Regulation of electron transport in photosynthesis

BIOCHEMICAL

Vol. 3 1, No. 4, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REGULATION OF ELECTRON TRANSPORT IN PHOTOSYNTHESIS Walter Department

Received

April

of Biology, Milwaukee,

results

et al., mole

of NADPH.

dependent

cyclic

phosphorylations (Black

the

or pseudocyclic

requiring

(Forti

ferredoxin These

1963). relative

However,

these

of other

photophosphorylation

between in this

pseudocyclic

production

suggest

paper

inhibit

either

These

operative

in the

would

(1963)

serve

found

reductase

in causing

show no inhibition suggest

the possielectron

to a cyclic

transor

of ATP.

This investigation was supported by Public Health Service General Support Grant 5 SO1 FR-5434 and Research Grant No. GM 13732 from National Institute of General Medical Sciences.

582

as

phosphorylation.

photosyn%hetic

from NADP reduction

et al.

compounds

or at least results

must be

photophosphorylation.

that

associated

stimulate

reactions,

electrons

Tagawa

ferredoxin-NADP

and the

or phospho-

there

acceptor

and noncyclic demonstrate

1961)

that

of ATP and NADPH.

cyclic

selectively

mechanism directs

and Jagendorf,

of NADP as an electron

same extracts

of a control

et al.,

observations

output

availability

extracts

which

of ATP and

1963)

of NADP photoreduction

system

moles

each

(Tagawa

reported

port

three

that

photophosphorylation

The experiments

bility

requires

out

ferredoxin

regulator

inhibition

have pointed

(Arnon

by either

a physiological

spinach

cycle

chloro-

each NADP reduced

and others

Calvin-Bassham

isolated

ATP may be generated

of the

that

(1957)

in

Additional

et al.,

some regulation

of one ATP for

Kandler

by the

two moles

boiled

University

to NADP photoreduction

production

However,

of CO2 fixed

suggest

coupled

in the

1958).

doxin

Marquette Wisconsin

15, 1968

Photophosphorylation plasts

W. Fredricks

Research the

BIOCHEMICAL

Vol. 3 1, No. 4, 1968

MATERIALS Transhydrogenase lated

by acetone

Pietro

and Lang

column

following

from genase

fractionation These

the procedure dialyzed

was further

ammonium sulfate.(Shin

were

prepared

glass

peaks

Chloroplast

preparations

used

the Cls

prepared

(1958)

the

using

omitted Hill blank

cuvette

genase

measuring

the decrease

cally

of Taussky at pH 8.0

of Lowry

et al.

(BSE)

"grana"

- 8.2. (1951)

Protein

through

= 24, A325nm = 15.8) of San Pietro

et al.

(1959)

directly

by measuring

of identical

and Avron

the increase

composition

except

the absorbance except

change

lacking

phosphate

were

to the

was followed reaction

were

procedure

mixtures

carried

estimated

by by the

out aerobiby the procedure

of Arnon

(1949).

RESULTS AND DISCUSSION Figure

1 shows that

BSE contains

a compound(s)

583

which

a

The transhydro-

at pH 8.0 according

reactions

by the

(TCPIP)

at 625nm against

TCPIP.

in the

in

NADP was

The trichlorophenolindophenol

All

and

medium.

concentrations

and chl orophyll

filtered

of Jagendorf

Photophosphorylation

(1953).

a Waring

was characterized

of Whatley

et al,

and Shorr

(BSE)

with

preparations

was measured

in inorganic

water cooled,

NAD as acceptor (1960).

extracts

(A260nm

procedure

1958).

composition

of Keister

method

and Lang,

of identical

procedure

the

was eluted

The transhydro-

spinach

fluid

to San

40 and 65% saturation

minutes,

NaCl buffer

a blank

Ferredoxin

in distilled

ten

to the

by noting

with

leaves

iso-

according

of purity.

Boiled

were

on a DEAE cellulose

between

fragments

was measured

reaction

state

1963).

was followed

San Pietro

reaction

(1963).

included

sucrose,

at 340nm against (cf

et al.

chloroplast

NADP photoreduction absorbance

of Shin

for

homogenates

in the ultraviolet

according

Tris,

separated

The supernatant

two absorbance

chloroplasts

were

was boiled

and centrifuged.

(1958),

proteins

spinach

by having

and Lang

spinach

et al.,

by homogenizing

and ferredoxin

of crude

by fractionation

The homogenate wool

reductase)

and used at this

purified

with

blender.

AND METHODS

(ferredoxin-NADP

(1958).

the column,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

strongly

inhibits

BIOCHEMICAL

Vol. 31, No. 4, 1968

Al

I

I

I

I

I

I

I

0.1

0

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

' 0.3

0.1

BSE(ml)

10

0

,

of BSE on NADP photoreduction. The reaction mixFigure 1. Curve A. Effect chloroplasts (Jagendorf and Avron, tures contained in a final volume of 3 ml: 1958) containing 155 ug of chlorophyll, partially purified ferredoxin equiv200 umoles of Tris-HCl (pH 8.01, 0.4 umoles of alent to 0.132 mg of protein, NADP and BSE as indicated. The reactions were carried out at room temperature of BSE on the TCPIP Hill in white light (1400 ft. can.). Curve B. Effect chlororeaction. The reaction mixtures contained in a final volume of 3 ml: plasts (San Pietro and Lang, 1958) equivalent to 75 ug chlorophyll, 200 umoles The reactions 0.19 umoles of TCPIP and BSE as indicated. Tris-HCl (pH 8.0), were carried out at 15' in white light (400 ft. can.). of BSE on transhydrogenase activity. The reaction mixtures Figure 2. Effect partially purified transhydrogenase contained in a final volume of 3 ml: equivalent to 2.59 mg of protein, an excess of isocitric dehydrogenase, BSE Tris-HCl (pH 8.0), 150; sodium as indicated and the following in umoles: The reactions were run at isocitrate, 10; MgC12, 10; NADP, 0.1; NAD, 1.0. room temperature.

photoreduction

of NADP (curve

In terms

B). thesis

(cf

ened

of the current

Vernon

associated

with

by the

and Avron, photosystem

observation reductase

suggesting

the

that

the

correlation

inhibition

charcoal. charcoal

inhibitor between

1965)

results

these selectively

2) that strongly

the

site

inhibition

since

inhibitor the

of both

unadsorbed

is the

a reaction

This

view

transhydrogenase by comparable is

ferredoxin-NADP

observed

when BSE is

appears

of BSE inhibit

in photosyn-

that

of transhydrogenase

reactions

components

transport

inhibited.

the

sensitive

of TCPIP (curve

suggest

inhibited

of NADP photoreduction The

photoreduction

of electron

I is

is

the

formulation

(Figure

ferredoxin-NADP

A further

A) but not

strength-

activity levels

of of BSE

reductase. activity treated

to be adsorbed neither

is

and

with by

NADP photo-

BIOCHEMICAL

vol. 3 1, No. 4, 1968

reduction

nor transhydrogenase

Photophosphorylation (Figure in

3, curve

activity. coupled

A),

but

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

to NADP reduction

degree

of

the case of NADP photoreduction.

basis

of another

inhibited

observation.

by BSE (Figure

stimulates

the

rate

Although

1, curve

of endogenous

inhibition

This

A),

also

is

is

disparity

less might

electron

transfer

in the absence

inhibited than

that

by BSE observed

be explained

on the

to NADP is

of added

photophosphorylation

strongly

NADP BSE strongly

(Figure

3, curve

B).

,-

I-

4 J 0.1

O

0.1

I

I

0

0.3

L

0.3

BS E(ml)

o”BSE(ml)ha

Figure 3. Curve A. Effect of BSE on photophosphorylation coupled to NADP photoreduction. Curve B. Effect of BSE on photophosphorylation catalyzed by The reaction mixtures contained in a final volume low levels of ferredoxin. of 3 ml: chloroplasts (Jagendorf and Avron, 1958) equivalent to 439 ug of chlorophyll, partially purified ferredoxin equivalent to 0.26 mg of protein, BSE as indicated and the following in umoles: 10; ADP, 8; K2wO4 I 5; W12, NaCl, 88; Tris-HCl (pH 8.0), 40. In addition reaction mixtures of curve A (but not curve B) contained 10 umoles of NADP. The reactions were run at room temperature in white light (1400 ft. can.). Figure 4. Curve A. Effect of BSE on PMS catalyzed photophosphorylation. The reaction mixtures contained in a final volume of 3 ml: chloroplasts (Jagendorf and Avron, 1958) equivalent to 103 ug chlorophyll, BSE as indicated, and the following in umoles: ascorbate, 15; PMS, 0.1; ADP, 8; K2HP04, 5; MgC12, 10; NaCl, 88; Tris-HCl (pH 8.0), 40. Reactions were run at room temperature in white light (1400 ft. can.). Curve B. Effect of BSE on photophosphorylation catalyzed by high levels of ferredoxin. The reaction mixtures contained in 3 ml: chloroplasts (Whatley.et al:, 1959) equivalent to 140 ug chlorophyll, partially purified ferredoxln equivalent to 1.66 mg of protein, BSE as indicated and the following in umoles: 10.5; Tris-HCl (pH 8.2), 90. M@12 > 6; ADP, 10.5;oK2HP04, The reactions were carried out at 15 In low intensity red light.

This (Black

latter et al.,

effect

is presumably

1963).

Hence

due to the

in Figure

presence

3, curve

585

of phosphodoxin

A addition

in BSE

of BSE results

in

BIOCHEMICAL

Vol. 31, No, 4, 1968

decrease

in electron

phorylation, doxin

but

transport this

Figure

high

catalyzed

levels

curve

the

system

I,

site

the

rylation

that

reductase

diverting

NADPH.

The relative

form

to the

this

hypothesis

compounds

amounts

Efforts the

are

inhibitor

is that

is destroyed Over

the

glutarate, of all

these

formaldehyde

which

involved

events

with

photo-

in photophosphoit

is

of ferredoxin-NADP produce

ATP but

regulated

in the cell.

of the

3,

that

hypothesis

which

not to con-

A corollary

inhibitor

made to identify

will

of

the

itself

to

reflects

inhibitor

ferredoxin-NADP

be reported This

acid.

conclusion

is

found

reductase

elsewhere,

has a low molecular

compounds

reported

compounds that

activity,

have been

by Keister

transhydrogenase

tested

the enzyme

synthetic

activity

organic

ascorbate,

compound

associated

activity

pathways

suggests

of ATP and NADPH are thus

being

inhibitor

to those

inhibited

This

(Figure

as a model

suggest

based

that

in part

weight,

anionic

in BSE.

the

on the

obser-

properties

and

by ashing. forty

addition

into

the

A) or relatively

of ATP and NADPH.

an organic the

by phospho-

of ferredoxin

As a working

the concentration

transhydrogenase experiments,

levels

is not

or PMS.

flow

currently

reaction,

vations

reaction

production

that

(PMS)(curve

At low

in BSE modulate

for

in phos-

on photophosphorylation

by BSE is a reaction

electron

is

effect

of phosphorylation.

sensitive

requirements

the relative

Using

the rate

by ferredoxin

suggested

little

B).

of inhibition

catalyzed

by a stimulation

methosulfate

(curve

inhibitor

decrease

of ATP.

by phenazine

of ferredoxin

a corresponding

reversed

BSE has very

B) BSE stimulated

although

is partly

production

4 shows that

reactions

to NADP with

effect

of a pseudocyclic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

activity.

methylglyoxal, is that has this e.g.

et al.

and acetaldehyde,

inhibitory Several

They include:

possess

grouping

vicinal

act

carbonyl

found

the

as inhibitors

activity of those

pyruvate,

and glyoxal.

has been

stimulates also

for

(1960).

diacetyl,

they

oxalate

tested

in tested

c+keto-

A common feature groups.

Every

to have some effect

reaction. at high

Two compounds, concentrations,

on

BIOCHEMICAL

Vol. 3 1, No. 4, 1968

perhaps

by virtue

carbonyl

of the

abiiity

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of two molecules

to simulate

the vicinal

configuration.

These reductase,

findings located

plays

a key role

control

of the

are consistent at a potential in the

activity

with branch

regulation of this

the

hypothesis

point

of electron enzyme the

that

in electron flow

relative

ferredoxin-NADP transfer

reactions,

in photosynthesis. output

By

of ATP and NADPH

is controlled. The author Miss

Judy

wishes

to acknowledge

the

excellent

technical

assistance

of

Kohlmann.

REFERENCES Arnon, Arnon, Black,

D.I., Plant Physiol., 24, 1 (1949). D.I., Whatley, F.R., az Allen, M.B., Science, 127, 1026 (1958). C.C., San Pietro, A., Limbach, D., and Norris, G., Proc. Natl. Acad. 37 (1963). sci., U.S., 0, Forti, G., and Jagendorf, A.T., Biochim. Biophys. Acta, 54, 322 (1961). Jagendorf, A.T., and Avron, M., J. Biol. Chem., 231, 27771958). Kandler, O., Z.Naturforsch., 12b, 271 (1957). Keister, D.L., San Pietro, A.,nd Stolzenbach, F.E., J. Bio. Chem., -'235 2989 (1960). Lowry, O.H., Rosebrough, N.J., and Farr, A.L., J. Biol. Chem,, 193, 265 (195 1) San Pietro, A., and Lang, H.M., J. Biol. Chem., 231, 211 (1958). Shin, M., Tagawa, K., and Arnon, D.I., Biochem. zt., 338, 84 (1963). Tagawa, K., Tsujimoto, H.Y., and hrnon, D.I., Proc. Natl. Acad. Sci., U.S., 49, 567 (1963). TaussG, H.H., and Shorr, E., J. Biol. Chem., 202, 675 (1953). Vernon, L.P., and Avron, M., Photosynthesis, AK Rev. Biochem., -'34 269 (1965). Whatley, F.R., Allen, M.B., and hrnon, D.I., Biochim. Biophys. Acta, 2, 32 (1959).

587