C-Type cytochromes of Desulfovibrio vulgaris amino acid composition and end groups of cytochrome C553

Vol. 38, No. 4, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

C-TYPE CYTOCHROMES OF DESULFOVIBRIO

VUIGARIS

AMINO ACID COMPCSITION AND END GROUPS OF CYTOCHROME Csaax Mireille (with

BRUSCHI and Jean IF. GALLxx

the technical

Laboratoire

assistance

de-Chimie

of Mireille

Bacterienne,

LE GUEN)

CNRS, Marseille,

France

and Karl Department

of Chemistry, Urbana,

Received

DUS University

Illinois,

of Illinois

U.S.A.

3, 1969

December

SUMMARY Cytochrome composition (12,000)

caaa of Desulfovibrio

and in molecular of the same organism.

in crystalline comprising the

form. about

Both

termini

amino

terminal

In addition

It

acid

freely

and the

of larger

and related

organisms.

l

protein

of a single residues

available; carboxyl

to cytochrome

cytochrome,

(9,100

This

consists

SO amino are

vulgaris

weight

differs 100)

terminal

weight,

ca

obtained

polypeptide

chain

a single

heme group.

and leucine position,

are marking

respectively.

caaa and to cytochrome

molecular

acid

cytochrome

can be easily

and bearing alanine

in amino

from

co a third

was detected

in 2.

c-type

vulgaris

INTRODUCTION Postgate Japan

first

the strict

in England reported anaerobe

(1954,

on isolation Desulfovibrio

1956)

and Ishimoto

and properties desulfuricans

et al.,

of cytochrome Hildenborough,

XThese researches were supported in part by grants-in-aid Martin D. Kamen from the National Institutes of Health the National Science Foundation (GB-7033X). XXPresent Athens,

address: Georgia,

Biochemistry U.S.A.

Department,

607

(1954)

University

in

ca from now

to Professor (HD-01262) and of Georgia,

Vol. 38, No. 4, 1970

renamed

BIOCHEMICAL

g. vulgaris

(NCIB 8303)

sulfate-reducing

bacteria.

contains

heme groups

Horio

several and Kamen,

1960;

and exhibits

a very

Its

amino

complete Recently,

having

Cytochrome

acid

that

caaa

molecular

sequence

In the

first

part

reduced

1967;

Drucker

same organism

wband

heme group

from

determined

of cytochrome

of an investigation

of the

1956). (Ambler,

chemical

and

are

very

ca by a lower less

and Le Gall,

casa.

c

peak at 280 nm,

cytochrome

the

1968).

cytochrome

and a substantially

we report

1969)

at 553 nm (Le Gall

(Bruschi-Heriaud

communication

and Campbell,

another

ca

1956;

(Postgate,

ca5a shows a higher

a single

residues

also

cytochrome

of the two cytochromes

distinguished

present

been

and related

Postgate,

potential

The spectra

potential

and the terminal

the

further

oxidation-reduction

1954;

has already

from

cytochrome

is

weight,

et al.,

1966),

cytochromes,

et al.,

low oxidation-reduction

1968).

except

and Campbell,

most c-type

(Ishimoto

Drucker

the maximum of its

similar

(Postgate

Unlike

we isolated

Bruschi-Heriaud,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

amino This

1967).

acid

work

structure

negative

composition constitutes of the

the

protein.

EXPRRIMRNTALPRGCRDIJRR8 Materials The chemicals used without tallized

used

further

preparation

cytochrome

c5aa was that

against

successive

distilled

Amino Acid Protein at 105-llO"

were

used

for

Biochemical

purification

described

by Le Gall

crystallizations,

water

of reagent

Carboxypeptidase

of the Worthington

The method

three

study

purification.

New Jersey.

After

in this

grade;

they

were

A-DFP was a 3x crysCorportion,

Freehold,

and crystallization

of

and Bruschi-Heriaud

(1968).

the cytochrome

was dialyzed

and lyophilized.

Analysis samples for

(about

24 hours.

0.5 mg) were Decomposition 608

hydrolyzed of tyrosine

in 0.5 ml of 6N HCL and methionine

was

Vol. 38, No. 4, 1970

BIOCHEMICAL

almost

completely

prevented

to the

HCl before

sealing

under

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by the

addition

of thioglycolic

the

tubes.

After

hydrolysis

were

carried

out

on a modified

analyzer

using

acid

(0.05$)

the HCl was removed

vacuum. Amino

model

acid

analyses

120B amino

program

acid

of Dus --et al.

Cysteine as cysteic tophan

of Amino

DNP-amino

acids

(1945,

were

Whatman No 1 filter

identified paper.

buffer,

of Carboxyl

those

Aliquots

and analyzed

acid

(Hirs,

oxidation

1956).

and Chambers

Trw-

(1949).

using

ratios

reaction

on the

The hydrazinolysis the modification

acids

were

micromole

systems

of 4:l (Levy,

as

of protein.

The

chromatography employed

(Murachi

on

were

t-amyl

and Neurath,

1960)

and

1954).

carboypeptidase

cytochrome outlined

using

0.5

was performed

by two dimensional

pH 6.0

of 1:70.

of the

of Spies

of DFP-treated

of native

ratio

performic

respectively

The solvent

determined

acetate

(1967).

, pH 6.5,

The digestion mixture

automatic

procedure

and the

withdrawn acid

enzyme was added for

at appropriate

in

3 hours. intervals

analyzer.

of Akabori

on the

609

were

was dissolved

out at 37'

et al.,

of Niu and Fraenkel-Conrat quantitatively

conditions

The protein

was carried

were amino

A was used for

The experimental

casa.

by Ambler

in 0.2 M N-ethylmorpholine a molar

elution

Terminus

A preparation

essentially

after

(PDNB) reaction

1949)

NH,OH in volume

digestion

sulfone,

4-dinitrobenzene

by Sanger

Analysis

buffer

Terminus

described

1.5 M phosphate

analyzed

by the method

The I-fluoro-2,

alcohol/2N

were

and methionine

was determined

Analysis

four

(1966).

and methionine acid

the stepwise

Beckman-Spinco

amino

(1952) (1955). acid

was applied The free

analyzer.

amino

Vol. 38, No. 4, 1970

BIOCHEMICAL

Determination

of Molecular

The approximate on a column

Weight

size

of the

of Sephadex

The exact

molecular

including

one protoheme

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

protein

was determined

G-75 following

weight

the procedure

was calculated

from

by gel

filtration

of Whitaker

the amino

acid

(1963). composition

IX group.

Results The yield in the

range

of pure

of 76 mg as compared

obtained

under

obtained

in crystalline

heme protein saturated

caaa p er kg of wet

cytochrome

the

to a yield

same conditions.

obtained

form. after

Acid With

ca

of the oxidized

recrystallizations

in the

presence

of

given

in

ammonium sulfate.

Fig.

Amino

was

csaa can be readily

1 shows a crystal

several

of cells

of 178 mg of cytochrome

Cytochrome

Figure

paste

Crystal

of cytochrome

Length

of the crystal:

cssa

1 after

three

approximately

crystallizations. 0.2

nm.

Composition the

exception

of tryptophan

the 610

amino

acid

composition

BIOCHEMICAL

Vol. 38, No. 4, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Amino Acid Cysteic

I

Composition

of Cytochrome

cssg

Acid

Aspartic

Acid

Threonine Serine Glutamic

Acid

1.7

2

6.2

6.3

7.0,

6

0.9

2.9

2.8

4.8

4.9

4.0*

1 6*

6.0

6.0

6.0 2.4*

6

Proline

1.5

1.9

Glycine

10.9

10.9

10.5

11

Alanine

12.9

13.0

12.2

13

1.9

2.0

5.2

5.2

Valine Methionine Methionine

2

2

2.0

5

Sulfone

4.9

Isoleucine

0.9

0.9

0.8

1

5.0

4.3

5

6.0

3.5*

6

Leucine

4.9

Tyrosine

5.9 trace

trace

1.0

1.0

Lysine

12.0

12.0

Argfnine

0.9

0.9

Phenylalanine Histidine

0 0.5*

1.

13.4

12

1.4*

1

Tryptophan-

0

Total

(2)

(1)

80

(3)

two separate analyses of hemeprotein in 6 N HCl, 24 hours at 110'; (1) and (2): (3) analysis of performate oxidized protein in 6N HCl, 24 hours at 110'; *not reliable after oxidation with performic acid; *extrapolated to zero time hydrolysis; -determined by the calorimetric method of Spies and Chambers.

Table

I was obtained

from acid

after

performic

oxidation.

acid

hydrolyxates Despite

+,a3

phan was furnished

by calorimetric

Assuming

weight

of cytochrome

result

of the gel

around

the presence cnaa is filtration

the

relatively

280 nm, no indication

peak of cytochrome

(1949).

of the protein

analysis of a single This

9,000. experiment

611

according protoheme value

which

agrees indicated

before high

for

absorption

presence

to Spies IX group

of trypto-

and Chambers the formula

very well 9,100.

and

with

the

However,

Vol. 38, No. 4, 1970

for

this

BIOCHEMICAL

molecular

weight

and Bruschi-Heriaud,

Terminal

soluble

and heme analyses

were

low

(Le Gall

1968).

terminal

alanine

DNP-amino surprisingly

protein

incurred

and no more than

recovery

acid

a known

6N HCl at 105-llO"

for

sustained

hydrolysis

micromole

this

step

alone

in chromatography,

paper

DNP-alanine

presence

however,

to correct

and subsequent

in the

ether

per micromole

In order

of synthetic

18 hours from

0.4

of DNP-alanine,

experiments.

amount

of DNP-alanine

losses

The yields

low,

in the best

No other

by the FDND method.

was detected.

during

identification,

was found

acid

was obtained

losses

the

iron

Residues

Amino

were

both

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

chromatographic

of cytochrome

a corrected

for

was heated

was only

of

with The

cssa. Including

64. yield

of about

also 75% may

be assumed. From hydraxinolyzates excellent

No other

yields.

Digestion

of cytochrome

of native

total

period

of 3 hrs.

which

suggested

amino

acids

cytochrome yielded

the sequence

c5s3 leucine were

csss

mainly

in

noted.

with

carboxypeptidase

leucine

..D....

was recovered

and alanine

A for

a

in proportions

Ala-Leu-OH.

Discussion From the 2.

vulgaris,

are

of cytsteine, apoprotein,

other

listed

distinctly

most types

the

comparison

necessary hand,

in Table

different of amino just while

one in cytochrome

with

acids.

cssa.

it

ca and cytochrome can be seen that

regard

to molecular

cytochrome

to link

cytochrome

residues

II,

Thus,

sufficient

complement nine

of cytochrome

the

ca has eight

for

attachment

of histidine On balance,

cssa single residues

these

weight

there

612

found

of

two heme proteins and content

contains

only

heme group

of

two residues

to the

of cysteine

of even three are

cssa

or more than

hems groups.

in cytochrome

seem to be at least

ca but one

On the only

Vol. 38, No. 4, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Comparison Cytochrome

Cytochrome (according

of Amino

II Acid

Compositions

ca and Cytochrome

of

csaa of D. vulgaris

ca to sequence

by Ambler,

Cytochrome

Asp Thr

12

6

5

1

Ser

6

6

Glu

5

6

Pro

4

2

GUY Ala

9

11

10

13

cys/2 Val

8

2

a

2

Met

3

5

Ile Leu

0 2

1 5

Wr Phe

3

6

2

0

His

9

1

LYS

20

12

Arg

1

1

Trp

0

0

107

80

Total histidyl

and one methionyl

iron

of either

et al.,

protein

residue

available

as was observed

for

for

other

chelation cytochromes

to each heme c (I-Jar-bury

1965).

The exact

number

is still

at

Horio

and Kamen,

1960;

It

interesting

or three,

is

caaa

1968)

that

of heme groups issue

(Ishimoto

Drucker the

present

amino

t

et al., acid

al.,

1954;

1967;

Drucker

sequence

613

in cytochrome Postgate,

cat

namely 1956;

and Campbell,

of cytochrome

two

1969).

ca (Ambler,

Vol. 38, No. 4, 1970

1968)

BIOCHEMICAL

revealed

only

two sequences

of the heme attachment two additional

however, strikingly

differ

and all

To underscore C

553 a comparison

the

the

vulgaris,

ca,

between

soluble

Table

is

given

c-type

Amino

Residues

Weight

Terminal

Residue

Carboxyl

Terminal

Midpoint

Potential

Isoelectric

Residue

Ishimoto

et al., 1956

Horio Drucker

and'Kamen, et al.,

heme

cytochromes is very

III. from

the same

intriguing

of 2.

ca

vulgaris:

Cytochrome

107

80

2-3*

1

12,000

9,000

Ala

Ala

Glu

Leu

10.5

Postgate,

a single

III

-205mV

Point

* See references:

which

ca and cytochrome

in Table

good yields,

Cytochromes

Heme Groups Molecular

contains,

heme attachment.

cytochrome

Cytochrome

Amino Acid

caaa has only

c-type

two proteins

of c-type

The protein

CyS-A-B-C-D-Cys-His

cytochrome

differences

in reasonably

Properties

type

characteristic

arrangement.

of a typical

of several

CyS-X-Y-CyS-His,

cytochromes.

of the

normal

makings

of the

Availability organism,).

in c-type

to cytochrome the

of the type

sequences

from

In contrast group

sites

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1954 1960 1967

614

between basic

casa

-100

and OmV

(78.6)

Vol. 38, No. 4, 1970

especially stood

in view

electron

instance, the

BIOCHEMICAL

apparently

transport

system

in D. vulgaris,

presence

of yet

Several

26,000. either both

of the

cytochrome

rudimentary

of the

2. gigas,

another

of its

cytochrome

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

sulfate-reducing

c which

properties

ca or cytochrome ca5a and the

poorly

csaa.

larger

we have detected

also

different

cytochrome

weight

of about

from those

The physiological

c-type

For

has a molecular

are

under-

bacteria.

and D. desulfuricans

cytochrome

other

and still

functions

are

still

of of

unknown.

References: Akabori,

S., (1952).

Ohno,

K. and Narita,

Ambler,

R. P.,

Methods

Ambler,

R. P.,

Biochem.

L.,

Bull.

in Enzymology, J. 109,

Vol.

J.,

Bull.

Drucker, Il., Woody, R. and Campbell, Abstracts 26; 823. H. and Campbell,

DUS, K., l4,

Lindroth, 41 (1966).

L. L., Pabst,

S.,

XI,

C. H. W., J. Biol.

Horio,

T. and Kamen,

SOC. Chem. Biol.

L. L.,

Fed. Proc.

J. Bacterial.,

R. and Smith,

Ishimoto, 4l,

Chem.,

M. D.,

M., Koyama, 537 (1954).

J.,

100

219,

(Chicago,

(19691,

R. M., Anal.

A. L.,

Niu,

174,

R. II., Saski, 57, 439 (19671.

Matsubara, U.S., Murachi,

Nature,

T. and Neurath,

C. I. 1955.

1967)

in press.

Biochem., T. P., Murphy, Acad. Sci.

611 (1956).

Biochem. Omura,

Paris

Biophys.

Acta,

T. and Nagai,

Y.,

48,

266,

(1961).

J. Biochem.,

Structure and Function Le Gall, J., and Bruschi-Heriaud, M., K. Okunuki, M. D. Kamen and I. Sekuzo, Eds., University Press and University Park Press, pp. 467 (1968). Levy,

214

442 (1967).

Harbury, H. A., Cronin, J. R., Fanger, M. W., Hettinger, S. N., Proc. Natl. A ., Myer, Y. P. and Vinogradov, u. s., 54, 1658 (1965). Hirs,

Japan 25,

47 P (1968).

Bruschi-Heriaud, M. and Le Gall, 49, 7 (1967).

Drucker,

Chem. Sot.,

Japan of Cytochromes, of Tokyo

(1949). M.

and Chain,

Il.,

J. Biol.

and Fraenkel-Conrat,

R. K., Chem.,

H., J. Amer.

615

Proc. 235,

Natl.

Acad.

99 (1960).

Chem. Sot.,

77,

5882

Sci.

Vol. 38, No. 4, 1970

BIOCHEMICAL

Postgate,

J.

R.,

Biochem.

J.,

Postgate,

J. R.,

Postgate,

J. R. and Campbell,

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

58,

xi

J. Gen. Microbial.,

(1954). 14,

L. L.,

Bacterial.

Sanger,

F.,

Biochem.

J.,

39,

507 (1945).

Sanger,

F.,

Biochem.

J.,

45,

563

Spies, Whitaker,

J.

R. and Chambers, J.

R., Anal.

35,

Rev. 30,

732 (1966).

(1949).

D. C., Anal.

Chem.,

545 (1956).

1950

616

Chem., (1963).

2l,

1249

(1949).