PMR characterization of alfalfa and soybean ferredoxins: The existence of two ferredoxins in soybean

Vol. 42, No. 2, 1971

PMR

BIOCHEMICAL

Characterization The Existence

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of Alfalfa and Soybean Ferredoxins: of Two Ferredoxins in Soybean

J. D. Glickson*, W. D. Phillips, C. C. McDonald, and M. Poe** E. I. du Pont de Nemours and Company Central Research Department*** Experimental Station Wilmington, Delaware 19898 Received December 10, 1970

Summary Observation resonance

of contact-shifted

spectra

of the oxidized

soybean ferredoxins

confirms

and reduced

previous

of each of these plant ferredoxins coupled.

The contact-shift

is best interpreted in approximately spectra

resonances

phylogenetic

The physical associated

with plants

molecular

weights

apparently

common

have been extensively

are about 10,500, to all,

As part of a general of the iron-sulfur

soybean preparation

sulfur

of plant ferredoxins

of plants. of the iron-sulfur characterized.

proteins (1) Their

structural

feature,

at the redox center of two atoms.

study of the electronic proteins

ferredoxins

that contact-shift

controls

and a prominent

is the presence

iron and two “inorganic”

structures

properties

and

exchange

of two distinct

the genetic

magnetic

that the two iron atoms

It is concluded

relationships

and chemical

of alfalfa

of the reduced

of the presence

may be useful for studying

nonheme

conclusions

equal concentration.

and for elucidating

forms

are antiferromagnetically

spectrum

in terms

in the proton

and geometrical

by proton magnetic

resonance

(PMR)

*

Preseti address: Division of Molecular Biophysics, Laboratory of Molecular Biology, University of Alabama, Birmingham, Alabama.

**

Present address: Merck Institute Rahway, New Jersey 07065.

***

Contribution

No. 1770. 271

for Therapeutic

Research,

Vol. 42, No. 2, 1971

spectroscopy, forms

BIOCHEMICAL

we have observed

of the ferredoxins

originate

from

from interactions

in the oxidized

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

contact-shifted

resonances in both redox spinach and parsley. (2) These resonances

with paramagnetic

as well as reduced

of contact-shifted

resonances

doxins are closely

related,

shift spectra

observed

forms

observed but differ

centers

of these proteins. in the spinach

in detail.

in the two-iron

present,

apparently,

The patterns

and parsley

ferre-

On the other hand, contact-

ferredoxins

appeared

qualitatively

different

from those encountered with the one-iron rubredoxin from (3) Clostridium pasteurianum, the four -iron high potential iron protein (4) from Chromatium, and the eight-iron ferredoxin from C. pasteurianum.

In this note the patterns redox forms

of alfalfa

of contact ferredoxin

of the spinach and parsley

shifts in the PMR

ferredoxins.

In contrast,

of ferredoxin

related

of the other plant ferredoxins,

distinctly

different

approximately

from soybean

ferredoxins

of the two

are shown to be very similar

shift spectrum tothose

spectra

(cultivar

to those

while the contactKent) is obviously

it is concluded

are present

(5)

that two

in the soybean preparation

in

equal concentrations.

Experimental Ferredoxins isolated

from

by the method

of the latter

protein

alfalfa@)

and soybean

of Keresztes-Nagy

was completed

0.01 M Tris*HCl

linear

gradient

concentrations an extinction techniques

coefficient similar

Elution

(3.0 x

against

0.15 M

was accomplished

0.01 M Tris.

with a

HCl pH 7.8.

Protein

spectrophotometrically at 422 ~Q.L, using (7) Reduction and spectroscopic of 0.79 cm liter/g.

to those employed Dithionite

the characteristic

associated Results

pH 7.8 buffer.

a column

were determined

were used here. yielded

fine) equilibrated

of 0.2 to 0.4 M NaCl,

Kent) were (7) purification

and Margoliash;

by passage through

90 cm) of Sephadex G-75 (Pharmacia; NaCl,

(cultivar

with spinach and parsley

reduced

alfalfa

ferredoxins

and soybean ferredoxins

g = 1.94 electron

spin resonance

both

absorption

(8)

with other plant ferredoxins.

and Discussion The 220 MHz PMR spectra

15”, 23”,

and 30°C.

are presented

of oxidized

alfalfa

in Figure

1. Resonance

272

ferredoxin

at 6”,

absorption

(2)

Vol. 42, No. 2, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ALFALFA OXIDIZED

4X0

Fioure of oxidized Tris.

5600

1.

alfalfa

DCl).

3200

2400

2000

H2 I600

800

1200

0 -200

400

Temperature

dependence

ferredoxin.

10% (w/v) in D20 at pD 7.8 (0.2 M

The region

from the spectrum. spectral

2800

FERREDOXIN / D20. pD 7.8

of the 220 MHz PMR spectra

of HDO absorption

(3-5 ppm) has been omitted

The -1 to 10 ppm regions

are derived

scans, while the 11 to 19 ppm regions

represent

from single 100 computer

accumulations. in the -1 to +8 ppm range is qualitatively diamagnetic averaging,

proteins,

from the background

absorption.

A similar

the ferredoxins interaction exchange

and will not concern

a broad resonance

emerges

from residual

the iron-sulfur

moiety

but unresolved

resonances

parsley

ferredoxins

of the contact-shift

region

to a single

of resonance

in the oxidized

forms

residues

chain.

to reside

thought

273

absorption

to bind

The seven other expected

for the oxidized upfield

was assigned

spinach

and

and to be indistinguish-

of the bulk of the protein.

of resonance

of

to a contact

atoms. (2) The resonance

of unit intensity absorption

proton

of two antiferromagnetically

of four cysteine

are believed

for

Upon computer

and was attributed

to the polypeptide

able from the resonance

in intensity

is observed

and parsley ferric

to one of eight B-CH2 protons

here.

in the +14 to +15 ppm region

paramagnetism

high-spin

to that observed

us further

corresponding

resonance

from spinach

coupled,

similar

of oxidized

Analysis alfalfa

50% molar

dependence

excess of solid sodium

dithionite.

1. Reduction

with a

and downfield was accomplished

for the upfield

regions

of the 220 MHz PMR spectra

10% (w/v) in D20 at pD 7.8 (0.2 M

accumulations

ferredoxin.

Spectral

alfalfa

Temperature

were the same as for Figure

Tris- DCl).

of reduced

Ficrure 2.

ALFALFA FERREDOXIN REDUCED / D20, pD 7.8

Vol. 42, No. 2, 1971

ferredoxin

BIOCHEMICAL

proceeds

of the low-field

along identical

region

the contact-shifted (14.5 ppm),

resonance

is plotted

ferredoxin ferredoxin,

oxidized

form.

intensity

side of Figure

dependence

+12 to +31 ppm,

The temperature

corresponding

of alfalfa

dependence

ferredoxin,

including

between 3,100 Hz (14.1 ppm) and 3,200 Hz

on the left-hand

resonances

The temperature

spectrum

is given in Figure

reduced

contact-shifted

lines.

of the PMR

The temperature alfalfa

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the PMR spectrum

of reduced

2.

region

The contact-shift

is much richer

dependences

of reduced

alfalfa

of the six resolved ferredoxin,

FERREDOXI

OXIDIZED

of the

than that of the

to one proton per molecule,

ALFALFA

3.

each of

are plotted

on

N

REDUCED 70007

3500r--

2500

30oo

-------

2ooot 0

Fiaure of oxidized

3.

Temperature

(left) and reduced

dependences (right)

alfalfa

,

,

,

IO

20 T

30

of the lowfield ferredoxin.

resonances

Only the

resonance

that occurs between 3,100 and 3,200 Hz in oxidized

ferredoxin

can be clearly

all of those displayed

identified

for reduced

with a contact-shift alfalfa

contact interaction. 275

ferredoxin

origin, appear

alfalfa whereas

subject to

Vol. 42, No. 2, 1971

BIOCHEMICAL

the right-hand

side of Figure

eight resonances residues

the two missing absorption

resonances

are located

range.

shifted resonances contact-shifted

of reduced

(increase

moved upfield

(2)

slowly.

moved down(2)

The six

were qualita-

with respect

parsley

to

distinguishable apparently

ferredoxin,

four

and four of which

ferredoxin

the contact -shift as well as quantitatively

and spinach ferredofins.

We suggest that these rather

striking

of the plant ferredoxins

differences

in the reduced

in contact-

form arise from

in angles about the C -S bonds of the cysteine residues, such B in turn being produced by differences in conformation of the

variations polypeptide

part of the protein.

These conformational

be consequences various

over

resonances,

with temperature

from those of the alfalfa

variations

ferredoxin

form was qualitatively

with

six contact-

but were readily

For parsley

of the reduced

shift spectra

ferredoxin,

for reduced

alfalfa

contact-shift)

Eight contact-shifted

moved downfield

of resonance

contact-shift)

(decrease

alfalfa

were observed

that

of reduced

spinach ferredoxin

dependence,

comparison.

of which rapidly

in the region

and two very slowly moved upfield.

and temperature

a full complement,

it is suggested

four of which rapidly

to the six of reduced

on quantitative

of four cysteine

resonances

spinach

were observed,

structure,

of the rest of the protein.

move downfield

resonances

similar

different

protons

For reduced

field with temperature

spectrum

upfield

and two slowly move upfield

the 6” to 30°C.

form,

contact-shifted

four rapidly

temperature

position

to the B-CH2 protons

of the carbon-bound

ferredoxin,

based on the presumed

As for the oxidized

Of the six resolved

tively

3. Again,

corresponding

are expected.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of sequence variations (9) plant ferredoxins.

Rather

subtle structural

the plant ferredozdns,

among the polypeptides

and compositional

thus, may be reflected

of resonance

absorption

of the reduced

temperature

dependence

of the contact-shift

of the reduced is presented

form

of a ferredoxin

in Figure

4.

276

would of the

differences

between

in the contact-shift

forms.

In support region

preparation

The striking

differences

feature

region

of this,

of resonance

of soybeans is that instead

the absorption

(cultivar

Kent)

of the six

Vol. 42, No. 2, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SOYBEAN KENT FERREDOXIN OXIDIZED

REDUCED

-I iooo-

20000

IO 20 30

0

IO 20 30

T ("Cl

Figure of oxidized

4.

Temperature

(left) and reduced

protons/ferredoxin indicated

(right)

molecule

to the right

Only those resonances

with a contact interaction or eight contact-shifted

responsible

temperature,

of

The intensity

data were treated

ferredotins

are

in equal concentra-

intensity

indices

are identified

origin. resonances

soybean ferredoxin. and six slowly optical

Numbers intensities

with associated

observed fourteen * Eight

shift upfield.

that only one kind of soybean ferredoxin and of normal

resonances

for resonance

of two alfalfa

the three other plant ferredoxins, for the reduced

of the lowfield

soybean ferredoxin.

of each resonance.

on the basis of the presence tion.

dependences

absorbance

for the reduced resonances rapidly

is present,

of

are actually

detected

move to lowfield

In addition, molecule

forms

under the assum.ptions

of molecular

weight 10,500

each of the fourteen

contact-

* Two resonances of Figure 4 have twice the intensities of the other ten and each is considered to be two coincident resonances of unit intensity. 277

with

Vol. 42, No. 2, 1971

shifted simplest

BIOCHEMICAL

resonances

corresponds

interpretation

there are actually tion present

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in intensity

is that instead

two different

to only one-half

of a single

ferredoxins

ferredoxin

species,

of approximately

equal concentra

in the preparation.

PMR would appear to offer a particularly physical

The

a proton.

differentiation

even to detection

between ferredoxins

of multiple

known what structural soybean Kent,

differences

although

acid sequences

ferredoxins

of different

approach species

of a given species.

to

and

It is not

exist between the two ferredoxins

such differences

of the polypeptide

could be smaller

sensitive

probably

chains.

reside

in the amino

Such differences

than the four points of amino

of

conceivably

acid heterogeneity

detected

by Benson and Yosunobu in the sequences of ferredoxin from Leucaena (10,ll) and as suggested by Keresztes-Nagy, Perini, and glauca trees (12,131 Relatively minor differences Margoliash for alfalfa ferredoxin. in sequences

could affect through

conformational

tions about Cg-S bonds of cysteine in the PMR spectra spectra,

by large differential phylogenetic

Studies are in progress a variety

orienta-

and these could be manifested

contact

thus, may be useful for studying

doxins and for elucidating including

residues,

perturbations interaction

genetic

controls

relationships

on a number

shifts.

These

of plant ferre-

of plants.

of other plant ferredoxins,

of soybean ferredoxins.

References 1. 2. 3.

J. C. M. Tsibris

and R. W. Woody,

No. 3, September

(1970).

M. Poe, W. D. Phillips,

J. D. Gliqkson,

and A. San Pietro,

Natl.

W. D. Phillips, W. Lovenberg,

4.

W. D. Phillips, Proc.

5.

Coord.

Natl.

Proc.

Nature

Natl.

Acad.

C. C. McDonald, C.- C. McDonald,

(1971). and

227. 574 (1970).

M. Poe, Acad.

Rev. 5

Sci. (U.S. 1, Jan.

M. Poe, J. F. Weiher, C. C. McDonald,

Sci. (U.S.),

M. Poe, W. D. Phillips, Proc.

Acad.

Chem.

67. 682 (1970).

C. C. McDonald,

Sci. (U.S.),

and R. G. Bartsch, and W. Lovenberg,

65. 797 (1970). 278

BIOCHEMICAL

Vol. 42, No. 2, 1971

6.

We are indebted Delaware

7.

to Dr. W. H. Mitchell

for supplying

the ferredoxin

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

us with alfalfa

of the University

of

(Medicacro sativa)

for

preparation.

S. Keresztes-Nag-y

and E. Margoliash,

J. Biol.

Chem.

241.

5955 (1966). 8.

(3. Palmer

9.

Effects

and R. H. Sands, J. Biol.

of rotation

{contact -shift

241, 253 (1966).

about CB-S bonds of cysteine

spectra

more completely

Chem.

in the iron-sulfur

in Refs.

residues

proteins

on

are discussed

2-5.

10.

A. M. Benson and K. T. Yasunobu,

J. Biol.

11.

A. M. Benson and K. T. Yasunobu,

Proc.

Chem. Natl.

244. 955 (1969).

Acad. Ski.

(U.S.)

$3. 1269 (1969). 12.

S. Keresztes-Nagy, 22

13.

F. Perini,

and E. Margoliash,

J. Biol.

Chem.

981 (1969).

We, incidentally, ferredoxins shift spectra

have not detected

in our preparation, for reduced

by A. San Pietro

by PMR a heterogeneity

but have observed

parsley

ferredoxin

prepared

and by J. A. Fee and G. Palmer.

279

different

of alfalfa contact-

independently