF THREE CRYSTALLINE COENZYME Blz PI~EPARATIONY
Figure 1 depicts the electron-spin resonance spectra of three crystalline coenzyme B,, samples. The signals were observed with samples at - 165”. Even at high gain (500X) sample M gave no signal. The other two samples showed similar complex signals 130 gauss wide; one component appeared to be a narrow line, peak-to-peak width 90 gauss, with a g value at about 2.04. The concentrations of unpaired electrons in these samples were calculated by comparing the double integrals of the signals with that of cupric ethylenediamine tetraacetate observed under identical conditions, and were found to be, PFl, 3.9 %, and DVPs, 1.2 % of the concentration of cobalt present. This indicates the
ESR STUDY
OF COEWYME
B,z
355
FIG. 1. Electrtrn-spin resonance spectra of crystalline coenzyme B,, preparations ML2, PFl, and IIVP~. Field modulation amplitude: IF gauss. This and subsequent spectra are recorded as derivatives of the actual absorption with respect to field strengt.11.
absence of unpaired electrons in the coenzyme HI2 component of the samples, which appear to contain small amounts of unidentified tram&ion elementSs or an organic free radical. IPITOTOLYYIS
in
OF COENZYME soLuTIorvs
ES,1
-1. solution of coenzyme HI., (PFl), 7.1 i-d4 deionized and deoxygcnatcd water,
prepared in the dark, was placed in an electron-spin resonance quartz sample tube and cooled to - 190” in liquid nitrogen. The frozen sample, kept in liquid nitrogen in a narrow unsilvered Dewar vessel, was exposed to illumination from a 300-w. General Electric Co. Refl&or Spotlight at a distance of 30 cm.; a barely det,ectable signal having a width of 2500 gauss centered around 9 = 2.a appeared after 3 min. exposure (Fig,
356
HOGENKAMP,
BARKER,
AND MASON
FIG. 2. Changes occurring during the photolysis of coenzyme Blz. A solution of coenzyme Br2 (PFl, 7.1 m&f in deoxygenated, deionized water, 0.2 ml.) contained in a quartz sample tube, 2 mm. i.d., was illuminated (A) at -195” by a 300-w. lamp at 30 cm., for 3 min., gain 100 X; (B) at 0” with the same apparatus at 135 cm. for 1 min., gain 109 X; (C) at 0” for 5 min., gain 100 X; (D) at 0” for 46 min., gain 25 X. The field marker pips are 100 gauss apart..
ESR STUDY
OF
COENZYME B12
357
Fro. 3. Identity of the electron-spin resonance spectra of vitamin B12r and photolyzed coenzyme B~z. (A) 4.5 mM,BIPr in deoxygenated deionized water, gain 3.2 X ; (B) coenzyme Bls, 7.1 mM, photolyzed at 0” for 30 min. at a distance of 25 cm. from a 300-w. G. E. lamp under anaerobic conditions, gain 3.2 X ; (C) solution (A) after 5 min. bubbling with 02, gain 100 X ; (D) solution (B) after bubbling with 02 for 15 min., gain 32 X. The double integrals of (A) and (R) were119sq. cm. and 122sq. cm. per 5 mM CO+~, respectively.
211). If, however, the sample was illuminated in the liquid state at O”, and at a distance of 135 cm. from the lamp, a signal with g = 2.015 with a peak-to-peak width of 100 gauss appeared within a few seconds of illumination (sample retooled to -165” for electron-spin resonance observation (Fig. 2B). Upon further illumination under these conditions at O”, the signal was partially submerged in a much more intense signal about 1000 gauss wide, consisting of a major line at g,,, = 2.2, and several lines 100 gauss apart and centered at approximately g = 1.96 (Figs. 2C and 2D). After 46 min. of illumination, no further change in the electron-spin resonance spectrum was observed (Fig. 20). Evidence that the signal at g,,, 2.2 was in fact causedby a Co+2-containing molecule closely related to vitamin Ble was obtained by comparison with the electron-spin resonance spectrum of authentic vitamin B12r, which was identical to that of the
photolysis product of coenzyme B,, (Fig. 3). After photolysis was complete, the concentration of unpaired electrons was equivalent to 9.4 PM Co+*. On the assumption that the paramagnetic species responsible for the absorption contained only one unpaired electron, this is equivalent to 132 % of the coenzyme RI2 originally present. The concentration of unpaired electrons of vitamin BIZr was 4.0 PM, equivalent to 89% of the vitamin Bn reduced. When the photolysis product or vitamin B1?, was exposed to oxygen for a few minutes, the characteristic spectrum vanished. PHOTOLYSIS
OF COENZYME CRYSTALS
B,?
A number of attempts were made to stabilize and detect organic free radicals formed from coenzyme Blz during photolysis, but these were unsuccessful, except during photolysis of coenzyme B,, crystals at 0”. When the crpst’als, in a quartz electron-
358
HOGENKAMP,
BARKER,
AND MASON
FIG. 4. The electron-spin resonance spectrum of illuminated coenzyme B~z crystals. Sample PFl illuminated for 27 min. under the conditions described in the text,.
On the other hand, the final state of cobalt in coenzyme B,, photolyzed under anaerobic conditions is identical with that of . . . cobalt m vltamm BIZr (Fig. 3), which has, been established as a cobaltous state (14). Accordingly, the photolytic decomposition of coenzyme Blz must involve an intraDISCWSSION molecular oxidation-reduction, or a homoThree crystalline samples of coenzyme lytic splitting of a carbon-cobalt covalency. B 12, prepared from different sources in The electron-spin resonance signal of photodifferent laboratories, displayed no electron- lyzed coenzyme BIz or of vitamin B,z, should contain S-line hyperfine splittings spin resonance spectra commensurate with the amount of cobalt in the samples. These corresponding to the nuclear spin of 76 of results suggest that coenzyme B,, is dia- cobalt-59; what appears to be at least a magnetic; this presumption is in agreement six-line hyperfine splitting is centered at with the chemical degradation studies and g = 1.96, but because the samples were someof the magnetic susceptibility measure- amorphous or polycrystalline, the interpretaments. However, it should be pointed out tion of these spectra in terms of g values that our failure to detect a signal does not must be ambiguous; in any case the spectra prove the diamagnetism of coenzyme B12, resemble that already reported for cobaltous since several structural features (spin-spin phthalocyanine (15, 16). The signal is a low-spin or spin-lattice interactions) may so broaden therefore consistent with cobaltous state, but the experiments cannot an electron-spin resonance spectrum that it cannot be distinguished from electronic give unambiguous evidence concerning t’he reaction. noise in the detection system (13). The mechanism of the photolytic small absorptions observed with two of the During the initial stages of photolysis of three samples (Fig. 1) suggest that a small either solutions of coenzyme B,Z (Fig. 2) amount of paramagnetic impurities, possibly or crystals of coenzyme Blz (Fig. 4), there is both free radical and transition element, is clear evidence of the formation of an organic free radical having an electron-spin present in these samples.
spin resonance sample tube, are illuminated with a 300-w. lamp at 15 cm., a narrow signal at g = 2.00, peak-to-peak width about 40 gauss, develops simultaneously with the broad signal of the photolysis product. This is shown in Fig. 4.
ESR
resonance spectrum centered at this radical may be involved in tion of the two nucleosides which identified as products of the cleavage (17).
STUDY
OF
g = 2.0; the formahave been photolytic
REFERENCES 1. ~)IEHL, H., VANDER HAAR, R. W., AND SEA I.OCK, R. R., J. Am. Chem. Sot. 72, 5312 (1950). 2. GRUN, F. AND MENASSE, R., Experientia 6, 263 (1950). 3. WOLLMANN, J. C., CUNNINGHAM, B. B., AND CALVIN, M., Science 113, 55 (1951). 1. HODGKIN, 1). C., KAMPER, J., LINDSEY, J., MACKAY, M., PICKWORTH, J., ROBERTSON, J. H., SHOEMAKER, C. B., WHITE, J. G., PROSEX, R. J., AND TRUEBLOOD, K. N., Proc. Roy. Sot. (London) Ser. A 242, 228 (1957). 5. BOEHM, G., FAESSLER, A., AND RITTMAYER, G., Zeit. A’aturforsch. 9b, 509 (1954). 6. BERNHAGER, Ii., GAISER, P., MULLER, O., MULLER, E., AND GUNTER, F., Riochem. 2. 333, 560 (1961).
COENZYME
B12
359
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