The late steps of anaerobic heme biosynthesis in E. coli: Role for quinones in protoporphyrinogen oxidation

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.

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