14N-ENDOR evidence for imidazole coordination in copper proteins

Vol. 108, No. 3, 1982 October 15, 1982

BIOCHEMICAL

14 N-ENDOR EVIDENCE

AND BIOPHYSICAL

FOR IMIDAZOLE

COORDINATION

Hiroshi Chemical

Research

Tohoku Received

IN COPPER PROTEINS

Yokoi

Institute

University,

RESEARCH COMMUNICATIONS Pages 1278-1284

of Non-aqueous

Katahira,

Sendai

Solutions, Japan

980,

30, 1982

August

SUMMARY: 14N-ENDOR studies of simple nitrogen-coordinated copper complexes in frozen aqueous solutions show that the nitrogen hyperfine constants, A,, and Al, of imidazole are'much more isotropic (R = A,,/A, = 1.05) than those of the other biologically-related ligand nitrogens. From this result, combined with 14N-ENDOR results of some copper proteins containing imidazoles as ligands, it is concluded that R < 1.10 for nitrogen hyperfine constants can be employed as an empirical criterion for demonstration of the existence of imidazole coordination in copper proteins. INTRODUCTION:

EPR and ENDOR techniques

al estimation ENDOR is ining

of

of a practical

ligand

formation

on the

on the that

copper

the

copper

By a careful literature, "*N-ENDOR

solution

stellacyanin, coordinates

with

ENDOR data

we have

recently

noticed

of copper

proteins

to examine

late

fundamental

this

fact

in

14N-ENDOR

copper(I1)

complexes

this

is

in

more data

frozen

to establish

with

nothing

except

two nitrogen proteins

we attempted

ligands. in the

isotropic.

an empirical

compar-

14N-ENDOR

are

aqueous

In-

can be obtained

fact

almost

exam-

accuracy.

a striking

on simple

1278

for

However,

on copper

detail,

0006-291X/82/191278-07$01.00/0 0 1982 by Academic Press, Inc. of reproduction in any form reserved.

high

observable

at least

Abbreviations: EPR, electron paramagnetic electron nuclear double resonance. Copyright All rights

(l-5).

tool

has revealed

of all

signals

are

a structur-

proteins

ligands

samples.

survey

der

paper

frozen

ion

with

which

for

effective

of nitrogen

spectra,

protein,

copper

interactions

coordination

for

in

as a most

(hf)

14N-ENDOR

ease even

sites

value

hyperfine

by analyzing ative

copper-binding

are useful

that

most In

or-

to accumu-

nitrogen-coordinated solutions. method

The purpose for

resonance;

estimation ENDOR,

of of

Vol. 108, No. 3, 1982

BIOCHEMICAL ISOELECTRIC

AND BIOPHYSICAL

FOCUSING

RESEARCH COMMUNICATIONS

+

‘a

Figure proteins minutes a)

2.

Induction of heat afte two-dimensional with [ 55 S]-methionine 23“C b) 37°C c)

The same heat

shock

a lower

M) ethanol

(.170

Effect various

resuspended

of ethanol ethanol

in fresh

proteins

shock

proteins. separation.

at 1.55

Methanol,

are

induced.

concentration pretreatment

concentrations

media

of [35S]-labeled pulse-labeled for

20

23OC

This (data

on heat for

lacking

Autoradiograms Cells were

ethanol 1343

20

not

induction

was not

observed

at

shown).

killing: minutes

and heat

Cells were

shocked

preincubated rapidly

for

at

filtered,

10 minutes

at

Vol. 108, No. 3, 1982

BIOCHEMICAL

10

Figure

tetraimidazole

(8)

have the

around

13 MHz are

following

8.0) hf

15 20 FREQLENCYVlHZ)

10

here

(Table

of imidazole than

the

constants

Copper

25

higher shape.

indicate

that

amides

All

BESOD-CNStellacyanin c oxidase

R

= 1.5

by preliminary

A,,

36

-c

36 46

38 55

1.06

35 45

36 47

1.03

17.1

17.6

1.03

H,,

results hf con-

at pH 6.3--

for

nitrogen

ENDOR experi-

Proteins

R = A,,/ALa

A,

1280

the

(.R = 1.05

obtained

a R 20.05 on the basis of A ?1 (MHz). b BESOD = bovine erythrocyte superoxide ' The 14N-ENDOR spectrum at the field too broad to be well-defined.

field,

the nitrogen

Nitrogen hf Constants (MHZ) of Copper as Determined by "N-ENDOR

b

ENDOR signals

extreme

in line

We have also

aqueous solutions

proton

are much more isotropic

Protein

Cytochrome

aqueous-glycerol

in frozen

on&y at the

2) clearly

of deprotonated

BESOD(native)

proteins

are broad

others'.

Table 1.

in a frozen

common features:

ohseryed

and I'N-.ENDOR spectra

stants

25

complex

and most copper

(2-5),

obtained

15 20 FREQUENCY(MHz)

RESEARCH COMMUNICATIONS

1. EPR and ENDORspectra of (A) [Cu(imidazole)~]2t and (B) [cu(NH~),,]~+ in frozen aqueous solutions: a, second derivative EPR spectra ((A), 9.334 GHz; (B), 9.330 GHz) with two magnetic field markers indicated by arrows ( (A), H, = 3305 G and H,, = 2675 G; (B), H, = 3312 G and H,, = 2693 G); b, ENDORspectra at the field setting of H,; c, ENDORspectra at the field setting of H,,.

copper(I1) solution

AND BIOPHYSICAL

ref.

5 5

1.20 2

1.04

dismutase. setting of H, was

3

BIOCHEMICAL

Vol. 108, No. 3, 1982 Table

2.

Nitrogen Copper(I1)

hf

Constants Complexes

Ligand Imidazole

AND BIOPHYSICAL

(MHz)~ of Nitrogen-coordinated as Determined by "N-ENDOR AL

A,,

6.3-8.0

40.3

42.3

1.05

39Ab

41.6b

1.05

Ammonia

10.0

31.7

39.1

1.23

Ethylenediamine

10.0

27.6

39.4

1.43

Glycine

10.0

29.3

35.8

1.22

34.8

41.3

1.19

7.0

a The data were determined mainly from b Data on a copper(I1) tetra-imidazole aqueous-glycerol solution (8).

ments.

Now, it

ligand

nitrogens

strains, 1.10);

is

this

also

nitrogen

way.

be some clue

to the

and imidazole

R

of imidazole

discrimination the

other

of Table

copper

fact

concerning

(20.2

without

smallest

in the

pyridine

because both

R

2.

the

in copper of view, structures

is

closest

This

fact

nitrogens

for

to may

of pyri-

the nitrogen

hf

criterion

imidazole

fOY

from those

of

By re-inspection

proteins.

we are led to the of

on it

configuration.

< 1.10

of

coordination

considerations

as shown in Table

of

(R <

of imidazole

nitrogen

in electronic

any

imidazole

can be used as an empirical

point

MHz).

R value

uniqueness

theoretical

reason,

similar

ligands

1 from this

copper-binding

following sites

in

proteins.

In native four

< 1.10

of a '"N-ENDOR signal

nitrogen

conclusions

R

value,

above are

copper

of the

although

The above experimental constants

to the

a proof for

positions

complex in a frozen

among biologically-related

Interestingly,

one in the

peak

may have the

provides

at present,

are now under

that,

coordinate

The reason

uncertain

imidazole

concluded

which

imidazole

as a ligand.

dine

R = *,,/A,

PH

Pyridine

is

RESEARCH COMMUNICATIONS

imidazoles

bovine

erythrocyte

belonging

superoxide

to histidines-44, 1281

dismutase 46,

61,

(BESOD), and 118

coordi-

Vol. 108, No. 3, 1982

nate

to the

results

in

copper

accompanied

this

around

is

strains

the

in

native

suggesting

CN- ion

Stellacyanin, "blue"

or "Type

a methionine

thioether,

though

stellacyanin

does not

also

shows that stellacyanin

are

This

contains

fact

with

and that

these

covalency

stellacyanin imidazole

nearly makes

it

the

above

the

coordination (12).

in

accordance

into

imidazole larger

is

contain

possible kinds

comparable

complex

in Table

coordinations

in

results. is

proteins

coordinated

by.a

there

cysteine Al-

is no doubt

Table

(11).

1 clearly

of ~35 and ~46 MHz for

to conclude

that

of imidazole that

it are these

in magnitude

1282

The

imidazoles.

and 1.04,

2.

classi-

(10).

(R = 1.03

bondings

cis

frequency,

and azurin,

constants

for

coordi-

R value

as ligands

the

the

methionine,

Interestingly,

are

with

hf constants

and two histidine

mention

same A,

seems reasonable

introduced

isotropic

two non-equivalent

agreement

the

hf

the

are much higher

two proteins

nitrogen

as also

in

has two imidazoles both

strains,

it

1" copper-containing

thiol,

it

latter

the

(5),

as plastocyanin

of the

imidaz-

nitrogen

forcibly

copper

CN- ion,

nitrogens

a somewhat

as well

Since

due to two imidazole

perturbs

way that

are

to CN- (5).

has almost

shows K = 1.06,

copper

BESOD-CN-

small

Therefore,

reaction

of the

for

the

with

The other

strongly

such a specific

in

nitrogen

CN- carbon

the

from

and

substitution

trans

its

are probably

that sphere

ly).

of s37 MHz

BESOD.

nitrogens

neighborhood

ion

criterion.

to the

the

copper

that

imidazole

a ligand

nitrogen

nitrogen

which

or adjacent

nation

fact

imidazole

BESOD-CN-,

that

in

remote

to the

empirical

is

RESEARCH COMMUNICATIONS

of CN- to BESOD, which

three

copper,

relatively

by the

as those

fied

of

due to one imidazole

supported

above

The addition

hf constants

may coordinate

that

(9).

the

by severe

imidazole

value

AND BIOPHYSICAL

arrangement

The nitrogen

probably

ole

ion

a planar

one CN- carbon

ion.

8lOCHEMlCAL

respective-

stellacyanin

coordination,

has two ligand as different nitrogen to those

in imidazoles,

as %30% in hf

constants

of a copper(I1)

of

Vol. 108, No. 3, 1982

Cytochrome

BIOCHEMICAL

c oxidase

of R = 1.03

signal dence

for

the

However,

iar

because

type,

than

half

are

(Table

those

the

density

other

azole

coordination

copper

proteins

However,

This

small

isotropic R

value

of imidazole

imidazole nitrogen

hf

of the

other

copper

for

constants

imidazole

the

of the

certain

eviin

oxidase

the

peculare

indicating

oxidase

nitrogen

is

is of a quite

proteins,

which

14N-ENDOR

coordination

coordination

the

on the

copper

The above

1).

reasons

RESEARCH COMMUNICATIONS

shows a nearly

of a kind

the

some specific

low spin

also

existence

oxidase.

AND BIOPHYSICAL

that

less there

has an extremely

atoms,

compared

with

proteins.

method

for

obtaining

by 14N-ENDOR

valuable is widely

information applicable

and to many copper-substituted

further

on imidto many other

metalloenzymes.

examination

or refinement

of this

method

will

ACKNOWLEDGEMENTS:

This

was supported

in part

by a Grant-in-

Aid

Research

be

needed.

for

tion,

Scientific Science,

M. Iwaizumi's

work

and Culture, group

for

No.354212 Japan.

many helpful

from

the

The author

Ministry is grateful

of Educato Prof.

suggestions.

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AND BiOPHYSICAL

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Van Camp, H. L., Sands, R. H., and Fee, J. A. (1981) J. Chem. Phys. 75, 2og8--2107. 9. Fee, J. A., and Gaber, B. P. (1972) J. Biol. Chem. 247, 60-65; Beem, K. M., Richardson, D. C., and Rajagopalan, K. V. (1977) Biochemistry 16, 1930-1936, and refs. therein. 10. Peisach, J., Levine, W. G., and Blumberg, W. E. (1967) J. Biol. Chem. 242, 2847-2858; Fee, J. A. (1975) Struct. Bonding (Berlin) 23, 1-60; Colman, P. M., Freeman, H. C., Guess, J. M., Murata, M., Norris, V. A., Ramshow, J. A. M., and Venkatappa, M. P. (1978) Nature 272, 319-324; Adman, E. T., Stenkamp, R. E Sieker, L. C., and Jensen, L. H. (1978) J. Mol. Biol. 123, 8.

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Hill, H. A. O., and Lee, W. K. (1979) J. Inorg. Biochem. 11, 101-113. McGarvey, B. M. (1967) In Transition Metal Chemistry: Electron Spin Resonance of Transition Metal Complexes, Carlin, R. L., Marcel Dekker, New York. ed., PP. 89-201,

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