The amino acid sequence of Scenedesmus ferredoxin

Vol. 32, No. 6, 1968

8lOCHEMlCAL

THE AMINO

ACID

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SEQUENCE

Koichi

Sugeno+

OF SCENEDESMUS

and

Hi roshi

FERREDOXlN*

Matsubara

Space Sciences Laboratory University of California Berkeley, Cal i fornia 94720

Received

August

The species

1967)

amino

acid

(Tanaka

et

and

alfalfa

5, 1968

higher

sequences

amino

from

the

plants,

acid

Scenedesmus

o

found

1965;

San

atoms

of

In

the

be

Pietro iron

and

two

Scenedesmus

ferredoxin,

ferredoxins

(Arnon,

Margol

have

illuminated

leave

been from

structure

report

(Matsubara,

1968),

similar

to

of

the

Black,

1965;

moles

of

as

in

those

inorganic

sulfide

al 1 other

San

Pietro

the

its plant

and

Margoliash,

were

found

of

interest

alga,

weight

of

absorption

spectrum

ferredoxins

(Arnon,

1966). mole

Two

of

chloroplast-type

1965;

Black,

of

a green

per

we1 l-characterized and

and

relationships.

molecular

and

Keresztes-Nagy

are

from

typical

al,

1968)

evolutionary

ferredoxin

be 11,500

et

species

and of

Tsunoda

Comparisons

different

functional,

bacterial

1967,

al,

established.

to

is

of

spinach,

identical

activities

spinach

chloroplasts.

one

less

* This investigation and Space Administration to the Space Sciences On

have

1967;

et

determined

1965;

ferredoxins

nearly

96, which

1966,

al,

from

Keresztes-Nagy

and

1966).

iash, The

1968)

isolated

(Matsubara

ferredoxins

was

and

et

spinach

primary

earlier

very

ferredoxins

Benson

structural,

ferredoxin to

as

al,

of

of

studied

Scenedesmus was

sequences

certain

1966;

such et

standpoint

We have

+

1964,

al,

(Keresztes-Nagy

the

of

from

the

than

Swiss

chard,

when

assayed

The those

of

total

higher

was

aided by grants and HE 11553-01 Laboratory, University

University

of

Osaka,

951

and by number plant

Scenedesmus the

photoreduction of

amino

ferredoxins.

NsG 479 by the by the National of Cal i fornia, Osaka,

were

Japan,

acid The

found

to

of

TPN with

residues amino-

was and

National Aeronautics Institutes of Health Berkeley.

Vol.

32, No.

6, 1968

BIOCHEMICAL

carboxyl-terminal

sequences

Leu-Phe, of

respectively,

Scenedesmus

AND

were This

ferredoxi

1968).

article

ferredoxin of

carried

out

of

of (OFd),

ication

CMCFd

reports

were

and

when

the

peptides

of

OFd

The

Anfinsen

and

the

paper

70

to

96,

and

the

revealed

the

complete

in

Figure

and

1.

separated

Sugeno, The

amino

Awl

A~P,~

Thr10

Th’is

value

is

intact amide

acid

in

data

in

canposition Glu6

Gin,,

good

agreement

ferredoxin are

the

seuquences

were

About

13 u moles

of

ion

purif-

CMCFd

exchange

with chromatography,

or

some

Sequence

subtilisin.

Cyanogen

bromide

described

by

cleaved

of

cleavage

Steers,

Craven

with

cyanogen

bromide,

other

from

I ON 1 to

69

and

the

cleavage,

from

These

tryptic of

amide

residues

and

Scenedesmus

residue

werlapping

chymotryptic

peptides

ferredoxin

previously

as

reported

shown (Matsubara

figure. from

Pro4

Gly7

AlalO

with

that

of

(Matsubara, four

chymotrypsin,

electrophoresis.

were

sequence

this

oxidized

chromatography.

deduced

Ser8

There

OFd

bromide

of

revised

paper

as

DISCUSS

derived

acid

by

peptides.

of

and

1968).

thermolysin

residue

cyanogen

amino

is

Scenedesmus groups.

by

distribution

1968)

or

paper

AND

(Matsubara,

and

10 u moles

essentially

by

from

al,

purified

these

acid

one

of

about

were

2 p moles

sequence

The

and

on

formic

obtained

peptides

sequence

0.65.

trypsin

et

with

out

peptides,

were

to

by

chromatography

About

were

acid

elsewhere

determination

hydrolyzed

in

(1965).

large

amino

(CHCFd)

(Matsubara

RESULTS Tko

= 0.62

trypsin,

carried

performed

peptides

described

protein

fragments

further

were

and

as

and

with

by

were

the

previously

peptide

necessary

was

of

hydrolyzed

determinations

complete

METHODS

A420/A276

fragments,

reported

n.

the

and

S-carboxymethylcysteinylferredoxin

peptide

as

AND prepared

had

hydrolysis

the

chymotrypsi

was

preparations

Preparation

COMMUNICATIONS

n.

ferredoxin

These

RESEARCH

Ala-Thr-Tyr-Lys-Val-Thr-Leu-Lys-Thr-Pro

MATERIALS Scenedesmus

BIOPHYSICAL

the

Cys6 direct

1%8),

glutamine

residues

952

sequence

is

Va15 amino

except and

for no

as

follows:

Met,

Ile3

acid

analysis

Lys4Hisl

Leu7

TyrkPhe3. of

the

number

of

asparagine

in

the

Vol.32,No.6,

1968

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS~

4

30 1 ~-Thr-Tyr-I1e-Leu-Asp-A1a-Aia-G1u-Glu-Ala-~-Leu-Asp-Leu-Pro-Tyr-Ser-Cys~Ar~T

40

t

f

60

50 Vai-Gl

,I

u-Ala-a-Thr-Val-Asp-e-Ser-&-

70

Gin-Ser-PheYLeu-Asp-Asp-Ser-Gin-Met~Asp-Gly-~-Phe~Val-Lcu-Thr-Cys-Val-Ala-Tyr-

1

J

I I

c :

80

90 Pro-Thr-Ser-Asp-Cys-Thr-lle-Ala-Thr-His-Lys-Glu-Glu-Asp-Leu-Phe

Figure

The

1.

amino

acid

h

sequence

of

Scenedesmus

ferredoxin.

The peptide bonds cleaved by trypsin in S-carboxymethylcysteinylferredoxin are shown by upward arrows, and those by chymotrypsin by dwnward arrows. Dotted-line arrows represent the positions relatively less susceptible to hydrolysis. The peptide bond cleaved by cyanogen bromide in oxidized The residues common to Scenedesmus, spinach ferredoxin is shown by a triangle. and alfalfa ferredoxi ns are under1 i ned.

seque

rice 0

10,172,

not The

19 of The

sequence

which

and

Legumi

in

explained base

other

is

those

two

than

nosae

fami

in 1 ies,

positions

by

one

base per

higher other,

they

considerations. angiosperms

29

The

do

from

.Spinach the

order

respectively,

a molecular

from

that

of

change

in

their

codon

simi plants

but

to

weight

of

sulfide.

Scenedesmus

two

each

corresponds

inorganic

0.27.

of to

acids

difference of

alfalfa

homologous

taxonomical donous

be

and

amino and

ferredoxins

Scenedesmus

the

96 iron

minimum

spinach

from

of

differs

can

between

clearly

total

including

average

0.41, of

The

between

and

alfalfa

lari

ty is

and

Tracheophyta, while

953

the

two

is

0.43,

and

those

the

ferredoxins

striking

as

alfalfa,

might

both are

Scenedesmus

in

the is

they

differs be

of

between

and

ferredoxin

other,

codons.

ferredoxins

between quite

ferredoxin,

respective

is

Scenedesmus each

spinach

are more

expected

from

are

dicotyle-

which

Chenopodiaceae in

of

the

green

and algae,

Vol.

32, No.

6, 1968

Chlorophyta,

Comparisons

plast-type in

BIOCHEMICAL

ferredoxins,

detail

by

lacking

in

Matsubara,

Margoliash,

1966;

the

(residue

(1968).

Cantor first

Sasaki

of

have

and Matsubara,

1967;

nor

methionine

can

and

recently

been

found

methionine

(Keresztes-Nagy

Matsubara

et

be

responsible

the

positions

chloro-

described

ferredoxin

containing

and

COMMUNICATIONS

bacterial

chloroplast-type

asparagine,

tryptophan

three

go),

and

chloroplast-type

one

gl),

of

(residues

32,42,49,54

and

al I of

four

20-21

and

65-66),

maintenance

of

It

is

positions

the 7,

apparently Benson

1966)

that

et

al,

1967,

for

electron-

invariant

and

observed in

1966,

in

its

1968).

et

ferredoxin. in

which

the

invariant

of al,

example,

12,

42

The

chloroplast-type

25,

of

64,

75

12,

and

g5),

(residues

92-93)

these

are

residues

72

are

for

and

the

the

(Tanaka

(Benson was

29

chloroplast-type

and and

equidistant ferredoxins

repetitive distribution

is

may

an et

et

1967;

sites

in

al,

1964,

1964,

Matsubara, length.

clostridial

ferredoxins

sequences

possibly

al,

residues,

al,

of

18,

at

consecutive et

positions

leucines

(Tanaka

26

in

at

There

proposed for

located

cysteines

ferredoxins first

954

six

(residues

groups

equidistant.

1968)

4,

of

35,

and

the

and

repetition

1967,

five

Asp-Asp 29-30

histidine

(residues

glycines

residues

For

proposed

the

7,

one

function,

repeated

was

regions

(Matsubara

residues

for

was

partially

homology

certain

spinach

It

seven

two

clostridial

1967).

36),

importance

approximately in

was

1568)

in

are

structure

molecule

Cantor,

the

positions

95

19 and

(residues

suggest

of

lysines

of

80),

of

four

(residues

groups

some

at

six

37,

positions.

a revised

and

77),

23,

molecule that

al,

the

Similarities

found

protein

64

and

3,

to

of 10,

leucines

Glu-Glu

seems

glycines

35,

recently

were

two

repetitive

1966;

Jukes

and

equidistant 77,

47

seven

(residues

interest

approximately and

of

three

(residues

44,

nes

the

of

39,

six

fact

40),

prolines

18,

tyrosi

This

(residue

four

72),

invariant.

ferredoxins,

arginine

three

cysteines

but

sequences

and the

RESEARCH

activity. In

47,

acid

aspects

is

and

neither

transfer

50

ferredoxin

amino

BIOPHYSICAL

evolutionary

Jukes

tryptophan

Evidently

the

and

Scenedesmus

and

of

AND

some be

were of

also

the

related

to

Vol. 32, No. 6, 1968

the at

repetitive

BIOCHEMICAL

structure

85,

position

is

of

under

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

molecule.

The

status

of

the

cysteine

residue

investigation.

ACKNOWLEDGEMENTS The

authors

this

investigation;

Mrs.

0.

wish

to

thank

Dr.

Tsuchiya

and

W.

Mr.

Dr.

J.

L.

T.

H.

Oswald

Jukes

for

Nerenberg

for

his

supplying

for

interest

Scenedesmus

their

technical

and

support

cells;

and

of to

assistance.

REFERENCES

Arnon,

0.

Benson,

Science,

I .,

A,

M.,

1532 (1966);

S.

Keresztes-Nagy,

S.,

Matsubara, Structure,

H., Jukes, Function

Matsubara, San

H.

Pietro,

A.

Sasaki,

R,

Steers,

E.,

M.

Margoliash,

Biol.

E.,

F.

and

Chem., T.

H.

and

K.,

Black,

C.

and

Matsubara, Craven,

G.

Proc.

Nat1

o Acad.

241.

5955

Sci 0,

5

Biol.

Chem., E.,

personal

(1966). (1968).

communication

370 (1968).

Fed.

R.

Proc.,

C.,

Annu.

H.,

Biochem.

R.

T.,

563 (1967).

Cantor, C. R., in Proteins,

and Evolution

Sugeno,

K.

Margoliash,

& and

J.

Sasaki, R. M. and Chain, 1725 (1968); Chem., 24 & 1732 --t 1%8).

and

Jr.,

(1965).

and Yasunobu, Biophys., 121,

Perini,

H.,

H., Biol. Chem.,

J.

and

Matsubara,

J.

1460

Mower, H. F. Arch. Biochem.

Keresztes-Nagy,

Matsubara, (1967); J. Biol.

a

and

K., Proc. Matsubara,

a Rev.

Brookhaven No. 21

Biology,

2, M.,

439

(1968).

Plant

Physiol.,

Biophys.

Res.

C.

in press.

Natl, Acad, Sci., H. and Sasaki, R.

392

Anfinsen,

Symposia in

(1968),

B.,

J.

s

155 (196.5).

Comnun., Biol.

28.

467

Chem.,

240,

(1967).

2478

(1965). Tanaka, Biophys. Tsunoda, Biochemical

Nakashima, M., Res. Commun., J.,

Whiteley, Conference

T., 16,

Benson, A., 422 (1964);

H. and (1967)

Yasunobu, p. 103;

Mower, H. Biochemistry, K. T., personal

955

F.

and 5,

Yasunobu, K. 1666 (1966).

Abstract of communication

Pacific (1967).

T.,

Slope

Biochem.