- Email: [email protected]
The flavin of Chromatium cytochrome c -552
Vol. 44, No. 2, 1971
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
THE FLAVIN OF CHROMATIUM CYTOCRROME c-552 Robert Hendriks and John R. Cronin Department of Chemistry Arizona State University Tempe, Arizona 85281 Received May 18, 1971 SUMMARY. A flavin obtained from Chromatium cytochrome ~-552 has been purified and characterized with respect to its neutral and acid absorption spectra, fluorescence-pH relationship, and behavior on partition and molecular exclusion chromatography. The chromatographic results are consistent with a flavin adenine dinucleotide type structure. Neutral and acid absorption spectra taken after degradation of the flavin to the riboflavin level differ in the near DV from those of riboflavin and are similar to those of flavins modified in the dimethylisoalloxazine ring system, such as the histidyl-riboflavin obtained from mammaliansuccinate dehydrogenase. However, cytochrome c-552 flavin when at the riboflavin level and histidyl-riboflavin are quite dissimilar when compared fluorometrically and by molecular exclusion chromatography. Cytochrome c-552 as purified firmly
bound flavin
prosthetic
of cytochrome ~-552 with of urea, it exhibits
a
genase flavin flavin
a neutral
to that of FAD band (2).
of the absorption spectrum to that of the succinate dehydropeptide raised the possibility
link
tion of properties,
(2).
in concentrated urea solution
the further
has been carried
(3).
the
of the
is not characteristic
of
In view of this unusual combina-
characterization
of the cytochrome c-552
out.
on the modified (4) "heterotrophic"
harvested by centrifugation c-552 was isolated
of a covalent link joining
On the other hand, lability
MATERIALSARDMRTHODS. Chromatium strain bottles
in the cold and when freed
in the maximumof the near ultraviolet
the succinate dehydrogenase flavin
flavin
is released on treatment
absorption spectrum similar
and the cytochrome protein
flavln-protein
The flavin
group (1).
saturated urea solution
except for a 20 nm blue-shift The similarity
from Chromatium has been shown to contain a
D was grown in one litet (5).
Cells were
and stored as a frozen paste at -20".
Cytochrome
and purified
mediumof Fuller
prescription
through the first 313
DRAE-cellulose elution
Vol. 44, No. 2, 1971
BIOCHEMICAL
AND BIOPHYSICAL
according to the procedure of Bartsch (6).
RESEARCH COMMUNICATIONS
Flavin was dissociated
in cold,
saturated urea (2) and freed of hemeprotein by passage through a Sephadex 625
equilibrated
column
with saturated urea.
The flavln-containing
fractions
were combined and the urea removed by passage through a Sephadex G-15 column equilibrated
with 0.1 M acetic acid.
again combined, dried on a rotary This material
is hereafter
Hydrolysis was carried
The flavin-containing
evaporator,
referred
TGA was removed by extraction
to as “2-552 flavin.”
(Sigma, Type II).
into ether.
The phosphate ester bond in flavin at room temperature for lo-12 hours
pH 5.0, containing
The reaction
adenine dlnucleotides
acid (TGA) at 38’ for 4 hours (7).
mononucleotides was cleaved by Incubation in 0.5 M acetate buffer,
1 mg/ml potato acid phosphatase
was stopped by heating the solution
boiling water bath for 10 min.
were
and stored in the dark at -20’.
of the pyrophosphate linkage in flavin
out in 10% trichloracetic
fractions
Precipitated
in a
protein was removed by centrifuga-
tiop and the supernatant then eluted from a Sephadex G-15 column. Paper chromatography of flavins
was carried out In descending fashion
in the dark at 36’ using the upper layer of a-butanol:acetic (4rlt5
v/v)
as the developer (8).
The riboflavin purified
acidrwater
peptide used for molecular exclusion chromatography was
from a tryptic-chymotryptic
branes (9).
Hietidyl-riboflavin
peptide (10) and purification
digest of beef heart mitochondrial was obtained by acid hydrolysie
of the flavln
by cation exchange chromatography (9).
RESULTSANDDISCUSSION. In Table 1 it can be seen that ~-552 flavin the same Rf value as authentic Figure 1 the ~-552 flavin
and FAD are seen to have the same elution
FADwere subjected to mild acid hydrolysis,
Whenc-552 flavln
from FMN in both chromatographic systems.
to riboflavin
altered
on paper chromatography. 314
In
volume and
products were obtained which were
products with acid phoephatase they were further values similar
exhibits
FAD when compared by paper chromatography.
when Sephadex G-15 chromatography was carried out.
indistinguishable
mem-
On treating
these
and displayed Rf The acid hydrolyzed,
Vol. 44, No. 2, 1971
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1 Paper Chromatography of Flavins
Sample
Rf
FAD c-552 flavin
.05 .05
FMN FAD after TCA hydrolysis ~-552 flavin after TCA hydrolysis
.13 .13 .13
riboflavin FAD after TCA hydrolysis and acid phosphatase treatment c-552 flavin after TCA hydrolysis and acid phosphatase treatment
.36 .37 .35
r
Fig. 1. Molecular exclusion chromatography of flavine. Flavine were applied to a 2 x 70 cm. column of Sephadex G-15 that had been equilibrated with 0.1 N acetic acid, eluted with 0.1 N acetic acid, and collected in 3.0 ml. fractions. c-552 flavin,a ; ~-552 flavin after TCA hydrolysis, 0; c-552 flavin after TCA hydrolysia and acid phosphatase digestion, 0; FAD,@ FMN, @; riboflavin, ; eucclnate dehydrogenase riboflavin peptide, a; hlstidylriboflavin, ; void volume marker: horse heart cytochrome c, 8. phosphatase treated ~-552 flavin of riboflavin
and well after
was eluted from Sephadex G-15 slightly
both hietidyl-riboflavin 315
and the riboflavin
ahead peptide
Vol. 44, No. 2, 1971
BIOCHEMICAL
from succinate dehydrogenase. an adenine dinucleotide. tographic
intensity-p8
These observations suggest that c-552 flavin
This view is further
identification
hydrolysate
relationship
the neutrals-552
in the near ultraviolet flavin
TCA hydrolysis
phosphatase digestion.
Both neutral
tion are shown in Figure 2. is also distinct
consequence of a blue-shift the neutral
as isolated
TCA
in the fluorescence
(11).
absorption band which is seen in
spectrum remains a characteristic
spectra obtained after
solution
of the neutralized
of a maximumat pH 2.8-2.9
for ~-552 flavin
is
supported by the paper chroma-
of adenine in ether extracts
and by the observation
The blue shift
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and also after
feature
in the
TCA hydrolysis
and acid
and acid spectra of the latter
prepara-
The spectrum of this material obtained in acid
from the corresponding riboflavin
spectrum as a
in the absorption maximum. The similarity
and acid spectra of ~-552 flavin
after
phosphatase treatment to those of histidyl-riboflavin dehydrogenase is apparent in Figure 2.
TCA hydrolysis
450
400
and acid
from mammaliansuccinate
Spectral alterations
WAVELENGTH
of
of this sort
500
600
(nm 1
Spectra were obtained in water Fig. 2. Absorbance spectra of flavins. (neutral) or 6N HCl (acid) using a Cary 14 spectrophotometer and quartz cells of 1 cm. light path. c-552 flavin after TCA hydrolysis and acid phosphatase neutral, @ and acid, 0; digestion: neutral, @ and acid, @; riboflavin: histidyl-riboflavin: neutral, @ and acid, @. Histidyl-riboflavin spectra have been replotted for comparison from Ghisla, -et al. (13) * 316
BIOCHEMICAL
Vol. 44, No. 2, 1971
AND BIOPHYSICAL
have been described for several derivatives involving
substitutions
of the dimethylisoalloxazine
at the 8-methyl position
have been replotted riboflavin
and histidyl-riboflavin
for comparison (13).
undergoes a transition
the material
of ~-552 flavin
and acid phosphatase treatment is shown in Figure 3.
sponding data for riboflavin
2
3
4
5
after Corre-
from succinate dehydrogenase
It can be seen that whereas histidyl-
to a less fluorescent
derived from c-552 flavin
system
(12).
The pH dependence of the fluorescence intensity TCA hydrolysis
RESEARCH COMMUNICATIONS
form with a pK of 4.5,
is similar
to riboflavin,
6
9
7
8
IO
showing a broad
II
PH Fig. 3. pH Dependence of flavin fluorescence. c-552 flavin, A; hiatidylriboflavin from succinate dehydrogenase,m; and riboflavin, 0. Samples of c-552 flavin after TCA hydrolysis and acid phosphatase digestion were added to 0.03 M buffer solutions. The buffers used were: pH 1.95, ICCl:HCl; pH 3.0.0, glycine; pH 4.00 and 4.93, acetate; pH 6.36 and 7.25, phosphate; pH 9.60 and 9.95, borate; pH 11.30, phosphate. The data for histidylriboflavin and riboflavin have been replotted for comparison from Ghisla, et al. (13). -region of maximal fluorescence which declines to half-maximal about pH 9.3. isoalloxasine with a similar
This finding
seemsto rule out a substitution
system by an imidasole group. broad maximumhas recently
intensity
at
of the dimethyl-
A fluorescence-pH relationship
been reported for the flavin
of beef
Vol. 44, No. 2, 1971
liver
BIOCHEMICAL
monoamineoxidase.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
This flavin
exhibits
spectral
like those of the succinate dehydrogenase flavin released only by proteolysis
in
The substituent
is an adenine
ring system is substituted
is presumably not an imidazole
group and, as judged by comparison with riboflavin
and histidyl-riboflavin
on molecular exclusion chromatography, it must be of relatively Further characterization
is
of the enzyme (14).
which the dimethylisoalloxazine
in the 8-methyl position.
very much
and, like that flavin,
Taken together these facts suggest that ~-552 flavin dinucleotide
properties
of this flavin
small size.
is in progress.
ACKNOWLEDGEMENTS. This research was supported by a grant from the U.S. Public Health Service
(AM 12908).
the University
of California
strain
The authors are grateful
to Dr. T. E. Meyer of
at San Diego for a shake culture
of Chromatium
D and for advice on growing the bacteria.
REFERENCES. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
R. G. Bartsch, Fed. Proc. 28, 43 (1961). R. G. Bartsch, T. E. Meyer and A. B. Robinson, in "Structure and Function of Cytochromes," K. Okunuki, M. D. Kamen, and I. Sekuzu, eds., University Park Press, Baltimore, Md., p. 443 (1968). E. B. Kearney, J. Biol. Chem. 235, 865 (1960). T. E. Meyer, personal communication. "Bacterial Photosynthesis," H. Gess, A. San Pietro, and L. P. Vernon, eds., The Antloch Press, Yellow Springs, Ohio, p. 508 (1963). R. G. Bartsch in "Methods in Enzymology," Vol. VI, S. P. Colowick and N. 0. Kaplan, eds., Academic Press, N. Y., p. 400 (1963). 0. A. Bessey, 0. H. Lowry, and R. H. Love, J. Biol. Chem.180, 755 (1949). F. M. Huennekens, D. R. Sanadi, E. Dimant, and A. I. Schepartx, J. Am. Chem. Sot. Is, 3612 (1953). J. R. Cronln, unpublished data. W. H. Walker and T. P. Singer, J. Biol. Chem. 2, 4224 (1970). R. Hendriks, unpublished data. P. Hemmerich, A. Ehrenberg, W. H. Walker, L. E. G. Eriksson, J. Salach, P. Bader, and T. P. Singer, FEBSLetters 2, 37 (1969). S. Ghlsla, U. Hartmann, and P. Hemnmrich, Angew. Chem. internat. Edit. 2, 642 (1970). E. B. Kearney, J. I. Salach, W. H. Walker, R. Seng. and T. P. Singer, Biochem. and Blophys. Res. Comm.42, 490 (1971).
318
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
THE FLAVIN OF CHROMATIUM CYTOCRROME c-552 Robert Hendriks and John R. Cronin Department of Chemistry Arizona State University Tempe, Arizona 85281 Received May 18, 1971 SUMMARY. A flavin obtained from Chromatium cytochrome ~-552 has been purified and characterized with respect to its neutral and acid absorption spectra, fluorescence-pH relationship, and behavior on partition and molecular exclusion chromatography. The chromatographic results are consistent with a flavin adenine dinucleotide type structure. Neutral and acid absorption spectra taken after degradation of the flavin to the riboflavin level differ in the near DV from those of riboflavin and are similar to those of flavins modified in the dimethylisoalloxazine ring system, such as the histidyl-riboflavin obtained from mammaliansuccinate dehydrogenase. However, cytochrome c-552 flavin when at the riboflavin level and histidyl-riboflavin are quite dissimilar when compared fluorometrically and by molecular exclusion chromatography. Cytochrome c-552 as purified firmly
bound flavin
prosthetic
of cytochrome ~-552 with of urea, it exhibits
a
genase flavin flavin
a neutral
to that of FAD band (2).
of the absorption spectrum to that of the succinate dehydropeptide raised the possibility
link
tion of properties,
(2).
in concentrated urea solution
the further
has been carried
(3).
the
of the
is not characteristic
of
In view of this unusual combina-
characterization
of the cytochrome c-552
out.
on the modified (4) "heterotrophic"
harvested by centrifugation c-552 was isolated
of a covalent link joining
On the other hand, lability
MATERIALSARDMRTHODS. Chromatium strain bottles
in the cold and when freed
in the maximumof the near ultraviolet
the succinate dehydrogenase flavin
flavin
is released on treatment
absorption spectrum similar
and the cytochrome protein
flavln-protein
The flavin
group (1).
saturated urea solution
except for a 20 nm blue-shift The similarity
from Chromatium has been shown to contain a
D was grown in one litet (5).
Cells were
and stored as a frozen paste at -20".
Cytochrome
and purified
mediumof Fuller
prescription
through the first 313
DRAE-cellulose elution
Vol. 44, No. 2, 1971
BIOCHEMICAL
AND BIOPHYSICAL
according to the procedure of Bartsch (6).
RESEARCH COMMUNICATIONS
Flavin was dissociated
in cold,
saturated urea (2) and freed of hemeprotein by passage through a Sephadex 625
equilibrated
column
with saturated urea.
The flavln-containing
fractions
were combined and the urea removed by passage through a Sephadex G-15 column equilibrated
with 0.1 M acetic acid.
again combined, dried on a rotary This material
is hereafter
Hydrolysis was carried
The flavin-containing
evaporator,
referred
TGA was removed by extraction
to as “2-552 flavin.”
(Sigma, Type II).
into ether.
The phosphate ester bond in flavin at room temperature for lo-12 hours
pH 5.0, containing
The reaction
adenine dlnucleotides
acid (TGA) at 38’ for 4 hours (7).
mononucleotides was cleaved by Incubation in 0.5 M acetate buffer,
1 mg/ml potato acid phosphatase
was stopped by heating the solution
boiling water bath for 10 min.
were
and stored in the dark at -20’.
of the pyrophosphate linkage in flavin
out in 10% trichloracetic
fractions
Precipitated
in a
protein was removed by centrifuga-
tiop and the supernatant then eluted from a Sephadex G-15 column. Paper chromatography of flavins
was carried out In descending fashion
in the dark at 36’ using the upper layer of a-butanol:acetic (4rlt5
v/v)
as the developer (8).
The riboflavin purified
acidrwater
peptide used for molecular exclusion chromatography was
from a tryptic-chymotryptic
branes (9).
Hietidyl-riboflavin
peptide (10) and purification
digest of beef heart mitochondrial was obtained by acid hydrolysie
of the flavln
by cation exchange chromatography (9).
RESULTSANDDISCUSSION. In Table 1 it can be seen that ~-552 flavin the same Rf value as authentic Figure 1 the ~-552 flavin
and FAD are seen to have the same elution
FADwere subjected to mild acid hydrolysis,
Whenc-552 flavln
from FMN in both chromatographic systems.
to riboflavin
altered
on paper chromatography. 314
In
volume and
products were obtained which were
products with acid phoephatase they were further values similar
exhibits
FAD when compared by paper chromatography.
when Sephadex G-15 chromatography was carried out.
indistinguishable
mem-
On treating
these
and displayed Rf The acid hydrolyzed,
Vol. 44, No. 2, 1971
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1 Paper Chromatography of Flavins
Sample
Rf
FAD c-552 flavin
.05 .05
FMN FAD after TCA hydrolysis ~-552 flavin after TCA hydrolysis
.13 .13 .13
riboflavin FAD after TCA hydrolysis and acid phosphatase treatment c-552 flavin after TCA hydrolysis and acid phosphatase treatment
.36 .37 .35
r
Fig. 1. Molecular exclusion chromatography of flavine. Flavine were applied to a 2 x 70 cm. column of Sephadex G-15 that had been equilibrated with 0.1 N acetic acid, eluted with 0.1 N acetic acid, and collected in 3.0 ml. fractions. c-552 flavin,a ; ~-552 flavin after TCA hydrolysis, 0; c-552 flavin after TCA hydrolysia and acid phosphatase digestion, 0; FAD,@ FMN, @; riboflavin, ; eucclnate dehydrogenase riboflavin peptide, a; hlstidylriboflavin, ; void volume marker: horse heart cytochrome c, 8. phosphatase treated ~-552 flavin of riboflavin
and well after
was eluted from Sephadex G-15 slightly
both hietidyl-riboflavin 315
and the riboflavin
ahead peptide
Vol. 44, No. 2, 1971
BIOCHEMICAL
from succinate dehydrogenase. an adenine dinucleotide. tographic
intensity-p8
These observations suggest that c-552 flavin
This view is further
identification
hydrolysate
relationship
the neutrals-552
in the near ultraviolet flavin
TCA hydrolysis
phosphatase digestion.
Both neutral
tion are shown in Figure 2. is also distinct
consequence of a blue-shift the neutral
as isolated
TCA
in the fluorescence
(11).
absorption band which is seen in
spectrum remains a characteristic
spectra obtained after
solution
of the neutralized
of a maximumat pH 2.8-2.9
for ~-552 flavin
is
supported by the paper chroma-
of adenine in ether extracts
and by the observation
The blue shift
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and also after
feature
in the
TCA hydrolysis
and acid
and acid spectra of the latter
prepara-
The spectrum of this material obtained in acid
from the corresponding riboflavin
spectrum as a
in the absorption maximum. The similarity
and acid spectra of ~-552 flavin
after
phosphatase treatment to those of histidyl-riboflavin dehydrogenase is apparent in Figure 2.
TCA hydrolysis
450
400
and acid
from mammaliansuccinate
Spectral alterations
WAVELENGTH
of
of this sort
500
600
(nm 1
Spectra were obtained in water Fig. 2. Absorbance spectra of flavins. (neutral) or 6N HCl (acid) using a Cary 14 spectrophotometer and quartz cells of 1 cm. light path. c-552 flavin after TCA hydrolysis and acid phosphatase neutral, @ and acid, 0; digestion: neutral, @ and acid, @; riboflavin: histidyl-riboflavin: neutral, @ and acid, @. Histidyl-riboflavin spectra have been replotted for comparison from Ghisla, -et al. (13) * 316
BIOCHEMICAL
Vol. 44, No. 2, 1971
AND BIOPHYSICAL
have been described for several derivatives involving
substitutions
of the dimethylisoalloxazine
at the 8-methyl position
have been replotted riboflavin
and histidyl-riboflavin
for comparison (13).
undergoes a transition
the material
of ~-552 flavin
and acid phosphatase treatment is shown in Figure 3.
sponding data for riboflavin
2
3
4
5
after Corre-
from succinate dehydrogenase
It can be seen that whereas histidyl-
to a less fluorescent
derived from c-552 flavin
system
(12).
The pH dependence of the fluorescence intensity TCA hydrolysis
RESEARCH COMMUNICATIONS
form with a pK of 4.5,
is similar
to riboflavin,
6
9
7
8
IO
showing a broad
II
PH Fig. 3. pH Dependence of flavin fluorescence. c-552 flavin, A; hiatidylriboflavin from succinate dehydrogenase,m; and riboflavin, 0. Samples of c-552 flavin after TCA hydrolysis and acid phosphatase digestion were added to 0.03 M buffer solutions. The buffers used were: pH 1.95, ICCl:HCl; pH 3.0.0, glycine; pH 4.00 and 4.93, acetate; pH 6.36 and 7.25, phosphate; pH 9.60 and 9.95, borate; pH 11.30, phosphate. The data for histidylriboflavin and riboflavin have been replotted for comparison from Ghisla, et al. (13). -region of maximal fluorescence which declines to half-maximal about pH 9.3. isoalloxasine with a similar
This finding
seemsto rule out a substitution
system by an imidasole group. broad maximumhas recently
intensity
at
of the dimethyl-
A fluorescence-pH relationship
been reported for the flavin
of beef
Vol. 44, No. 2, 1971
liver
BIOCHEMICAL
monoamineoxidase.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
This flavin
exhibits
spectral
like those of the succinate dehydrogenase flavin released only by proteolysis
in
The substituent
is an adenine
ring system is substituted
is presumably not an imidazole
group and, as judged by comparison with riboflavin
and histidyl-riboflavin
on molecular exclusion chromatography, it must be of relatively Further characterization
is
of the enzyme (14).
which the dimethylisoalloxazine
in the 8-methyl position.
very much
and, like that flavin,
Taken together these facts suggest that ~-552 flavin dinucleotide
properties
of this flavin
small size.
is in progress.
ACKNOWLEDGEMENTS. This research was supported by a grant from the U.S. Public Health Service
(AM 12908).
the University
of California
strain
The authors are grateful
to Dr. T. E. Meyer of
at San Diego for a shake culture
of Chromatium
D and for advice on growing the bacteria.
REFERENCES. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
R. G. Bartsch, Fed. Proc. 28, 43 (1961). R. G. Bartsch, T. E. Meyer and A. B. Robinson, in "Structure and Function of Cytochromes," K. Okunuki, M. D. Kamen, and I. Sekuzu, eds., University Park Press, Baltimore, Md., p. 443 (1968). E. B. Kearney, J. Biol. Chem. 235, 865 (1960). T. E. Meyer, personal communication. "Bacterial Photosynthesis," H. Gess, A. San Pietro, and L. P. Vernon, eds., The Antloch Press, Yellow Springs, Ohio, p. 508 (1963). R. G. Bartsch in "Methods in Enzymology," Vol. VI, S. P. Colowick and N. 0. Kaplan, eds., Academic Press, N. Y., p. 400 (1963). 0. A. Bessey, 0. H. Lowry, and R. H. Love, J. Biol. Chem.180, 755 (1949). F. M. Huennekens, D. R. Sanadi, E. Dimant, and A. I. Schepartx, J. Am. Chem. Sot. Is, 3612 (1953). J. R. Cronln, unpublished data. W. H. Walker and T. P. Singer, J. Biol. Chem. 2, 4224 (1970). R. Hendriks, unpublished data. P. Hemmerich, A. Ehrenberg, W. H. Walker, L. E. G. Eriksson, J. Salach, P. Bader, and T. P. Singer, FEBSLetters 2, 37 (1969). S. Ghlsla, U. Hartmann, and P. Hemnmrich, Angew. Chem. internat. Edit. 2, 642 (1970). E. B. Kearney, J. I. Salach, W. H. Walker, R. Seng. and T. P. Singer, Biochem. and Blophys. Res. Comm.42, 490 (1971).
318