BIOCHEMICAL
Vol. 78, No. 1, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE LATE STEPS OF ANAEROBIC HEME BIOSYNTHESIS IN E. COLI: ROLE FOR QUINONES IN PROTOPORPHYRINOGEN OXIDATION J.M.
Jacobs
and N.J.
Jacobs
Department of Microbiology Dartmouth Medical School 03755 Hanover, New Hampshire
Received
July
20,
1977
SUMMARY The anaerobic oxidation of protoporphyrinogen with fumarate as electron acceptor in cell-free extracts of E. coli is inhibited by ultra-violet irradiation. The activity of Trradiated extracts is restored by addition of menadione and the restored activity is blocked by the electron-transport inhibitor 2-heptyl-4-hydroxy quinoline-Noxide. These observations suggest that quinones are required as electron transport carriers at this late step in the pathway of anaerobic heme biosynthesis. These findings have important implications both for the mechanism of anaerobic heme synthesis and for the physiology of cytochrome biosynthesis in anaerobic microorganisms.
INTRODUCTION In mammalian biosynthesis, is
linked
the
and yeast
mitochondria,
oxidation
of protoporphyrinogen
directly
transport
to molecular
system
(l-4).
synthesize
cytochromes
mechanisms
for
shown
fumarate
this
that
this
oxidation
tween
is
in c.
unidentified
carrier
In this
communication,
identified
carrier
0
of
1977 by Academic reproduciion in any
exist
6).
(7,
(5,
found
system
by the electron
suggesting
in the anaerobic
electron
a quinone. Press, Inc. form reserved.
429
that
and the
transport
(HQNO),
evidence
oxygen
electron
We also
in heme
of the
the
bacteria
studies
have
acceptor
for
the coupling fumarate inhibitor
chain that
be-
re2-heptyl-
involvement
transport
suggesting
electron
independent
Our previous
anaerobic
8).
we present is
mediation
and anaerobic
growth,
oxidizing
enzyme was blocked quinoline-N-oxide
without
anaerobic
an important
coli
4-hydroxy
Copyright AI1 rights
must
step
to protoporphyrin,
many facultative
during
the protoporphyrinogen
ductase
oxygen
Since
step
the penultimate
this
of an (9). un-
Vol. 78, No. 1, 1977
MATERIALS
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
AND METHODS
Sonic extracts containing both particulate and soluble fractions were prepared from E. coli grown anaerobically on a complex medium containing 0.2% glu?ose and 0.6% fumarate as previously described (8). The oxidation of protoporphyrinogen directly to protoporphyrin in anaerobic cuvettes was followed spectrophotometrically in a reaction mixture containing Tris buffer (pH 7.6), EDTA and glutathione as described (8). Extracts were treated with U.V. light for 60 min. with a Blak-Ray long wave lamp (3666 X) (Ultra-Violet Products, San Gabriel, Calif.) at a distance of 6 cm. During irradiation, a thin layer of extract in a petri dish was cooled in an ice bath and gently stirred (10). Solutions of menadione (Sigma Chemical Company, St. Louis, Missouri) and HQNO were dissolved in alcohol, and 0.05 ml amounts were added to extracts along with other reagents of the reaction mixture (8). Control experiments showed no effect of this amount of alcohol alone. Other methods and chemicals have been described previously in detail (8, 9).
RESULTS AND DISCUSSION
cant
Irradiation
of cell-free
inhibition
of anaerobic
as electron for
acceptor
is
extracts (Fig.
added
with 2).
tract
by menadione
tract,
we tested
Fig.
inthe
as electron
electron
2, HQNO also
extracts
is
the
similar
the effect
results carrier
the fumarate
reductase
is completely
between
of irradiated restored
of menadione
restored
We previously
untreated found
inhibits
extracts
the activity
does
irradiated
the protoporphyrinogen
in untreated
alone
2).
to the of the
If
that
exexHQNO
oxidizing (9).
As shown
in
restored
to irradiated
role
quinones
of menadione.
clearly
reductase
acceptor
to the activity
activity
fumarate.
the activity
(Fig.
fumarate
little
of added
addition
a signifi-
with
exhibit
absence
activity
between
causes
mixture,
of HQNO.
transport
markedly
light oxidation
of protoporphyrinogen
by the addition
These
U.V.
extracts
of fumarate,
whether
and fumarate
electron
oxidation
the oxidation
To determine
system
These
In the absence
not cause
blocked
1).
to the reaction
fumarate
with
protoporphyrinogen
(Fig.
protoporphyrinogen
menadione
extracts
suggest
an obligatory
the protoporphyrinogen enzyme.
Treatment
430
for
oxidizing of extracts
system with
U.V.
as and light
Vol.
78,
No.
1, 1977
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
g”r
20
40
60
80
100 120
140 160 180 200
Time (mm) Figure 1. Effect of U.V. irradiation on the anaerobic oxidation of protoporphyrinogen to protoporphyrin. The assay procedure and contents of the reaction mixture are described in the Methods section. All extracts were present at a concentration of 60 mg per 3 ml. reaction mixture. Protoporphyrinogen (123 nmoles) was added to each cuvette, and potassium fumarate (260 *moles) was added where indicated. Extracts were treated with U.V. light as indicated in the Methods section.
UV Treated Extract
Fumarale + Menadlone
90
HUN0
Time (mini Figure 2. Effect of menadione on the anaerobic oxidation of protoporphyrinogen with fumarate as electron acceptor in extracts treated with U.V. light. Assay conditions as shown in Fig. 1. Where indicated, the following were added to the reaction mixture: menadione (1.25 umoles); fumarate (260 pmoles); HQNO (0.15 mg). All extracts were treated with U.V. light.
is
known
adding light
to
destroy
menadione specifically
is
quinones
(10,
a clear
indication
destroys
an
11).
Restoration that
intermediate
the
of treatment
electron
431
activity with transport
by U.V. carrier
Vol. 78, No. 1, 1977
rather
than
The effect
by menadione
is not
extracts.
lated
this
with
a requirement
(10,
11).
the
of U.V. for
probable
reductase
uracil
menaquinone Although and fumarate electron
different
may be operative Further
quires
we have tested involvement
of replacing published
in anaerobic
observations).
Possibly
the
involvement
probably
takes of soluble
involve
other this
than
than
function
menaquinones
fumarate. of anaerobic
the nature
pro-
of the reaction
in the E. flmembrane to those
as electron
protoporphyrinogen
place
and
of menaquinones
serve
other
similar
the
in-
acceptor.
the mechanism
nucleotides
an in-
our results
importance
other
and
heme biosynthesis
may also
acceptors about
is
protoporphyrinogen, (8),
pro-
of mena-
acid
shown in
of NAD and NADP and have found
of pyridine
for
dehydrogenase
processes
Carriers
Inecperiments
and fumarate
acid
show the
oxidizing
fumarate
porphyrinogen
involvement
Therefore,
and quinones
the effect
between
Dihydroorotic
For instance,
protoporphyrinogen
carrier
as a carrier
both
conditions.
oxidation.
by menadione
substitute
as electron
clearly
remain
have corre-
can presumably
anaerobic
where
questions
does in un-
electron
to the
(12).
such as nitrate
elucidation.
without
similar
between
electron
toporphyrinogen
restoration
of uracil.
with
as it
subsequent
and fumarate
growth
reconstituted
microorganisms
the dihydroorotic
in anaerobically
acceptors,
between
is
parallel
findings
HQNO inhibits
involvement
biosynthesis,
our
the activity
as obligatory
in E. coli
as carrier
or the fumarate
in -E. c.
in the biosynthesis
anaerobic
that
menadione
found
system
in similar
menaquinone
between
an interesting
under
and the
and fumarate
fumarate
dicate
since
menaquinone
as a carrier
termediate
for
artificial,
menaquinone
toporphyrinogen quinone
of HQNO suggests
In our experiments
the natural
oxidizing
Investigations
effect
This
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the protoporphyrinogen
reductase.
treated
BIOCHEMICAL
Fig.
1,
no evidence
acceptors
capable
oxidation
(un-
between
proto-
entire
reaction
within
the cytoplasmic
components.
re-
membrane,
Vol. 78, ho. 1, 1977
BIOCHEMICAL
From a physiological in anaerobic aerobic
heme synthesis
directly
with
electron
transport
These obligatory chromes likely our
contrasts
oxygen,
observations anaerobic
often that
exhibit a partial
observation
biosynthesis
that
which
aerobic
without
chain
is
the
RESEARCH COMMUNICATIONS
interesting
sharply
oxidation In these
mitochrondria.
it
viewpoint,
protoporphyrinogen
yeast
AND BIOPHYSICAL
that
with occurs
systems,
involvement
this
step
the mechansim in mammalian
this
enzyme
of carriers
for and
reacts
in the
(l-4). also
have implications
bacteria.
Those
anaerobes
a growth
requirement
explanation
for
vitamin
K is
for
for
this
the physiology
that
contain
vitamin
K.
requirement
apparently
required
of
cytoIt
seems
can be found for
the anaerobic
of heme.
ACKNOWLEDGEMENT
This work was supported BMS 74-0249 and PCM 76-80478.
by National
Science
Foundation
Grants
REFERENCES 1. 2.
Sano, S. and Granick, S. (1961) J. Biol. Chem. 236, 1173-1180. Paulson, R. (1976) J. Biol. Chem. 251, 3730-3733, Poulson, R. and Polglase, W.J. (197r FEBS Letters 40, 258-260. 43: Poulson, R. and Polglase, W.J. (1975) J. Biol. Chem. 250, 12691274. 5. Jacobs, N.J. (1974) In "Microbial Iron Metabolism" (J.8. Neilands, ed.) pp. 125-148, Academic Press, New York. 6. Lascelles, J. (1964) "Tetrapyrolle Biosynthesis and Its Regulation" Benjamin, New York. 7. Jacobs, N.J. and Jacobs, J.M. (1975) Biochem. Biophys. Res. Commun. 65, 435-441. 8. JqJ;ob;rgN.J. and Jacobs, J.M. (1976) Biochim. Biophys. Acta 9. 10. 11. 12.
Eabs, N.J. and Jacobs, J.M. (1977) Biochim. Biophys. Acta gg, 141-144. Dunphy, P.J. and Brodie, A.F. (1971) "Methods in Enzymology" XVIII, (D.B. McCormick and L.D. Wright, eds.) pp. 407-461, Academic Press, New York. Hatchikian, E.C. (1974) J. Gen. Microbial. 3, 261-266. Newton, N.A., Cox, G.B. and Gibson, F. (1971) Biochim. Biophys. Acta 244, 155-166.
433
in
Vol. 78, No. 1, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE LATE STEPS OF ANAEROBIC HEME BIOSYNTHESIS IN E. COLI: ROLE FOR QUINONES IN PROTOPORPHYRINOGEN OXIDATION J.M.
Jacobs
and N.J.
Jacobs
Department of Microbiology Dartmouth Medical School 03755 Hanover, New Hampshire
Received
July
20,
1977
SUMMARY The anaerobic oxidation of protoporphyrinogen with fumarate as electron acceptor in cell-free extracts of E. coli is inhibited by ultra-violet irradiation. The activity of Trradiated extracts is restored by addition of menadione and the restored activity is blocked by the electron-transport inhibitor 2-heptyl-4-hydroxy quinoline-Noxide. These observations suggest that quinones are required as electron transport carriers at this late step in the pathway of anaerobic heme biosynthesis. These findings have important implications both for the mechanism of anaerobic heme synthesis and for the physiology of cytochrome biosynthesis in anaerobic microorganisms.
INTRODUCTION In mammalian biosynthesis, is
linked
the
and yeast
mitochondria,
oxidation
of protoporphyrinogen
directly
transport
to molecular
system
(l-4).
synthesize
cytochromes
mechanisms
for
shown
fumarate
this
that
this
oxidation
tween
is
in c.
unidentified
carrier
In this
communication,
identified
carrier
0
of
1977 by Academic reproduciion in any
exist
6).
(7,
(5,
found
system
by the electron
suggesting
in the anaerobic
electron
a quinone. Press, Inc. form reserved.
429
that
and the
transport
(HQNO),
evidence
oxygen
electron
We also
in heme
of the
the
bacteria
studies
have
acceptor
for
the coupling fumarate inhibitor
chain that
be-
re2-heptyl-
involvement
transport
suggesting
electron
independent
Our previous
anaerobic
8).
we present is
mediation
and anaerobic
growth,
oxidizing
enzyme was blocked quinoline-N-oxide
without
anaerobic
an important
coli
4-hydroxy
Copyright AI1 rights
must
step
to protoporphyrin,
many facultative
during
the protoporphyrinogen
ductase
oxygen
Since
step
the penultimate
this
of an (9). un-
Vol. 78, No. 1, 1977
MATERIALS
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
AND METHODS
Sonic extracts containing both particulate and soluble fractions were prepared from E. coli grown anaerobically on a complex medium containing 0.2% glu?ose and 0.6% fumarate as previously described (8). The oxidation of protoporphyrinogen directly to protoporphyrin in anaerobic cuvettes was followed spectrophotometrically in a reaction mixture containing Tris buffer (pH 7.6), EDTA and glutathione as described (8). Extracts were treated with U.V. light for 60 min. with a Blak-Ray long wave lamp (3666 X) (Ultra-Violet Products, San Gabriel, Calif.) at a distance of 6 cm. During irradiation, a thin layer of extract in a petri dish was cooled in an ice bath and gently stirred (10). Solutions of menadione (Sigma Chemical Company, St. Louis, Missouri) and HQNO were dissolved in alcohol, and 0.05 ml amounts were added to extracts along with other reagents of the reaction mixture (8). Control experiments showed no effect of this amount of alcohol alone. Other methods and chemicals have been described previously in detail (8, 9).
RESULTS AND DISCUSSION
cant
Irradiation
of cell-free
inhibition
of anaerobic
as electron for
acceptor
is
extracts (Fig.
added
with 2).
tract
by menadione
tract,
we tested
Fig.
inthe
as electron
electron
2, HQNO also
extracts
is
the
similar
the effect
results carrier
the fumarate
reductase
is completely
between
of irradiated restored
of menadione
restored
We previously
untreated found
inhibits
extracts
the activity
does
irradiated
the protoporphyrinogen
in untreated
alone
2).
to the of the
If
that
exexHQNO
oxidizing (9).
As shown
in
restored
to irradiated
role
quinones
of menadione.
clearly
reductase
acceptor
to the activity
activity
fumarate.
the activity
(Fig.
fumarate
little
of added
addition
a signifi-
with
exhibit
absence
activity
between
causes
mixture,
of HQNO.
transport
markedly
light oxidation
of protoporphyrinogen
by the addition
These
U.V.
extracts
of fumarate,
whether
and fumarate
electron
oxidation
the oxidation
To determine
system
These
In the absence
not cause
blocked
1).
to the reaction
fumarate
with
protoporphyrinogen
(Fig.
protoporphyrinogen
menadione
extracts
suggest
an obligatory
the protoporphyrinogen enzyme.
Treatment
430
for
oxidizing of extracts
system with
U.V.
as and light
Vol.
78,
No.
1, 1977
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
g”r
20
40
60
80
100 120
140 160 180 200
Time (mm) Figure 1. Effect of U.V. irradiation on the anaerobic oxidation of protoporphyrinogen to protoporphyrin. The assay procedure and contents of the reaction mixture are described in the Methods section. All extracts were present at a concentration of 60 mg per 3 ml. reaction mixture. Protoporphyrinogen (123 nmoles) was added to each cuvette, and potassium fumarate (260 *moles) was added where indicated. Extracts were treated with U.V. light as indicated in the Methods section.
UV Treated Extract
Fumarale + Menadlone
90
HUN0
Time (mini Figure 2. Effect of menadione on the anaerobic oxidation of protoporphyrinogen with fumarate as electron acceptor in extracts treated with U.V. light. Assay conditions as shown in Fig. 1. Where indicated, the following were added to the reaction mixture: menadione (1.25 umoles); fumarate (260 pmoles); HQNO (0.15 mg). All extracts were treated with U.V. light.
is
known
adding light
to
destroy
menadione specifically
is
quinones
(10,
a clear
indication
destroys
an
11).
Restoration that
intermediate
the
of treatment
electron
431
activity with transport
by U.V. carrier
Vol. 78, No. 1, 1977
rather
than
The effect
by menadione
is not
extracts.
lated
this
with
a requirement
(10,
11).
the
of U.V. for
probable
reductase
uracil
menaquinone Although and fumarate electron
different
may be operative Further
quires
we have tested involvement
of replacing published
in anaerobic
observations).
Possibly
the
involvement
probably
takes of soluble
involve
other this
than
than
function
menaquinones
fumarate. of anaerobic
the nature
pro-
of the reaction
in the E. flmembrane to those
as electron
protoporphyrinogen
place
and
of menaquinones
serve
other
similar
the
in-
acceptor.
the mechanism
nucleotides
an in-
our results
importance
other
and
heme biosynthesis
may also
acceptors about
is
protoporphyrinogen, (8),
pro-
of mena-
acid
shown in
of NAD and NADP and have found
of pyridine
for
dehydrogenase
processes
Carriers
Inecperiments
and fumarate
acid
show the
oxidizing
fumarate
porphyrinogen
involvement
Therefore,
and quinones
the effect
between
Dihydroorotic
For instance,
protoporphyrinogen
carrier
as a carrier
both
conditions.
oxidation.
by menadione
substitute
as electron
clearly
remain
have corre-
can presumably
anaerobic
where
questions
does in un-
electron
to the
(12).
such as nitrate
elucidation.
without
similar
between
electron
toporphyrinogen
restoration
of uracil.
with
as it
subsequent
and fumarate
growth
reconstituted
microorganisms
the dihydroorotic
in anaerobically
acceptors,
between
is
parallel
findings
HQNO inhibits
involvement
biosynthesis,
our
the activity
as obligatory
in E. coli
as carrier
or the fumarate
in -E. c.
in the biosynthesis
anaerobic
that
menadione
found
system
in similar
menaquinone
between
an interesting
under
and the
and fumarate
fumarate
dicate
since
menaquinone
as a carrier
termediate
for
artificial,
menaquinone
toporphyrinogen quinone
of HQNO suggests
In our experiments
the natural
oxidizing
Investigations
effect
This
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the protoporphyrinogen
reductase.
treated
BIOCHEMICAL
Fig.
1,
no evidence
acceptors
capable
oxidation
(un-
between
proto-
entire
reaction
within
the cytoplasmic
components.
re-
membrane,
Vol. 78, ho. 1, 1977
BIOCHEMICAL
From a physiological in anaerobic aerobic
heme synthesis
directly
with
electron
transport
These obligatory chromes likely our
contrasts
oxygen,
observations anaerobic
often that
exhibit a partial
observation
biosynthesis
that
which
aerobic
without
chain
is
the
RESEARCH COMMUNICATIONS
interesting
sharply
oxidation In these
mitochrondria.
it
viewpoint,
protoporphyrinogen
yeast
AND BIOPHYSICAL
that
with occurs
systems,
involvement
this
step
the mechansim in mammalian
this
enzyme
of carriers
for and
reacts
in the
(l-4). also
have implications
bacteria.
Those
anaerobes
a growth
requirement
explanation
for
vitamin
K is
for
for
this
the physiology
that
contain
vitamin
K.
requirement
apparently
required
of
cytoIt
seems
can be found for
the anaerobic
of heme.
ACKNOWLEDGEMENT
This work was supported BMS 74-0249 and PCM 76-80478.
by National
Science
Foundation
Grants
REFERENCES 1. 2.
Sano, S. and Granick, S. (1961) J. Biol. Chem. 236, 1173-1180. Paulson, R. (1976) J. Biol. Chem. 251, 3730-3733, Poulson, R. and Polglase, W.J. (197r FEBS Letters 40, 258-260. 43: Poulson, R. and Polglase, W.J. (1975) J. Biol. Chem. 250, 12691274. 5. Jacobs, N.J. (1974) In "Microbial Iron Metabolism" (J.8. Neilands, ed.) pp. 125-148, Academic Press, New York. 6. Lascelles, J. (1964) "Tetrapyrolle Biosynthesis and Its Regulation" Benjamin, New York. 7. Jacobs, N.J. and Jacobs, J.M. (1975) Biochem. Biophys. Res. Commun. 65, 435-441. 8. JqJ;ob;rgN.J. and Jacobs, J.M. (1976) Biochim. Biophys. Acta 9. 10. 11. 12.
Eabs, N.J. and Jacobs, J.M. (1977) Biochim. Biophys. Acta gg, 141-144. Dunphy, P.J. and Brodie, A.F. (1971) "Methods in Enzymology" XVIII, (D.B. McCormick and L.D. Wright, eds.) pp. 407-461, Academic Press, New York. Hatchikian, E.C. (1974) J. Gen. Microbial. 3, 261-266. Newton, N.A., Cox, G.B. and Gibson, F. (1971) Biochim. Biophys. Acta 244, 155-166.
433
in
