Reactions and interconversion of met and dimer hemocyanin

Vol. 86, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages

14, 1979

February

628-634

REACTIONS AND INTERCONVERSION OF MET

ANDDIMERHEMOCYANIN Richard

S. Himmelwright,

Nancy C. Eickman

Department Massachusetts

Received

December

21,

I. Solomon*

of Chemistry Institute

Cambridge,

and Edward

of Technology

Massachusetts

02139

1978

methemocyanin of Busycon canaliculatum is shown to SUMMARY: The regenerable be EPR-nondetectable. The small EPR signal present in met preparations is a nonregenerable binuclear cupric unit accounting for Q, 6% of the active sites. A variety of anions are found to bind to met with the reactions being complicated by both reduction and competitive binding of buffer. The metastable dimer hemocyanin is shown to rearrange either directly to EPR-nondectable met or through an EPR-detectable regenerable binuclear cupric form obtained on reaction of dimer hemocyanin with azide. These results combined with previous results on half met derivatives are used to support the presence of an endogenous protein bridge between the two coppers in hemocyanin. INTRODUCTION: unrelated

chemical

with

shows a broad ions

(5).

This

with

the small

sites

obtained

along

exhibited

and exhibit general

the mollusc

(3,4).

with

a rationale

by these

for

binuclear

of a protein

ligand bridge * To whom all correspondence

interconversion the different

in the dimer should

Copyright All rights

@ !979 by Academic Press, Inc. of reproduction in any form reserved.

derivatives forms.

be sent.

0006-291X/79/Q30628-07$01.00/O 628

coupling showing

between that

two cup-

the regener-

is EPR-nondetectable,

due to only

In addition,

cupric

EPR-nondetectable

however,

canaliculatum

to the

magnetic

Dimer hemocyanin,

Busycon

in the met preparation.

state

by

associating

evidence

being

the

prepared

have been reports

presents

observed

leading

there

are

ground

concerning

due to weak dipolar

communication of

very different

(1,2),

EPR signal

of oxyhemocyanin

agreement

methemocyanin

EPR signals

and reactions

sented,

derivatives

methemocyanin

intense

methemocyanin

cyanin

is

mollusc

able

ties

there

of arthropod

EPR signals

ric

reactions

While

properties. nature

The met and dimer

% 6% of non-regenerable reactions

of dimer

of met and dimer ground

state

in terms

magnetic

hemo-

are

pre-

proper-

of elimination

BIOCHEMICAL

Vol. 86, No. 3, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS AND METHODS: Hemocyanin from the marine snail, Bus con canaliculatum, was obtained by ultracentrifugation (6). Met (7,8) wand dlmer 9 hemosn were prepared according to published procedures. EPR of frozen solutions were recorded at 77K using a Varian E-9 spectrometer operating at 9.1 GHz. Optical spectra were taken on a Cary 17 spectrophotometer. RESULTS AND DISCUSSION: (7)

or incubation

with

the appearance can only tion

for

of the signal with

generated

and absorption

a stoichiometric

is observed

and only

slight

ed to a small EPR-detectable are action is ity

percentage

in that

demonstrated

by addition

of the EPR signal

The effect

EPR signal

they ions

coupled

cupric

of ligands

are found field charge

transfer

titration

binding

bind

observed

of acetate.

with

with

ved at Q, 360 nm and the binding

site

at pH = 6.3

629

This

phosphate

is

are sites interThis

the

(Figure

intens1C) is

g = 2 signal).

2, 4, ll),

SCN-, N02-, Nj

on changes

and SCN-

in the ligand

and the appearance

N3- at pH = 5.0 acetate

constant

While

[Amax = 360 nm (N3-)].

(4).

is

(10).

spectrum

based

buffer

the

dependent

broad

re-

in EPR in-

that

and that

to the protein.

references

then is

nonregenerable

sites

660 nm (SCN-)]

in the CD spectrum However,

is

can be ascrib-

has been investigated.

azide

integra-

methemocyanin

methemocyanin

(g = 4 signal,

to the active

band associated

for

the resulting

[hmax = 710 nm (N3-),

of methemocyanin

as previously

increase,

signal

when peroxide

the regenerable

cupric

azide

centers

observed

pH and anion

methemocyanin

to reversibly

transitions

binuclear

such as N3- (cf.

and CN- on the regenerable

on double

No loss

EPR-detectable

is buffer,

does not

of this

in the met preparation

These

in

at pH = 7.0 phosphate is

observed

of excess

results

90% of the oxyhemocyanin

peroxide

obtained

are

based

oxy concentration.

Therefore,

and nonregenerable.

buffer)

H202

When the methemocyanin

of H202,

with

of sites

the copper

of weakly

amount

buffer.

between

that

at 280 nm.

with

The intensity

present,

of the EPR intensity

and the

interesting,

1A).

copper

upon treatment

in pH = 5.0 acetate

EPR-nondetectable

(Figure

to the original

reduction

by treatment

at 37°C (pH = 5.0 acetate

6 f 3% of total

when compared

tensity

added

N3- (8)

of methemocyanin

of a weak EPR signal

account

treated

Generation

buffer

of a

An optical is

complicated

is due to competitive buffer

of N3- is calculated

one band

is obser-

to be 1.5 mM-1 .

Vol. 86, No. 3, 1979

.

1500

BIOCHEMICAL

.. <.

1700

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

.

.

2700

3500

3100

Gmlss

Fi sure 1. EPR spectra (pH = 5.0 .lM acetate buffer) of (A) Methemocyanin; (6) Methemocyanin after addition of stiochiometric amount of H202; (C) Methemocyanin after addition of 50 fold excess NaN3. Relative receiver gains are indicated.

Reaction stitution cyanin

of N02- and CN- with

at the active as shown by small

obtained.

primary

shifts

At pH = 5.0 acetate

NaN02 to met produces though

site.

nitrite generation

chemistry

a clear under

of NO (12).

met does

At pH = 8.0,

not

proceed

N02- reversibly

in the d-d bands buffer, solution acid

by simple binds

ligand

to methemo-

with

no new EPR signal

however,

addition

of loo-fold

of dimer

hemocyanin

conditions

The direct

630

is reaction

complex,

(Figure one would

of NO with

sub-

being excess

25).

Al-

expect

met does,

in

Vol. 86, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

.

E

xl.

.

V

.,

1300

‘*

1700

2500

2900

G8USS

3300

Fisure 2. EPR spectra (pH = 5.7 .lM acetate buffer) of (A) Methemocyanin; (B) Methemocyanin after addition of 100 fold excess NaN02; (C) Dimer hemocyanin prepared by the action of NO on deoxy; (D) Dimer hemocyanin after addition of loo-fold excess NaN3 (dimer-N3-); (E) Dimer-N3after addition of 10 fold excess H202. Relative receiver gains are indicated.

fact,

cause

Finally, ions

half produces

A one electron ric

addition

a one electron met-N02dimer

reduction,

resulting

in a 100 fold

hemocyanin

reduction of NaCN (1:l

perhaps

to half CN-:Cu)

excess through

met can also

in production of NaN08 under

631

anaerobic

an oxidation

via

be accomplished

to met at pH = 8.0

met-CN-(13).

of half

tris

met-N02-. condit-

nitrous

acid.

by stoichiomet-

buffer

yielding

half

Vol. 86, No. 3, 1979

Figure action

2C presents

Figure

excess

in the presence

this

is

also

or allowing

The large

a binuclear

g = 4 signal

and the broad

g = 2 signal

converted

half

addition EPR spectrum

EPR-nondetectable

buffer

for

24 hr shows

anin.

This,

coupled

has been converted reactions original

with dimer

methemocyanin. but is

dimer-N3 - forms nondetectable

with

other is

anions

dialyzed

After

ligand

by treatment of Busycon met.

dimer-N3-

.and thiocyanate.

field with

EPR spectral

transitions H202.

are

Therefore,

canaliculatum

The chemistry

preparation,

are metastable

of met and dimer

Scheme I. Scheme Dimer

Met

632

I

in the met the Further,

that

is found

dimer-N3to undergo

Finally, also

features

both

un-

of methemocy-

it

present

with

eliminates

indicates

such as cyanide

no dimer

(The resid-

In contrast

characteristic by H202,

after

of the

to pH = 5.7 acetate

hemocyanin

dialysis,

2E).

form produced

In addition,dimer

immediately

indicate

loss

preparation).

in

dimer-N3-

of oxyhemocyanin.

transitions

the regeneration

to met.

the methemocyanin regenerated

on dialyzing

field

of this

H202 to the dimer-N3-

form obtained

shown

and is associated

dimer

in regeneration

of a

to that

at g % 2 (Figure

and results

ligand

Addition

in complete

intensity

by the

g = 2 region

Dialysis

features

excess

of 02.

and broad

EPR-detectable

of 10 fold

obtained

EPR spectrum

72 hr results

in the original

preparation,

the

for

has % 20% of the original

and the nonregenerable

the

derivative.

spectral

met obtained

hemocyanin

amounts

g = 4 signal

to sit

to the dimer

dimer-N3-

changes

cupric

the preparation

of dimer

of trace

of NaN3 to the dimer

2D (dimer-N3-).

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the EPR spectrum

of NO on deoxy

100 fold

ual

BIOCHEMICAL

if

the

converts

to

are observed

and oxyhemocyanin the original

dimer

and convert

to EPR-

hemocyanin

is summarized

and

in

BIOCHEMICAL

Vol. 86, No. 3, 1979

The met and dimer but

exhibit

coupled

very

with

indicate

per(II)'s cular

with

site

must

be ascribed

rivatives

Group

(13,14).

Br-

require

and I-)

the Cu(II)-Cu(1) Group

ability

coppers coordination

(half

a second

ligand

the h(II)-Cu(I)

apart,

position

(half

coppers

where

2.5

copper(II).(l5)(See

Scheme

\ /L\ /

cut11

\Cu(Ir(LZ\U(I)

protein

ligand

633

CH3C02-,

on ligthe

force

the

open an additional For dimer

II

/\

HALF

de-

modes of L2 lig-

\“,I< ‘\,(I<

\/

to the

modes which

SCN-) have

Scheme II).

MET-L1

R=Endogenous

N3-,

leaves

A\

/

L, = N02-,

TIMER

,cu(I~R/uiII~

\cu(r;fLi\

the

between

- 4 i depending

MET

HALF

coupling

Thus, L2 ligands

which

molein

The bridging

at > 5 i. bridge

as their

in bridging

L2 = CN-,

at the Cu(I1).

cop-

of an EPR signal

where

ligand

to be between met-L2

of dimer

met [Cu(II)~~*Cu(I)]

met-L,

one exogenous

an endogenous on the

A com-

can now be related

2 half

dimer,

two tetragonal

The lack

1 and Group

separation

rupturing

the

met and dimer

separation

2 derivatives

to bind

ands place

only

contains

to antiferromagnetic

Group

for

copper(II

between

sites

The EPR spectrum,

(Amax = 710 nm) observed

met also

1 derivatives

coordinate

active

properties.

are similar.

between

between

cupric

two tetragonal

that

energies

observed

magnetic

weak interactions

then

binuclear

of met (Amax = 680 nm) to that

indicating

The difference

differences

and.

changes

contains

transition

met hemocyanin two coppers.

state

region

relatively

orbital

contain

of the d-d bands

the active

small

both

ground

of the d-d spectral

shows only

cl-,

different

the position

that

parison

derivative

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MET-L2

hemocy-

Vol. 86, No. 3, 1979

BIOCHEMICAL

anin,

the 6 i Cu-Cu separation

polar

coupled

in analogy

EPR spectrum

to Group

(5)

2 half

would

magnetism

a Cu-Cu separation

met derivative genous tween

bridge

is

provide

< 4 i,

can reform

the two copper(I

obtained

from

computer

requires

that

the endogenous

met forms.

or carboxylate with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Protein

an effective

in analogy providing

residues

Thus if

to the half the pathway

bridge

pathway

be broken phenolate

for

antiferro-

the Cu-Cu distance

met Group for

of the di-

containing

superexchange

of < 4 i.

and eliminating

simulation

1 series,

superexchange

in the

the

endo-

coupling

be-

the EPR signal.

ACKNOWLEDGEMENTS: We are grateful to the National Institute of Arthritis, Metabolism,' and Digestive Diseases of the U.S. Public Health Service (AM20406) for support of this research. Acknowledgement is made to the Alfred P. Sloan Foundation (E.I.S.) and the Whitaker Health Sciences Fund (N.C.E.) for Research Fellowships. REFERENCES: 1.

Makino, N., van der Deen, H., McMahill, P., Gould, D.C., Moss, T.H., Simo, C., and Mason, H.S. (1978) Biochim. Biophys. Acta 532, 315-326. 2. Lijnen, H.R., Witters, R., and Lontie, R. (1978) FEBS Letts. 88, 358-m. K., Blaton, V., and Lontie, R. (1965) Arch. Intern. Physiol. 3. Heirwegh, Biochem. 73, 149-150. R., DeLey, M., and Lontie, R. (1977) 4. Witters, Structure and Function of Hemocyanin, J.V. Bannister, Ed., pp. 239-244, Springer-Verlag, Berlin. 5. Schoot Uiterkamp, A.J.M., van der Deen, H., Berendsen, H.C.J., and Boas, J.F. (1974) Biochim. Biophys. Acta 372, 407-425. 6. Waxman, L. (1975) J. Biol. Chem. 250, 3796-3806. Felsenfeld, G. and Printz, M.P. (1959) J. Amer. Chem. Sot. 81, 6259-6264. ii: Witters, R. and Lontie, R. (1975) FEBS Letts. 60, 400-.403. 9. van der Deen, H. and Hoving, H. (1977) Biochemistry 16', 3519-3525. 10. An additional broad signal is sometimes seen in the g = 4 region for methemocyanin preparations. We find this signal to be variable and find that oxy can be regenerated from met without loss of intensity in this region. 11. M$alii;l, P.E. and Mason, H.S. (1978) Biochem. Biophys. Res. Comm. 84, 12. 13. 14. 15.

Coti0n;F.A. and Wilkinson, G. (7972) Advanced Inorganic Chemistry, Third Edition, pp. 354-366, Wiley Interscience, New York. Himn-elwright, R.S., Eickman, N.C. and Solomon, E.I. (1978) Biochem. Biophys. Res. Comm. 84, 300-305. Himmelwright, R.S., Eickman, N.C. and Solomon, E.I., in press, J. Amer. Chem. Sot. For the met apo form where only one copper is present at the active site the endogenous ligand remains coordinated and again only one exhangeable position is present on the Cu(I1).

634