Vol. 81, No. 1, 1978 March 15,1978

BIOCHEMICAL

PREPARATION

AND CHARACTERIZATION A SINGLE

Richard

S. Himmelwright,

Nancy

ACTIVE

C. Eickman,

Institute

Cambridge, December

OF MET APO HEMOCYANIN:

COPPER(I1)

Massachusetts

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 243-247

SITE

and Edward

I.

Solomon*

of Technology

Massachusetts

02139

28,1977

SUMMARY: A new derivative of Busycon canaliculatum hemocyanin has been prepared for which one copper has been removed from the binuclear active site of the holoprotein and the remaining copper has been oxidized with a variety of small molecule oxidizing agents. This met apo derivative [( )...Cu(II)] binds a number of ligands; EPR spectra of several forms are reported and compared to those obtained for a singly oxidized (half met-L) derivative [Cu(I)...Cu(II)L]. The site of the oxidized copper for both forms is found to be quite similar in structure but shows large differences in ligand binding ability. INTRODUCTION: copper

unit

intense

The unique in hemocyanin

absorption

have made it structure

met

bands

difficult

in

the active studies 4 of the

(singly

cyanin

oxidized5)

[Cu(I)...

site. effects

the

where

tightly

ordinates

in

site

a d,2- y2 ground

with

preparation

the plane

met apo-L,

site

and reversibly

* To whom all

the

of

perturbation

of Busycon L = N3-,

bound

but

contains

binds

correspondence

should

of

spectroon the half

canaliculatum

CH3C02-,

CN-,

ligand

tetragonal

copper(I1) [(

we L co-

copper(I1)

communication

ligands

hemoetc.],

reports

of a new hemocyanin

a single

a variety

detailed

exchangeable

This

state.

and

and geometric

through

ligand

binuclear

regions)

electronic

Recently,

EPR studies

which

uv and visible

of an approximately

and initial

tive,

near

derivative

Cu(II)L,

have shown that

the

to determine

of

scopic

electronic properties of the l-3 (the lack of an EPR signal

in

the

derivathe

active

)...Cu(II)L].

be sent 0006-291X/78/0811-0243$01,00/0

243

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 81, No. 1, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

\ \ \ \

---

5

18 (hr)

llll

Figure 1. polyphemus

15 TIME (hr)

Copper removal hemocyanin.

from

Busycon

canaliculatum

and Limulus

MATERIALS AND METHODS: Hemocyanin was purified from the hemolymph of the marine snail, Busycon canaliculatum, and the horseshoe crab, Limulus polyphemus, by ultracentrifugation. The 6 pellets were redissolved in 0.1 M tris, 0.1 M CaC12, pH 8.5. The half apo protein was prepared by careful dialysis of the hemocyanin against a buffered 0.05 M cyanide solution while monitoring copper content and optical absorption at 345 nm. The met apo derivative is obtained through oxidation of the copper by incubation in a tenfold excess of NaNO at pH 6.3, 0.1 M phosphate buffer. Oxidation of the copper ha ii also been achieved by anaerobic treatment at pH 8.0, 0.1 M tris with NaO2 dissolved in DMF, by autooxidation at 37OC, or by addition of peroxide at pH 8.0, 0.1 M tris. Regeneration7 of the half apo form to oxyhemocyanin is accomplished by anaerobic treatment with Cu(CH3CNJ4C104. Copper content was determined by atomic absorption using a Perkin Elmer 360 spectrometer equipped with a graphite furnace. EPR spectra of frozen solutions at -100°C were obtained using a Varian E-9 spectrometer operating at 9.1 GHz. RESULTS AND DISCUSSION: removal cyanin

from

by dialysis

percent

of

a function that

copper

oxygen

binds

against

the

the

ratio

extent It

of oxygen

1 02. -2 Cu in both

binding

244

ability

copper

represent

has previously

As seen in Figure oxygen

of

polyphemus

The curves

and the

of dialysis. in

results

and Limulus

cyanide.

remaining

hemocyanins.

parallels

1 shows the

canaliculatum

of length

and mollusc content

Busycon

Figure

lA, for

the

the

binding been

the

hemo-

as shown'

arthropod copper

Limulus.

In

BIOCHEMICAL

Vol. 81, No. 1, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MET APO

HALF

I

.

3000 FIELD(Gour~l

0

2

3400

MET

03

Figure 2. EPR spectra of met apo derivatives. (A) pH = 5.7 acetate buffer, .OlM NaN02; (B) pH = 5.7 acetate; (C) pH = 8.0 tris buffer, prepared by oxidation with Na02. Figure acetate

3. EPR spectra of half met derivatives. (B) pH = 5.7 acetate buffer, .OlM NaN02;

Figure

lB,

binding

though,

it

is obtained

demonstrates

that

is seen

when 50% of one copper

active

This

shows no EPR signal,

in a +l

oxidation

We are half

able

50% of the against

the

result

removed

yielding

a half

consistent

the

using with 2A.

sites

excess

Double

buffer

2B.

of increasing

single

a number

with

of oxygen 8

from

This the

apo protein.

the

of

small

active molecule

NaN02 produces

of

to a change

The EPR spectrum pH (0.1

245

the

shows that

Removal leads

the

copper

integration

are oxidized.

acetate

shown in Figure

loss

has been removed.

selectively

of Busycon,

to oxidize

Incubation

shown in Figure

complete

copper

being

state.

apo derivative

agents.

sis

site

almost

copper

is

binuclear form

that

(A)pH = 5.7 buffer.

the in

nitrite the

given

in Figure

M tris,

pH 8.0

site

of this

oxidizing EPR signal approximately by dialyEPR signal

as

2C shows buffer);

this

Vol. 81, No. 1, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I EPRPARAMETERS

of MET APO and HALF MET DERIVATIVES

MET APO pH = 5.7 acetate, pH = 5.7 acetate pH = 8.0 tris HALF MET PH = 5.7 PH = 5.7

pH change

have via

acetate, acetate

is

achieved

.OlM

NaN02

2.309 2.314 2.200

2.097 2.083 2.082

124 131 220

.OlM NaM02

2.302 2.318

2.096 2.080

125 141

reversible.

Regeneration

by reduction

also

and metal

been able

autooxidation

reincorporation.

to generate

the high

at elevated

an EPR spectrum

in

2C.

Finally,

the

with

hydrogen

peroxide.

treatment

As previously dized

form

bound

at the

half

comparison make

is

to the that

similar.

site met

the

derivatives

analogous

same copper

structure

play

the

a variety

of

of

the parallel

forms

in Table in both role

246

tightly

I.) forms

3 for

point

to

are extremely This

shows that

and that

in electron the

of

in Figure

The first

or that

by

oxi-

ligands

apo forms.

a significant formation

singly

met

is oxidized

shown

The EPR spectra

are presented

are given

that

can be oxidized

[Cu(I)...Cu(II)L].

the EPR spectra

does not

and site

with

Na02,

with

apo protein

we

apo derivative

and with

we have prepared

holoprotein

(EPR parameters

either Cu(1)

active

pH met

2D) identical

half

reportedl,

of the

two relevant

(Figure

is

Further,

temperatures,

obtaining Figure

to oxyhemocyanin

two coppers

the

delocalization in the

Vol. 81, No. 1, 1978

active

site

greatly

met-L

is met

against

Cu(1)

stants

the

an active L-Cu(I1)

part

Detailed

site

in binding

EPR spectrum

spectroscopic

Anions

the

in

studies

Thus, the

ligand

suggests

ligand,

cupric

the of

ligand the

of one

met apo-L bind

however, the

the

through

in

derivatives

all

presence

of

con-

Cu(1)

plays

a Cu(I)-

the half

is binding

to

by competition

due to ligand ion

and

tightly

binding

that

perhaps

perturbations

of the that

be displaced

the met apo forms,

strongly

(The strong

demonstrate

ability.

by dialysis.

are not

or removal

between

increases

This

which

difference

In

large

site.

bridge.

on the

active

to

environments

and can only

ligands.

RESEARCH COMMUNICATIONS

of a Cu(1)

binding

can be removed

leads

of

significant

derivative

ligands

ligand

presence

ligand

stronger

bound

tion

in

AND BlOPHYSlCAL

similar

by the

The most

the half

the

have very

affected

copper. half

8lOCHEMlCAl

variamet-L

to the

Cu(II).)

are presently

underway. We wish to thank Dr. Anna Ghiretti-Magaldi for ACKNOWLEDGEMENTS: helpful discussions. Acknowledgement is made to the Donors of the Petroleum Research Fund, administered by the American Chemical Society, for support of the initial phases of this research and to the Alfred P. Sloan Foundation (E.I.S.) and the M.I.T. Health Sciences Fund (N.C.E.) for research fellowships. We are grateful to the National Institute of Arthritis, Metabolism, and Digestive Diseases of the U.S. Public Health Service (AM20406) for continuing support of this research program. REFERENCES: 1. Lontie, R., and Witters, R. (1973) Inorganic Biochemistry, pp.344-358, ed. G.L. Eichhorn, Elsevier Scientific, New York. 2. Lontie, R., Vanquickenborne, L. (1974) Metal Ions in Biological Systems, Vo1.3, pp.183-200, ed. H. Sigel, Marcel Dekker, Inc., New York. 3. van Holde, K.E., and van Bruggen, E.F.J. (1971) Subunits in Biological Systems, pp.l-53, eds. Timasheff, S.N., and Fasman, G.D., Marcel Dekker, Inc., New York. 4. Himmelwright, R.S., Eickman, N.C., and Solomon, E.I., submitted for publication to Biophys. Biochem. Res. Comm. 5. Schoot Uiterkamp, A.J.M. (1972) FEBS Letters 20(l), 93-96. 6. Engelborghs, Y., and Lontie, R. (1973) Eur. J. Biochem. 39, 335-341. 7. Lontie, R., Blaton, V., Albert, M., and Peeters, B. (1965) Arch. Int. Physiol. Biochim. 73, 150-152. 8. This is probably true in general for molluscan hemocyanins based on previous observations on Octapus vulgaris hemocyanin: Salvato, B., Ghiretti-Magaldi, A., and Ghiretti, F. (1974) Biochemistry 13, 4778-82. 247