Vol. 68, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PRIMARY PHOTOCHEMICAL PROCESSES IN BACTERIORHODOPSIN by K. J. Kaufmann and P. M. Rentzepis Laboratories, Murray Hill, New Jersey

Bell

07974

and Walther Stoeckenius Department of Biochemistry and Biophysics of California, San Francisco, California

University

94143

and School of Applied Cornell University, Received

November

Aaron Lewis and Engineering Physics Ithaca, New York 14850

24, 1975 SUMMARY

Experiments in the picosecond range indicate that the rate formation of the first intermediate in the pptoreaction cycle of bacteriorhodopsin is approximately 1011 set . A transient at 580 nm has been observed and is tentatively attributed to the excited singlet state.

of

INTRODUCTION Halophilic

bacteria

halobium,

when grown

in the light

specialized

regions

50%

of the total

which

consists

membrane

These

the

to utilize

across

bR560

*

the

membrane

membrane

membrane These

known

energy

and synthesize

Halobacterium

patches

tension,

can occupy contain

to produce

a proton

more than

bacteriorhodopsin

protein

as the purple

produce

via

a Schiff

membranes,

enable

gradient

ATP (2,3).

exists dark,

which

to an opsin-like

patches,

photon

Bacteriorhodopsin * and bR In the 570'

cell

bound

species

and at low oxygen

area.

of retinal

base (1). cells

in their

such as the

it

in two relatively

stable

forms,

has an absorption

maximum at

The intermediates in the photoreaction cycle of bacteriorhodopsin have been designated bK5y0, bLsFO, bMA12, bN529, bOGhO (Fig.1). The subscripts indicate the maxima of the calculated absorption spectra; they vary slightly with temperature and. possible other experimental conditions.

1109 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 68, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

BACTEAIORl4DDOPSIP1 510 ntn

ALL Tams n

17

-&

/

t

,64&m, INTERMEDIATE

hu

cssoK fllnl INTERMEDIATE

I &rn)

I

,556nml INTERMEDIATE

INTERMEDIATE

MdL,

INTERMEDIATE

Figure 1.

Schematic of light see reference 6.

560 nm and the retinylide conformation.

adapted cycle,

for complete details

chromophore is apparently

In the light

in the 1%cis

the absorption maximumshifts

to 570 nm

and the conformation of the chromophore has an all-trans Both of these forms of the bacteriorhodopsin reaction

cycle after

configuration

(4:,5).

appear to undergo a photo-

a single photon is absorbed.

Several intermediates

in the cycle have been identified.(6), Data reported in this paper are for the bR

5-w

(Fig. 1).

Similar

to

and invertebrate

vertebrate

cycle only

rhodopsin, the

spectra of the first

photoproduct of bacteriorhodopsin

shift

absorption of the chromophore; this is followed

in the visible

by thermal intermediates which are blue shifted. pigments the chromophore in bR570 iS indication

that an isomerization

no additional

all-tranS

shows a red

Unlike the visual retinal

(5); there is no

occurs and bacteriorhodopsin

energy input to complete its reaction

bovine rhodopsin, it returns to the bR

570

cycle.

state within

requires Unlike

a few

milliseconds. Bacteriorhodopsin

in its native

state in the membranecan

be isolated without any apparent change of its structure The particle

size of such a purple membranepreparation

1110

or function is small

(7).

BIOCHEMICAL

Vol. 68, No. 4, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

l* 0.06

l .

0.06

d

-0.02

.

i j1

l~lllllllll1llll 0 40

60

120

160

200

240

200

tfpsecl

Figure 2.

Kinetics

enough that it shows very little of the light repetitive

of absorption at 635 nm.

light

scattering.

The reversibility

induced change, absent in bovine rhodopsin allows measurementson the samessmple. The apparently native

state of the chromoprotein makes an extrapolation in vivo events much more reliable --

from -in vitro

to

than the detergent preparations

of visual pigments. METHODS ANDMATERIALS A double beampicosecond spectrometer was used to measure time resolved absorption spectra, for apparatus see reference 8. in distilled

a complete description

of this

Suspensions of the isolated purple membrane

water were used at a concentration

of about 1 mM(molecular weight 26,000).

of bacteriorhodopsin

The sample was light

adapted as well as shielded from the laser flashlamps. RESULTS The kinetics

of the light-induced

spectral changes at 635 m

for a purple membranesuspensions in H20 show a rise in absorbance which is pulse limited

(< 6-10 picoseconds) (Fig.

in the difference

spectrum of light-adapted

and is due to the formation of the earliest so far,

2); 635 nm is the maximum bacteriorhodopsin intermediate

570

observed

bK590. The absorbance remains constant throughout our

1111

bR

Vol. 68, No. 4, 1976

BIOCHEMICAL

0.2

AND BIOPHYSICAL

-

RESEARCH COMMUNICATIONS

. .

0.1

l

.

d d 4

o-

-0.2

-

L

1 600

I

I

620

640

I 660

I

1

I

I

1

680

600

620

640

660

A (nm)

Figure 3.

Difference spectrum taken 13 psec after exictation Solid line (-) represents microsecond difference

(0). spectrum (6).

measuring time of 300 psec, which is consistent with the previously observed microsecond lifetime

of bK5go (6,9).

Stronger evidence that the

absorbance change is due to the formation of bK comes from the 590 excellent agreement of the difference spectrum taken at 13 psec (Fig.

3)

and that obtained in the microsecond range at l°C, and at lower temperatures

(6,9).

Suspending the membranesin D20 has no detectable effect on the kinetics electric

of the absorbance at 635 nm. Similarly,

vector of the interrogating

than parallel

beam is perpendicular

to that of the exciting

635 nm also remained unaltered;

when the

beam, the kinetics

however, the optical

rather observed at

density changes

are smaller by almost a factor

of two.

this dichroic

remains unchanged 300 psec after

ratio

.of Q 1.8:l

Within experimental error

excitation. We have also observed a fast transient near the isosbestic The

decay

positive

time

point in the difference

is 'L 15 picoseconds.

spectrum at 580 nm (Fig. 4).

A saturation

O.D. change at 630 nm and the transient

1112

decrease in absorbance

study of both the decrease at 580 nm

BIOCHEMICAL

Vol. 68, No. 4, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I

t (wea

are

depicted

change

Figure

4.

Kinetics

in Fig.

5.

The saturation

indicates

fashion

that

the

to excitation.

reliably

whether

as the

longer

of transient

the transient lived

curve

chromophore

From the

for

responds

data

(bK

the positive

in a rather

shown,

has the

intermediate

at 580 run.

we cannot

O.D.

normal

determine

same excitation

dependence

590)’

DISCUSSION The ultrafast rhodopsin in the with

it

is

molecular bK590 to the

time

at

in its

temperature

(bathoform)

further

base

This 590’ intermediate

emphasized

stretching

in both

bovine

enhances in bovine

the

visual

by resonance

the its is

degree

the

very

pigments.

C=C bonds

formation involve

of

formation. similar

This (10,ll)

which

and a protonated

(prelumirhodopsin)

does not 1113

and its

Raman data,

of the

bathorhodopsin

rhodopsin

during

of the

that

inter-

possibility

limits

and kinetics

frequency

thought

the

previously

such as a triplet,

place

can take

of bacterio-

intermediate

of this

excludes

state

change

has been detected

that

prelumirhodopsin

the

first

severely

characteristics

show a decreased

for

room temperatures

spectral

is

-1

The stability

excited

reorientation

similarity

bK

nitrogen

set

which

590 ’

(6,9).

a long-lasting

rise

11

due to the

bK

resolution

at liquid

of 10

apparently cycle,

microsecond

short

Schiff

is

photoreaction

mediate that

occuring

rate

of the a complete

and first cis-trans

Vol. 68, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

*X=580

0.2

nm nm

oA=630

0

0

0 0

0

0 0

0

I,,,,,,,,,,,,, 0

10

20

PULSE

Figure

5.

The time of bovine

in bacteriorhodopsin

is

We believe 580 nm is

other

intermediates not

this

is

after

work

of the

exciting

excited

singlet

bK590 excited

(12)

all-trans

retinal

the fast

causes. which

It

in absolute

the could

in

that

At present,

it

rise

extend

and requires

the

1114

be due to

of one of the since

possibility high

time

the

might the

part the

we

that concentration absorb

transient

formation

visible

to be almost

of bKYgO.

might

concentration

we attribute

would

spectrum

small

near

however,

at relatively fast

chromophore

we have observed

An alternate

of its

of bR570 which

absorption

the

(4,5).

be a photoproduct

is present

and because pulse.

such a conformational

furthermore,

possibility

darkness. which

and,

for

transient

must be present

to ?r 15 picoseconds, singlet

60

UNITS)

of bacteriorhodopsin;

discount

due to bK590, excitation

SO

to be short

rhodopsin

that

we cannot

entirely

did

appears

due to a photoproduct

at present

40 (ARBITRARY

Saturation of optical density changes coincident with the excitation at A = 580 nm (0) and 13 psec after excitation at A = 630 nm (0).

isomerization. rearrangement

30

ENERGY

time

light to the of

of the bR570

same as that

BIOCHEMICAL

Vol. 68, No. 4, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES

(1) (2)

Oesterhelt,

D. and Stoeckenius,

W., Nature

Oesterhelt,

D. and Stoeckenius,

W., Proc.

New Biol. Nat.

Acad.

233,

149 (1971).

Sci.

USA -70

2853 (1973). (3)

Danon,

A. and Stoeckenius,

W., Proc.

Nat.

Acad.

Sci.

USA 2,

1234

L.,

Eur.

J. Biochem.

(1974). (4)

Oesterhelt,

D.,

Meentzen,

M. and Schuhmann,

40, 453 (1973). (5)

Kung-Chung Pasadena,

Yeh Jan,

L.,

Thesis,

California

Institute

of Technology

1974.

(6)

Lozier, R. H., in press.

(7)

Oesterhelt, D. and Stoeckenius, W., in Methods in Enzymology, Volume XXXI, Biomembranes Part A; Fleischer, S. and Packer, L., Eds.; Academic Press, New York, 1974, pp. 667-678.

(8)

';;;;;;,

(9)

Stoeckenius,

Bogomolni,

R. A.,and

T. L. and Rentzepis, W. and Lozier,

P. M., R. H.,

Stoeckenius,

W.,

Chem. Phys.

Lett.

J. Suprsmolecular

Biophys.

2,

J.

337

Structure

2,

769 (1974). (10)

Lewis, A., Stoeckenius,

Spoonhower, W., Proc.

(11)

Lewis,

(12)

Busch, G. E., Applebury, M., Lamola, A. A. and Rentzepis, Proc. Nat. Acad. Sci. USA 69, 2802 (1972).

A. and Spoonhower,

J., Bogomolni, R. A., Lozier, R. H. and Nat. Acaa. Sci. USA 2, 4462 (1974). J. Proc.

1115

Feb.

Exp.

Biol.,

in press. P. M.,