BIOCHEMICAL

Vol. 91, No. 2, 1979 November

Pages 575482

2B,l979

ACTIVE

SITE

STRUCTURE OF BACTERIORHODOPSIN OF ACTION Henry

Chemistry

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

October

L. Crespi Division, Argonne,

and John Argonne Illinois

AND MECHANISM

R. Ferraro

National 60439

Laboratory

13, 1979

SUMMARY. Pressure experiments with freeze-dried bacteriorhodopsin indicate that water is an essential part of the chromophore. This observation is combined with already known information on (a) the pH dependence of proton pumping, (b) the secondary protein-chromophore interaction with lysine-40, and (c) the proton transfer in the initial photochemical step to give a detailed structure of the active site and a mechanism for proton pumping which is consistent with the bacteriorhodopsin polypeptide sequence. The bacteriorhodopsin driven it

proton

pump

is photochemically

mediate

observed

(2,3).

Subsequent

species

absorbing

in

(1).

40 usec.

of HaZobacterium

halobium

When bacteriorhodopsin

(bR)

transformed at

610 nm (K 610) reactions

at

Recently

Lewis

L and M, here

complex

is then

slow,

as

about

10 msec.

30-50

usec.

medium

in about We have

in the of

a diamond

subjected anvil

application 0 to

of pressure.

20 kbar,

(freeze-dried

the

(5).

the

bR under

absorption

thermally in

complete

will

time

observed

maximum

no pressure)

to

is

in the

to high response

increased

from

0006-291

external

pressure

the

range

540 nm

An absorption

The U.S. Government's right to retain a nonexclusive royalty-free to the copyright covering this paper, for governmental purposes,

57s

original

of bR to

in

500 run.

Copyright All rights

the

at physiological

blue-shifts about

an inter-

of

appear

1 shows the

As pressure

of

and at 412 nm (M412)

bacteriorhodopsin

Figure

transfer

formation

Reformation

cycling

A proton

proton

(4) have

"Xl'.

light,

to an inter-

to the

2 psec,

called

freeze-dried

cell

concomitant

and coworkers

between

is

with

550 nm (Lsso)

absorbs

a few picoseconds

proceed

mediate

temperatures

in

is a liqht-

license in and is acknowledged.

x/79/220575-08$0i.00/0

@ 1979 by Academic Press. Inc. of reproduction in any form reserved.

BIOCHEMICAL

Vol. 91, No. 2, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A. NO PRESSURE

D.PRESSURE RELEASED

4&I

4;O

560

5!b

6&I

650

nm

Optical spectra of freeze-dried natural F* ( H) bacteriorhodopsin (bR) showing the sequential involved in going from the starting material no

hydrogen abundance color changes at pressure, blueshifted bR at pressure, and bleached bR upon release of pressure. After standing three hours under ambient conditions, the bleached bR showed a slight recovery of absorption at 560 nm. Bleached bR is also observed when the entire sequence of pressure application and release is conducted in the dark. grows

maximum

in

(from

a minimum

often

total

at the

expense

end of

of about

its

However,

50°.

at ambient

release

instant,

of pressure

extensive,

Absorption

sample.

and

appears

at

370 nm

at 500 run. obtained

in a parallel with

D20 for deuterated

eye and also

We observed to stand

(in

situ

2, if

at room

after

576

hours

material

pressure

low relative

bleached

temperature,

(6).

eight

done at an consistently

at the

or no reversibility

(very the

experiment

absorption

more

little

conditions

as shown in Fig. humidity

is

The partially

to the

allowed

relative

complete

band were

spectrum.

samples

there

exchanged

color

to 24 hours

high

the

results

showed more

all

of

Upon total

nm.

of the

bacteriorhodopsin

average

390-400

of '~3 kbar),

bleaching

Similar with

at

sample recovery

red with

release)

up

humidity). is kept of color

at is

BIOCHEMICAL

Vol. 91, No. 2, 1979

1.0.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

1 ‘Ii, bR PRESSURE

0.0

RELEASED

I 500

I 400

300

600

nm Bacteriorhodopsin recovers its natural 560 nm absorption pressure-bleached material is exposed to 100 percent relative This experiment was done with fully humidity at room temperature. deuterated t21i) bR. The same result obtains with 1~ material. Studies tion

are

cause

of the

consistent

high

pressure

with

this

Suzuki

denaturation.

pressure

favors

and the

formation

bR appears release

disruption

about

of pressure,

restore

region

this

relaxation

cussed kinetics rhodopsin.

in

effects

this

form

the

takes

of the

Md12 intermediate that

and Hess using the

that

high

bonding

release

native

pressure-

to overall

pressure

its

3 kbar

protein

distorts

the

of water

(8).

available

to

conformation,

so

place.

of dehydration

by Korenstein

observed

is no longer to

solu-

pressure-denatured

related

high

water

molecule

detail

The concluded

that causing

of the

to a bleached

The possible

indicate

the not

one

out

and ionic

Although

in

above

(7) point

by hydration,

chromophore,

Upon release

Pressures

(7).

bonds.

may be a specific

the

of protein

of hydrophobic

of hydrogen

Our results

hydration.

result

and Taniguchi

to be renatured

effect

region

the

denaturation

on bR films (9),

577

who measured

air-dried

photochemical

has been dis-

films reaction

the

decay

of bacterio"is

only

Vol. 91, No. 2, 1979

influenced

BIOCHEMICAL

by the

microenvironment

suggest

that

the

state

event.

When bacteriorhodopsin

shift

in

color

there

is

a further

general ment

bleaching is

involved

dried

progressive of water

release

in

the

reaction

there

under

subjected

due,

we suggest

structure

of the

to

a result

and Hess. that

active

blue

to pressure

presumably,

pressure,

of Korenstein

native

and

is a progressive

is

shift

of pressure,

system"

a conformation-controlling

material

blue

observations

upon

is

is dried

and when the

the

of the

of hydration

redistribution with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in

a

agree-

Because

of the

a water

molecule

center.

DISCUSSION The molecular of

mechanism

bacteriorhodopsin

is

underlying

of

fundamental

a recent

publication,

bioenergetics.

In

data

suggesting

such a molecular

light

causes

the

the

Schiff

appears

complex.

consistent

with

Figures the

between groups

the

s-amino

group

transfer

in

quent

and 12.51

(b) with

interaction

the

kinetics

of proton

evidence

lysine-401

and

(e) with

the

part

of the

active

present center.

side

is then

our

presented

protein-chromo-

Schiff

(c)

with

the

observation

that

A space-filling

578

water

place

and guanidino

the

observation

here.

takes

from

the

which

concerning

bond

(3),

by

is consistent

a secondary

in

of high

mechanism

carboxyl

of a proton

that

chain

[pumping

step

of

reprotonated

scheme

pumping

photochemical

area

(2) presented

suggestion

This

for

(2),

and appearance

acid

a detailed

implicates

hydrogen

initial

detail

the

et al.

and results

pumping.

which

the

[the of

in

proton

pH dependence

pH 2.5 (21,

phore

of this

here

function

They proposed

acid

data

3 and 4 describe

mechanism

(a) with

existing

in

Lewis

amino

We suggest

pumping

importance

of an amino

deprotonated

base

proton

mechanism.

deprotonation

the

pK (arginine);

the

base

(d) with external is

to the of proton the

medium,

an essential

(CPK) model

subse-

is not

Vol. 91, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

C”3 L

R@H-

CH-CH-i--R,

I

ll CH;

PyCH-C-CH-CH-N-R, +H,N-R,

N"2

C-NH2 / /N R3

f$-CH-C-CH-CH-N-R,

I R4

XqeO

R,,--COOH

+

"20

$

C"3

I

NH2

I

H--$--R2 C"3

C-NH2

I R~CH-C-CH-CH-N-R,

I

/

/N R3

KSlO

H2N--A2

NH2

7

,iSNH

,p; I

2

t .F

\

IIf

kyl

c55o

R//l 3

0

0

I R4

3

I

F$-COO-tH+

hi,,, -

A proposed mechanism for proton pumping by bacterioStructure bKS70 represents the resting state. Rlr Rzr R3 and R4 indicate surrounding protein, and R5 is the remainder of the retinylidene group. A secondary interaction (hydrogen bond) between the Schiff base and a lysine e-amino group is shown, the guanidinylium group is in a planar configuration, and there is a salt bridge from a carboxyl group to the guanidinylium group. It has been shown (10) that the favored structure of the guanidinylium group has the positive charge at the NH position. In KSlO, the methyl group at position 13 of the retinylidene is given a "cisoid" conformation, which severely crowds the guanidinylium group, destroying its planarity. The reduced basicit of the non-planar guanidinyl group leads to the formation of H30x bound to the carboxyl group, while the guanidino group goes to an unprotonated state. Form L is not shown in detail, as the change from K would involve only a relaxed retinylidene and perhaps slightly changed protein positions. Fi%$A.

Intermediate X is formed when the Schiff base proton is trans erred to the a-amino group of lysine-40. -9. The guanidino group is unprotonated, and the carboxyl group may ionize or give up water and then ionize. Intermediate M forms when the guanidino group takes the proton from the lysine-40. Ionization of the carboxyl group is complete; the system is ready to accept a proton from the cytoplasm and reform bR3T0. inconsistent

with

in the proton involved

this

scheme.

pumping action

in reprotonation

Data implicating

(11)

of the

could Schiff

579

mean that base.

a tyrosine this

group

residue is

Vol. 91, No. 2, 1979

In the

Fig.

BtOCHEMICAL

3 we show a possible

photochemical

group

that

step.

drives

the

We have

shown in Fig.

13,

the

but

formation

thrust

and decreases any positive

charge

transfer

intact

secondary

mediate

L is

similar

retinylidene,

carboxyl

group

interaction

X is

lysine-40 group

the

external

medium.

Once the is Schiff

40 to the transfer ionization

the

guanidinylium

group

change

a 90'

twisted

ion

state),

a still-

c-amino

retinylidene

will

K is

"salt", the

in

also

the

group

of

group.

be relaxation

be some movement

(for

excited

Intermediate

with

could

localizes

guanidinylium

a carboxyl

there

shown in

removed,

in

proton

Inter-

of the

protein

due to

be some ionization

of the

carboxyl bRSTO.

described

4.

of

The retinylidene

intermediate from

proton

is

(13).

group This

base,

is complete,

proton

to

arginine

group

side

of

M is

guanidino and the

(14),

from

lysine-

formed

group,

the

system

may be an example

and Williams

and

to cascade

Intermediate

mechanism

by Vallee

Schiff

the

c-amino

to the

in

s-amino

up its

with

the

is

L, the

in a position

through

lysine-40

the

of giving

associated

basic) of

in

process

basic)

(most

Fig.

a proton

originally

(least

arginine

to reform

the

a second

base

state

is

proton

of the

assumed

has accepted

carboxyl

chain

in

changed

and there

the

entatic

base

conformation

of

con-

of the

at position

"salt."

relaxed

poised

group,

redistribution,

Intermediate

the

guanidino

could

light-induced

facilitated.

to K, but

there

the

conformation

be further

Schiff

group

this

(12)

retinylidene

methyl

that

NH group

in

an open question.

of

and a conformationally

lysine-40,

the

the

transfer

in the

still

planarity If

proton

change is

is

RESEARCH COMMUNICATIONS

for

of the

argument the

near

could

an unprotonated

the

3 a rotation

polarization

then

charge

transfer

basicity.

a sudden

proton

proton

distorts

its

mechanism

The conformation

of our

change

example,

AND BIOPHYSICAL

is

of the

whereby

the

on

BIOCHEMICAL

Vol. 91, No. 2, 1979

destablized actions

state elsewhere

The recently polypeptide peptide mined

favored

in the

protein.

published

chain

retinal

proton

donor

with

structure

bridge

the

(15) model

can be fitted

and Unwin

and the

(l),

Arg-224 In

moiety. to the

by a number

sequence

is consistent

by Henderson

a salt the

is

(K)

AND BIOPHYSICAL

addition,

Schiff

base,

The poly-

here.

helical

array

deter-

and Arg-224

to interact

is positioned

as proposed

inter-

bacteriorhodopsin

Asp-96

a position

Tyr-26

favorable

presented

to the

in

of

of the

such that

is

RESEARCH COMMUNICATIONS

form

with to act

by Konishi

as a

and Packer

(11).

ACKNOWLEDGEMENTS We thank

Ms. Ursula

Halobacterium.

This

Office

Energy

of Basic

U. S. Department

Smith

for

assistance

work was performed Sciences,

Division

under

with the

the

culture

auspcies

of Chemical

of

of the

Sciences,

of Energy.

REFERENCES 1.

2. 3. 4. 5. 6. 7.

a.

Osterhelt, D., and Stoeckenius, W. (1978) Proc. Natl. Acad. Sci. USA 70, 2853-2857; Henderson, R., and Unwin, P. N. T. (1975) Nature 257, 28-32; Stoeckenius, W., Lozier, R. H., and Bogomolni, R. R. (1978) Biochim. Biophys. Acta 505, 215-278. Lewis, A., Marcus, M. A., Ehrenberg, E., and Crespi, H. L. (1978) Proc. Natl. Acad. Sci. USA 75, 4642-4646. Peters, K., Appleburg, M. L., and Rentzepis, P. M. (1977) Proc. Natl. Acad. Sci. USA 74, 3119-3123. Lewis, A., personal communication. Ferraro, J. R., and Basile, L. J. (1974) Appl. Spectr. 28, 505516. We have attempted to denature bR by warming the freeze-dried material in vacuum (~10-4 Torr) for up to one hour at 80-85", but observed no marked color change. Suzuki, K., and Taniguchi, Y. (1972) in The Effect of Pressure on Organisms, Symposia of the Sot. for Exptl. Biol. (Academic Press, New York), Vol. 26, pp. 103-124; Visser, A. J. W. G., Li, T. M., Drickamer, H. G., and Weber, G. (1977) Biochemistry 16, 4879-4882; Murphy, R. B. (1978) Experientia 34, 188-189. The slight deuterium isotope effect involving exchangeable hydrogen probably indicates that the compression of the hydrophobic region about the chromphore is resisted by the hydrogen bonding in the protein.

581

Vol. 91, No. 2, 1979

9. 10. 11. 12. 13. 14.

15.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Korenstein, R., and Hess, B. (1977) Nature 270, 184-186. Yavari, I., and Roberts, J. D. (1978) Biochem. Biophys. Res. comm. 83, 635-640. Konishi, T., and Packer, L. (1978) FEBS Lett. 92, l-4. Salem, L. (1979) Accts. of Chem. Res. 12, 87-91. Favrot, J., Sandorfy, C., and Vocelle, D. (1978) Photochem. Photobiol. 28, 271-272. Campbell, I. D., Dobson, C. M., and Williams, R. J. P. (1978) Molecular Movements and Chemical Reactivity, in Adv. Chem. Phys., R. Lefever and A. Goldbeter, Eds. (Interscience), pp. 55-95. Orchinnikov, Yu. A., Abdulaev, N. G., Feigina, M. Yu., Kiselev, A. V., and Lobanov, N. A. (1979) FEBS Lett. 100, 219-224.

582