Easily polarizable N+H…N hydrogen bonds between histidine side chains and proton translocation in proteins

Vol.

95, No.

2, 1980

July

31, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 902-908

EASILY POLARIZABLE N+H- "N HYDROGENBONDS BETWEEN HISTIDINE SIDE CHAINS AND PROTON TRANSLOCATION IN PROTEINS. Pushti

Prakash

Rastogi,

Wolfgang

Kristof

and Georg Zundel

Physikalisch-Chemisches Institut der Universitat 41, D-8000 Miinchen 2, Miinchen, Theresienstr. West-Germany. Received

June 18,198O

SUMMARY: Histidinium perchlorate having protecting groups at theU-amino anda-carboxylate group is studied by IR spectroscopy as function of the addition of protected histidine molecules. An intense coptinuous absorption arises, indicating that the N+H*-•N e N.--H N formed are easily polarizable hydrogen bonds. From the integral absorbance of a band the concentration of the histidine-histidinium the concentration of the easily polarizable hydrogen complex, i.e. It is shown that the absorbance of the conbonds is determined. tinuum increases in proportion to the concentration of the easily polarizable N+H-**N $ N-**H+N bonds. Finally, it is discussed that via such an easily polarizable histidine-histidinium hydrogen bond a proton translocation in the active center of ribonuclease A may occur.

INTRODUCTION The presence larizabilities within

is

various

in proteins

polarizabilities hydrogen histidine

are easily

B+H*.*B

AH-.-B

SH.s-S--G

with

polarizable

(L-glu)n,

The large

0006-291X/80/140902-07$01.00/0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

(2).

$

N+H...N$

$

902

po-

of protons

It

was already or A ** *HA+

bonds between

with

Large

side

proton

the homoconjugated

N*..H+N

bonds between case of OH**.O-F

N***H+N bonds with

(L-cys),

proton

proton

B-**H+B

(3) and in the

-S.* *HS bonds with

(L-tyr)n.

(1)

(3)-(g).

by IR studies

(L-his)n

large

g A-***H+B

bonds in the case of N+H...N residues

very

translocation

mechanism

homoconjugated

were proved

-0 . ..HO bonds with

bonds with

to the

a Grotthus

-A and heteroconjugated

chains

(9),

bonds showing

a prerequisite

a system via

shown that AH...

of hydrogen

and OH*--0-F

polarizabilities

(L-lys), -O***HO

are indi-

Vol.

95, No.

cated the

in

the

IR spectra

polymer

at

the

in

this

dine

BIOCHEMICAL

2, 1980

systems

films

side

protecting

groups

N* .*H+N

studied

at

in

light

With scattering

evaluation.

bonds

formed

solutions,

the 'X-amino

COMMUNICATIONS

(1).

sometimes

quantitative

r"

are

RESEARCH

absorptions

above,

hindering

N+H-.*N

chains

BIOPHYSICAL

by continuous

discussed

occurs,

paper

AND

Therefore,

between

using

histi-

histidine

and&-carboxylate

with

groups.

RESULTS AND DISCUSSION Fig.

1

shows

IR

protected

histidine

histidine.

This

to

the

spectra as

from

about

the

wave

number

indicate

that

polarizable

the

structure

characteristic

of

by FERMI

resonance

hydrogen

bonds

Fig. to

the

Fig.

shows

3000

N+?-l**.N$ bonds

with

1

show

0:1-o; 1.5:1

Of

the

of

that the

t-BOC-his-O:
dm-3.

of protected

arises

in

smaller

wave

Continua

of

formed

length

region

(IO).

of

histidine

the

spectra,

numbers this are

type

vibrations the

absorbance

easily

and is in

is

caused these

(II)-(14).

addition of

the

of

0.5O:l.O;

histidine

continuum

+ the corresponding solutions: ----

type

cm-'

transitions

on

The band-like

2700-1900

this

with

protonated

addition

bonds

fundamentel

in acetonitrile *-*. mole

the

of medium

combinational

solution

IR spectra perchlorate .-

N. **H+N

bonds

the

with

studied.

the

addition

toward

hydrogen

histidinium

-.

cm

in

of

the

of

a continuum -1

observed

2a and table

1

that

region

hydrogen

doublet

of

solutions

extending whole

IM solutions

function

figure

histidinium

of

-e-e-

hydrc1.O:l.O;

below

Vol.

95, No.

2, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

..P t -

Fs -

008

; 006

F :

006

‘5 a04

E 004 2 2 002 a 0-:I 00

2 008 5

h

a Q a02 0.0 G:: & or0 0

a5

FO

m oh

Fig.

1-5

rdm

20

In~o~e/“sol,

0

0

I

01

cone

02

03

04

of NH+ ..N

0.5

cmplex

0 IM/drn3J

-2a) Absorbance of the continuum plotted against the various molar ratios of histidine to histidinium perchlorate. b) Absorbance of the continuum plotted as function of addition of (NH- . *N) + complex formed with increasing histidine to a 1-O EI solution of histidinium perchlorate.

a mole

ratio

of

concentration,

whereas

base

the

This

demonstrates

intensity

histidinium

the

less

with

of

the

that

of

continuous

i.e.

can be estimated

proportional

addition

the

to

an excess

concentration

by evaluating

of

the

histidine of

further

formation formation

the

amount

absorbance

The

occurs.

complexes,

than

no complete

complexes

tidinium bonds

increases

1

free

increases.

histidine-

of

the

histidine-his-

of

N+H...N$

N...H+N

characteristic

bands

NH stretching

vibrations

in

spectra.

In are

the

region

observed.

At

protonated mation, the

3160

histidine whereas

cm -1

3360

NH groups

which

are

arises.

The of

vibration

of histidine occurs

cannot

be used

in to

not

amide

the

the

NH stretching decreases

cm -1

latter

vibration

mation

the

the

residues

at

bonds

cm -1

3500-3100

the

is

groups

the

in

the

superimposed

same position. the

not

involved

for-

with

formation.

the

of N- ..H+N

on the the

the

complex

NiHS..NF

Therefore, complex

of

vibration

and furthermore,

molecules

determine

with

NH stretching

involved

band

vibration

stretching

stretching complex

for-

band

3360

at

cm

-1

vol.

95. No.

2, 1980

EIOCHEMICAL

AND

BIOPHYSICAL

0 N

P .

c

d

RESEARCH

co N .

c

d

P .

.

7

7

d

d

COMMUNICATIONS

Vol.

95. No.

2, 1980

BIOCHEMICAL

The decrease

of

formation.

This

Therefore,

a ring

which

is

dinium

band,

caused

determined.

equilibrium

For

a mean value tinuum

as function

This

figure

in

shows

proportion

to

N.. . H+N hydrogen

at

complex the

of

listed

(see

can be given.

of

this

that

the

the bonds

the

of

of

the

the

complex

col.),

and as

of

the

in

2b.

increases

polarizable

histidinium

con-

fig.

continuum

easily

inte-

calculated

plotted

the

histithe

the

last

is

evaluated

From of

well.

of

The absorbance

absorbance

between

is

residues

tab.

complex

very

complex

concentration

formation

the

formation.

the

COMMUNICATIONS

cm -1

1625

concentration

formation

K are

K = 1.20

evaluated

be

imidazole

with band

the

constants

cannot

protonated

this

RESEARCH

indicates

vibration

vanishes of

8lOPHYSlCAL

3160 cm -1

at

however,

by the

absorbance

was

band

stretching

and which

gral

the

AND

N+H**-N

and histidine

resi-

dues. CONCLUSIONS The IR results between easily

show

histidinium

via

polarizable

hydrogen via

may be part a Grotthus

Riiterjans

the

of

these

the

active

that

a proton

bond

occurs

of

types

hydrogen

N- *.H+N

residues

bonds

in

medium of

bonded

bonds

proteins

are

length.

hydrogen

systems

formed

Thus,

bonds

protons

and these

conducting

protons

mechanism. and Witzel

(15)

have

histidinium-histidine

such

N+H..*N;=2

and histidine

can be translocated bonds

that

center

of

hydrogen ribonuclease

translocation with

EXPERIMENTAL

the

shown

bonds

this

it

easily

mechanism

that

may be present

A. Thus,

via

catalytic

by NMR measurements

seems

possible

polarizable in

this

in

hydrogen

enzyme.

PART

Substances: The protected methyl-ester

amino (t-Boc-his-OMe)

acid

N-tert was

butoxycarbonyl-l-histidine-oobtained

906

from

the

Max-Planck-

F

Vol.

95, No.

Institut

fiir

salt

of

Biochemie,

the

methanol, HC104

BIOCHEMICAL

2, 1980

was

the

RESEARCH

W.Germany.

obtained

an equimolar

and removing

BIOPHYSICAL

Martinsried,

compound adding

AND

The perchlorate-

by dissolving

amount

solvent

at

of

t-Boc-his-OMe

an aqueous

reduced

COMMUNICATIONS

in

solution

of

pressure.

IR-measurements: For

all

thickness

measurements of

The specific a cell

with

15 pm.

The windows

IR bands adjustable

measurementswere meter,

acetonitrile

of

the

layer

performed

Bodenseewerke

was of

the

solvent

used

IR

cell

could

thickness

as solvent were

at

made of

be compensated

in

the

with

a Perkin-Elmer

Perkin-Elmer,

tiberlingen,

a layer NaCl.

by using

reference

beam.

The

325 spectrophotoW. Germany.

ACKNOWLEDGMENTS Our thanks are due to the Humboldt Foundation for a grant conferred on Dr. P.P. Rastogi. The protected amino acids were made available to us by Prof. Wiinsch and Prof. Moroder, MaxPlanck-Institutefor Biochemistry, Martinsried, West Germany. Furthermore our thanks are due to the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie for providing the facilities for this work.

REFERENCES 1.

G. Zundel, in: The Hydrogen Bond - Recent Developments Theory and Experiments, P. Schuster, G. Zundel and C. Sandorfy eds., Vol. II, North Holland Publ. Co., Amsterdam 1976, pp. 683-766.

2.

G. Zundel and E.G. Weidemann, First European Biophysics Congress, Vol. IV, E. Broda, A. Locker and H. SpringerLederer eds., Wiener Med. Acad. 43-47.

3.

G. Zundel

and J.

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R. Lindemann

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