Proton translocation in hydrogen bonds with large proton polarizability formed between a Schiff base and phenols

BIOCHEMICAL

Vol. 99, No. 3,198l April

AND

BIOPHYSICAL

RESEARCH

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15, 1981

Pages

804-812

PROTON TRANSLOCATION IN HYDROGENBONDS WITH LARGE PROTON POLARIZABILITY FORMED BETWJ3ENA SCHIFF BASE AND PHENOLS. + Pushti Prakash Rastogi and Georg Zundel Physikalisch-Chemisches Institut der Universitat M.iinchen; Theresienstr. 41, D-8000 Miinchen 2, West-Germany. Received

February

18,1981

SUMMARY: OH***N $ 0-m ..H+N hydrogen bonds formed between N-all-transretinylidene butylamine (Schiff base) and phenols (1:l) are studied by IR spectroscopy. It is shown that both proton limiting structures of these hydrogen bonds have the same weight with Schiff base minus pKa phenol) = 5.5. A pKa (50%) = (pK, protonated With the largely symmetrical systems, continua demonstrate that these hydrogen bonds show great proton polarizability. In the Schiff base + tyrosine system in a non-polar solvent the residence time of the proton at the tyrosine residue is much larger than that at the Schiff base. In CH CC1 these hydrogen bonds show, still proton polariza &1114 y, i,e., however, $he position of the proton transfer equilibrium OH-.-N + 0 *s-H N is shifted to and fro as function of the nature of the environment of this hydrogen bond. Consequences regarding bacteriorhodopsin are discussed.

INTRODUCTION N-all-trans-retinylidene studied

by Kristoforov,

visible

spectroscopy.

plexes hydrogen excess (I)

are formed,

Zvonkova They

of p-cresol,

OH---N

$0-s

they

phoric

group

at

that

the

s.H+N (II) (I)

rhodopsin

Schiff

are

with

two proton

Present address: Department Lucknow U.P., India.

formed

Copyright 0 1981 by Academic Press, Inc. AN rights of reproduction in any form reserved.

the

to

804

the proton

donor

were IR and

With

com-

in the

very

large

and the proton the base.

protonation

of Chemistry,

0006-291X/81/070804-09$01.00/0

(1) using

molecule.

bonds shifts that

complexes

base - p-cresol

1: 1 complexes

the p-cresol

1:2 complexes

suggested of

in

- p-cresol

and Evstigneeva

found

whereby

bond is present

result

+

butylamine

groups

in the

From this

of the

chromo-

must take

Lucknow University,

part.

Vol. 99, No. 3,198l

We have B,H-B2 ures

BIOCHEMICAL

shown (2)-(7)

and (II)

proton

side

Thus,

of

Recently

ated

by the

observed

and suggested

NH+.. .B-

with

chromophore

and Lemke

On the basis

of this

a study

result

Mohler

bonds the

retinyl

Schiff

group.

results

amino acid

observed

(8) pro(9),

base is modul-

Furthermore,

that

(11)

different

and Navangul

they

reconstitution the Schiff

residues,

be of particular

base

whereby

they

that

Fin-

an equilibrium

the pKa value

suggest,

an

significance.

depends on the nitration

that

of

of this

this

at the the

residue.

residue

parti-

system.

on the nature

a Schiff

proton

and Oesterhelt

these

on a change of

in the chromophoric

Thus, between

i.e.,

large

between

during

of bacteriorhodopsin

26 residue,

cipates

of

bond should

Oesterhelt

ally,

struct-

to one or to the

bacterioopsin

two protonable

hydrogen

(I)

limiting

such hydrogen

an anionic

with

on the basis

may interact

type

show very

Fischer

of the

with

pH-changes

(IO)

proton

equilibria

by Blatz,

spectrum

interaction

the

environments

the dark

postulated

the absorption

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bond.

of bacteriorhodopsin,

that

both

the case of local

RESEARCH

bonds of

weight,

by their

investigating

as earlier

posed,

in

the hydrogen

chromophores

hydrogen in which

(II),

may be shifted

other

BIOPHYSICAL

have noticeable

polarizabilities.

tyr

that

'T" B;...H+B2 (I)

AND

of OH***N $

base and phenols

O-**.H+N

bonds formed

seems of particular

interest.

RESULTS AND DISCUSSION N-all-trans-retinylidene phenols ccl*

(1:l)

solutions,

summarized

were studied using

in the

butylamine

(Schiff

as function

of the

IR spectroscopy.

table.

80.5

All

base)

with

pKa of the

systems

various phenol

and results

in are

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Vol. 99, No. 3,19Bl

AND

BIOPHYSICAL

RESEARCH

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Table Data Schiff

base

followins

with

of

the

Schiff

base

+ Phenol

A PK~~'

PKa

ohenols 1

protected

2

tyrosine

11.13

1:l

Complexes

R complex

% proton

formation

transfer

3

4

xx)

-4.14

95

-10~

KPT

KpT

5

6

7

9.2

0.10

+0.99

phenol

9.92

(14)

-3.93

96

2,4-dichlorophenol

7.30

(14)

-1.91

86

15.5

0.18

io.74

2,3,5-trichlorophenol

6.43

(14)

-0.44

96

18.0

0.22

+0.66

pentachlorophenol

4.74

(14)

+1.25

98

27.2

0.37

+0.43

2,4,6-trinitrophenol

0.81

(14)

+5.18

47.4

0.90

+0.045

pKa

x)

xx)

pKa

of

the

Schiff

value

of

tyrosine

The Schiff

taken

at

gen bonds in these

base observed complexes sorbance of the

the Schiff proton

in

~01.6.

complexes

complex

formation

of solutions

equilibria

this

in

band is

indicated

base -. HCl system,

(~01.4)

These values

oKa of the protonated of the Schiff base is

using

fig.lg,

in ~01.5

are plotted Schiff taken

shown for

the

Schiff

The integrated integrated

absorbance

proton

and the equilibrium of

theApK,,

to

of

transfer

constant

the phenols.

amounting

ab-

100% transfer

The percentage

as function

806

integrated

the various

in which

ref.(l2)

hydro-

of the protonated

base minus pKa of from

phenols.

O-.**H+N

from the

by an arrow.

base occurs. given

the pure

the OH.**N g

vibration

band was calibrated

base is

of

are determined

the C=N stretching -1 at 1655 cm . This

to the Schiff

to the Schiff

ref.(l2)

ref.(13)

equilibria

in fig-lb-lf, of

from

spectra

The proton-.transfer

of

from

the absorbance of the OH stretching vibration -1 (see fig.3). This band was 3600 or 3540 cm

from the

absorbance

taken

from

the phenols

calibrated

5.99

base - phenol

were determined of

base

>99

5.99,

i.e. The pKa and

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Vol. 99, No. 3,1981

.I ;r

AND

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RESEARCH

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I

\ ---T&F

Fig.

YC=N band of N-all a proton is present an arrow).

1

trans retinalidine at the N atom

butylamine indicated

(band

when with

a) Schiff base + protected tyrosine (1:l) ( V C=N band masked by the intense band of the protecting groups, b) Schiff base + phenol (l:l), c) Schiff base + 2,4d) Schiff base + 2,3,5-tricholodichlorophenol (1:1), phenol (i:ll, e) Schiff base + pentachlorophenol (l:l), f) Schiff base + 2,4,6-trini'rophenol (l:J), q) Schiff base + HCl (1:l).

the pKa values In fig.2a of

the

the

+ phenol

of

base occurs

of

atApKa

the Schiff

phenols

2500 cm

bonds o-...

of the

begin -1

according

with

and extend

are taken

(PT)

is

to Huyskens show that

50% transfer

plotted

of

with

the proton

from ref.(ll). as

function

(151,

-log

KpT

the

Schiff

base

to the Schiff

= 5.5.

of

the Schiff

spectra

of

Schiff

base + 2,3,5

towards

These continua

H+N bonds in these

the complexes

structure smaller

characteristic demonstrate systems

with

in fig.3b those

IR continua in the region

wave numbers. for (2)-(6)

show large

807

trichlorophenol

system

base shows that

a band-like

is particularly (6).

transfer

base + pentachlorophenol

or the pure

continua

in ~01.2

Both plots

Ccl4

50%

IR spectra

a comparison

feature

therhpK,. in

given

proton

and in fig.2b,

systems

In the

phenols

percentage

ApK,,

as function

and

of the

phenol that proton

This

of

and

3c

the pure

occur.

These

3000 spectral

- amine hydrogen the OH***N Z polarizabilities.

Vol. 99, No. 3,198l

BIOCHEMICAL

2

numbers of

(c)-(f)

b,

~~~c&-I

is

the protons

of

of

fluctuate

shows the

at

in

demonstrates

izability This

tentials ing.

The position

be determined band of

in

of this

the protecting

it

as

groups

base + protected having

groupings.

the

transfer

808

large

and

bonds.

systems

the

tyrosine

protecting

In this

spectrum

intense.

but

This

polar-

above.

the proton

discussed

equilibrium

in

re-

tyrosine

this

discussed

shape of

base systems

(fig.la).

weight,

not very

polarizability

with

since

proton

0-q **H+N bonds between

than with

system

both

is

tyrosine

proton

the proton

that

is much less

the OH.+.N g

- Schiff

fact

the Schiff

but

in good agreement

in tyrosine

constant)

of asymmetry

i.e.,

base show still

is

theApKa

bonds have noticeable

hydrogen

of

observed that

the

andOt'-amino

is much smaller

result

degree

spectrum

still

and the Schiff

with

ethylester),

is

of

equilibrium

hydrogen

these

itsa-carboxylate

a continuum sult

the

1 .

AUK a

these

systems

(N-acetyl-L-tyrosine groups

transfer

the

in good agreement

these

Fig.3a

in fig.

systems

Percentage proton transfer as function (UK, protonated base - pKa phenol)

structures

i.e .,with

the

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AMa

a)

result

limiting

RESEARCH

b

(proton

This

BIOPHYSICAL

A*a

a

Fig.

AND

po-

the follow-

cannot

directly

?/C=N is masked by an intense TheApK,

value

in this

sys-

BIOCHEMICAL

Vol. 99, No. 3.1981

AND

BIOPHYSICAL

Wave number Fig.

3

IR spectra

of

solutions

(layer

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cm-’

thickness

50 )Im)

a) Solutions in CH2C12; *** pure Schiff base (0.5 --pure protected tyrosine (0.5 M), Schiff (0.5 M) + protected tyrosine (0.5 M)

M), base

b)

Solutions in CC14; ... pure Schiff base (0.5 M), --pure 2,3,5 - trichlorophenol (0.5 M), Schiff base (0.5 M) + 2,3,5 trichlorophenol (0.5 M)

c)

Solutions in CC1 . m.. pure Schiff --- pentachloropffenol (0.2 Ml; -+ pentachlorouhenol (0.5 ".)

base (0.5 M), Schiff base (0.5

M)

Vol. 99, No. 3,198l

tern,

BIOCHEMICAL

however,

amounts

the non-polar Thus,

proton

in the

the proton Schiff

I:1

BIOPHYSICAL

to -4.34.Usinq limiting

mixture

this

structure

RESEARCH

value,

is much larger

at the

fiq.2a

OH. ..N

in CH2C12 solution

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shows that

has much larger

the residence

tyrosine

than

at

weight.

time

the N-atom

of

of the

base.

III.

CONCLUSIONS With

retinylidene

50% transfer

of

Schiff

polarizability With tem is

when these

regard

much larger

cal

of

Schiff This Schiff

are

at the

still

If

between

Schiff

of this

largely

This

polarizability.

Thus,

is

due to the

bond can be shifted

to

base + tyrosine

time

local

is

of the

hydrogen

by the

present proton

is

bond shows, property,

polarity

fields,

the tyrosine

sys-

the OH.**N e

Due to this

influenced

proton

symmetrical.

system

residence

residues.

equilibrium

show large

Schiff

this

The OH-.*N.$

base and tyrosine

the

tyrosine

proton

in

1:1 in CC14,

= 5.5.

systems

the

interest.

the poof the

the proton

residue

as to

lo-

in this the

base. explains

the result

base + p-cresol o-. ..H+N

structure

O-..

(16)-(19)

that

of p-cresol)

in

.H+N, since in hydrogen environments

the

of Kristoforov

system

equilibrium

caused by polar

structure

systems

systems,

a,~pK~"%

in these

low polarity,

environments.

hydrogen

at

to bacteriorhodopsin

of particular

in a solvent

sition

occurs

bonds found

0-a **H+N bond formed

however,

base + phenol

the proton

o-. . .H+N hydrogen

F

AND

equilibria

it

additional

favor

of

et

al.(l)

p-cresol

the polar

that shifts

proton

in the the OH-.-N

limiting

is known from many publications bonds with (in

great

proton

ref.(l)

addition

are shifted

in favor

B;*"H+B2.

810

(5) (6)

polarizability of large of

amounts

the polar

BIOCHEMICAL

Vol. 99, No. 3,1981

Oesterhelt the residue

and Lemke (11) tyr

Our molecular rhodopsin thesis of

step

could

model built

this

the

is

residue

than

since, the

hydrogen

bacteriorhodopsin structure. of bacterio-

may suggest

becomes deprotonated show that

this

Schiff

time

in the

tyrosine

when the environment

residence

the

of this

of the proton at

the

hypofirst

residue

base when the

bonds is much greater

at the Schiff

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the structure

by the protonated

too polar,

base - tyrosine

to

Our results

formed,

with

et a1.(20)

residue

photocycle.

M412

bond is not

according

tyrosine

RESEARCH

in the chromophoric

by Ovchinnikov

be reprotonated

mediate

have shown that

26 may be involved

evaluated

that

AND BIOPHYSICAL

interhydrogen

in Schiff tyrosine

base.

EXPERIMENTAL PART N-all-trans-retinylidene the procedure tyrosine, Serva,

given

i.e.,

IR-measurements:

solvents

For all

Retinal,

as well

ethylester

measurements

thickness

were compensated

according

to

as the protected were purchased

a cell

was used.

by a cell

with

beam. The measurements

spectrophotometer,

Uberlingen,

was prepared

from

West Germany.

50 um layer

in the reference Elmer

in ref.(l).

N-acetyl-L-tyrosine

Heidelberg,

windows with

butylamine

model 325,

with

sodium chloride

The IR bands of

adjustable

layer

were performed

Bodenseewerk

the

thickness

with

a Perkin

Perkin-Elmer,

West Germany.

ACKNOWLEDGMENTS Our thanks are due to the Humboldt Foundation for a grant conferred on Dr. P. P. Rastogi. Furthermore our thanks are due to the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie for providing the facilities for this work.

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