BIOCHEMICAL

Vol. 78, No. 2, 1977

AND BIOPHYSICAL

AN EPK SIGNAL FROM THE "IldVISIBLE"

COPPER OF CYTOCHROME OXIDASE

C.H.A. Seiter, S.G. Angelos, Department of Chemistry, University Los Angeles, California Received

July

12,

RESEARCH COMMUNICATIONS

and R.A. Perreault of Southern California 90007

1977

SUMMARY: Sulfide is both an inhibitor and a slow reductant of oxidized cytochrome c oxidase. When the enzyme is exposed to sulfide for short times (one minute or less) and frozen, the resultant electron paramagnetic resonance (EPR) signals show $yarly: low spin heme a, low spin heme a3, th$+usual "EPR detectable" Cu signal (g,, = 2.17, gL = 2.03), and a new cu signal superimposed on the same region, with (gLL% 2.19, gA= 2.05). This new signal presumably arises because the antiferromagnetic coupling postulated to exist between the iron atom of heme a3 and this copper is disrupted when heme a3 is driven to a low spin state by sulfide. The implications of this result with respect to models of the 02-binding site and redox geometry of oxidase are briefly discussed. Several

studies

as a ligand

for

(1,2,3)

cytochrome

reduced

forms

of the

hundred

times

more

it

as a slow

acts

incubation

compared

state

all

That

is,

indicating

that

and the

simultaneous

are

reduced

undetectable under here in the

these

redox the

the

for

enzyme;

in the

reductive

atom of the

conditions,

(The of Wever

spectrum

effect et al.,

(2),

This

a broadening but

these

in a

and detectable in the

that

coupled

spectrurm.

explains

sufficiently

components

imply

antiferromagnetically to the

can be

spectrum,

has been abolished,

of those (S,6),

long

can be prepared

are oxidized

coupling

titrations

contribute

is

enzyme

Additionally,

compound

binding

and

several

(4).

heme a3 signal

antiferromagnetic

oxidized

to oxidase

heme a3-SH

enzyme

of sulfide

at sufficiently

the

of a low-spin

to both

heme proteins

for

that

behavior

binds

the

constant

of the

presence

in

except

centers

presence

copper

EPR spectra

other

of reduction

a3-Cu

rapidly

experimentally.

to most the

unusual

strongly

than

The "on"

rate

binds

and specifically

centers

(1,2).

all

It

the

(l),

agent

redox

to the

in which

by EPR.

enzyme

reducing

times

confirmed

oxidase.

strongly

seen to be reduced fast

have

investigators

effect

the

which usually

pair

should, is

observed

of the Cu region concerned

Vol. 78,No.

themselves not

2, 1977

with

explore

the

BlOCHEMlCAL

the

heme binding

implications

AND BIOPHYSICAL

properties

for

of a series

antiferromagnetic

MATERIALS

RESEARCH COMMUNICATIONS

of

ligands

and did

coupling).

AND METHODS

The method of Sun --et al. (7) for purification of cytochrome oxidase dispersed in Triton X-100 was used, with the addition of several hours dialysis against EDTA at 4'C to remove "adventitious" copper. The absence of such copper was checked by a series of studies at different temperatures and microwave power settings (8). A cytochrome oxidase preparation using sodium deoxy cholate according to the method of Yonetani (9) gave virtually identical results spectroscopically, and the Triton results were chosen for presentation only because of the greater purity of the preparation when examined by gradient-gel electrophoresis in the presence ofSDS and 6 M urea (10). Detergents and other chemicals were purchased from Sigma Chemical Company and used as received, Heavy layer beef heart mitochondria used in the preparation were the kind gift of Dr. Y. Hatefi of Scripps Clinic and Research Foundation of La Jolla, California. The enzyme was stored fresh sulfide solution was enzyme. The variable rates (1,ll) correlate with the is kept on ice for several

under liquid nitrogen and sulfide titration with carried out rapidly upon thawing of the stored of reduction of the enzyme reported occasionally loss of specific activity noLed if the enzyme hours.

tPR spectra were recorded on a Varian E-12 spectrometer with an Air Products liquid helium cryogenic system, under noted in the figure ligands.

equipped the conditions

RESULTS AND DISCUSSION Figure oxidase

and the

4'C for the

1 shows a comparison

spectrum

normal

Figure

2 displays

treated

with

has taken (The

resting

copper

temperature,

place. signal

partially

treated

the

spectrum

which

of reduction

are

more rapidly

sulfide

copper which

is

of Palmer

--et al.

reduction

temperature by incubation

0-f

from (12)).

enzyme of the

enzyme

and pH dependent. at room

2.).

has a clearly obscured

discernible

by the

low

spin

762

g1=

2.05

and a less

heme a3 signal

of

usually

displaced

that

at

region

is

against

Extensive

can be removed

the

heme region

notation

cytochrome

with

to present

10 min.

for

of resting

has appeared

oxidase

The kinetics

as in ref.

than

(CuA in the

resting at 4'C

oxidase

scaled

signal

Cu2+ signal

sulfide

The new signal g,,s2.19,

new copper

the

is

rather

more prominently, A large

EPR spectrum

of cytochrome

The spectrum

40 seconds.

emphasized. the

EPR spectrum

of the

at g

Y

clear = 2.22.

BIOCHEMICAL

Vol. 78, No. 2, 1977

Fig.

1.

EPR spectra

of cytochrome

modulation Dashed

amplitude line:

buffer,

and buffer

Triton

pH 7.0.

in presence

typically

at 5'K.

G, Microwave

enzyme,

Solid

of 0.01

CuA and CUB as defined signals

oxidase

= 8.2

Native

phosphate

AND BIOPHYSICAL

in

Microwave frequency

X-100 line:

suspension, enzyme

Signals

in small

power

GHz.

0.1

M

in same detergent for

at g = 6.0

amounts

= 10 ml?l,

= 9.307

M Na2S in solution

text.

present

RESEARCH COMMUNICATIONS

40 seconds. and 4.3

in oxidase

are

preparations

(2, 5, 8, 16).

The signal

is

notable,

fine

splitting

lack

of hyperfine

electron

A,,,

as is

and for

paramagnetic

its

splitting

delocalization

heme a3.

changes,

%5 8 of the

Fe3+ of heme a3, (this

investigation

the

broadening in this

of the

two metals

to that

of other

in this

study are

breadth

(13,14),

center

is

CuA signal,for

lack

at the

low

may be attributed,

formational

broadening

the

and the Assuming

CUB (i.e.

should

the

heme-thiol somewhat

breadth

Consistent of the

derivatives abnormal

low (15).

to the

(12))

is within

electron-electron

evidence spin

The

induces no gross con-

be orientation-dependent,

laboratory).

of 5'K.

hyper-

CuA, to extensive

undetected

extensive

copper

to proximity

sulfide

previously

demanding

temperature

as for

linewidth

that

of resolved

dipolar a point

for

heme a3-SH The g values

the signal for

under

proximity compared CUB obtained

compared to those for copper proteins

763

in

Vol. 78, No. 2, 1977

Fig.

2.

Conditions for

which

of the

having is

A point

cyanide. spin

is the

intact.

cyanide,

bound

to reconcile

the

bridged

compound

in the

close

from enough

inhibitors.

in other

oxidases.

2)

A study

(~1.5'K)

this

for

site

histidine

same configuration studies

to form

an effective

In this

case

the

with

a3-CuB

employing no new low

both

an affinity

cyanide state,

of a histidine That

EPR data.

COU-

antiferromagnetic

to a low-spin

the

those

has as high Since

(12,17)

of 02 reduction

enzyme produces

the

coupling.

and CUB with

geometry

studies

the enzyme

suggestion

through

CO binding

that

which

and 02 binding

compound

a (gL - Z)/(g,,-

to the

oxidized

heme a3 exclusively

heme a3 iron

inhibitor

derived

allowed

CUA, but

temperatures

binding

isolated

for

recurring

low

respect

suggesting

eliminates

to drive

of the

with

to the

Sulfide,

of copper

at very

of sulfide

(16),

can be shown

the

exposure

way.

interest

contrast

obviously

between

under

of great

Cyanide

is

sulfide

to oxidase

to that

CUB signal

is

heme a3 signals

pling

that

only

similar

of the

resolution

by oxidase

1 except

a g,, similar

at least

linewidth

improved

as in Fig.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

10 minutes.

general (13), ratio

BIOCHEMICAL

as for

and sulfide it

is

difficult

ligand-bridge is,

in this

model

it

is most unlikely that the a3-CN 2+ while the a3-SH to CUB would be coupled would

(18,Ig),

A more plausible

not. is

that

two-metal-center I orbitals

the

a3 iron binding

of a cyanide

764

explanation, and CuB are

site

for

at the O2 site

O2 and would

BIOCHEMICAL

Vol. 78, No. 2, 1977

mediate the

the

antiferromagnetic

heme a3 iron

electron-spin

to a low-spin

coupling,

supporting

the

coupling,

idea

state,

Thus

the

AND BIOPHYSICAL

whereas would

EPR data

of an initial

sulfide,

provide

while

no such

presented

two-electron

RESEARCH COMMUNICATIONS

here

reduction

also pathway

driving for

may be taken

as

of oxidase-bound

o* (20,Zl). ACKNOWLEDGEMENTS: Corporation,

American

of Health

(Grant

supported

this

Dr.

We gratefully

Y. Galante

Chemical

HL 19392). work,

acknowledge Society Dr.

B. J.

(PRF-G)

support

and the

M. D. Kamen and Dr.

and we acknowledge

and Dr.

the

numerous

of Research National

Y. Hatefi

inspiring

Institutes have generously

discussions

with

Errede.

REFERENCES: t : 3. 4. 5. 6. 7.

8. 9.

10. 11. 12. 13. 14.

15. 16. 17. 18.

19. 20. 21.

Nicholls, P. (1975) Biochim, Biophys. Acta 396, 24-35. Wever, R., Van Gelder, B.F., and Dervortanix D.V. (1975) Biochim. Biophys. Acta 387, 189-193. Wilson, D.F., Erecihska, M. and Owen, C.S. (1976) Arch. Biochem. Biophys. 175, 160-172. Nicholls, P. (1976) Biochim. Biophys. Acta 430, 13-29. Beinert, H., Hansen, R.E., and Hartzell, C.R. (1976) Biochim. Biophys. Acta 423, 339-355. Hartzell, C.R., and Beinert, H. (1976) Biochim. Biophys. Acta 423, 323-338. Sun, F.F., Prezbindowski, K.S., Crane, F.L., and Jacobs, E.E. rrg68) Biochim. Biophys. Acta E, 804-818. S.P.J., Falk, K.-E., Lanne, B., and Vanngird, T. Aasa, R., Albracht, (1976) Biochim. Biophys. Acta 422, 260-272. Yonetani, T. (1960) J. Biol. Chem. E, 845-852. Capaldi, R.A., Bell, R.L., and Branchek, T. (1977) Biochem. Biophys. Res. Comn. 74, 425-433. Gilmour, M.r? Wilson, D.F., and Lemberg, R. (1967 ) Biochim. Biophys. Acta 143, 487-499. Palmer, G., Babcock, G.T., and Vickery, L.E. (1976 ) Proc. Nat. Acad. Sci. 73, 2206-2210. Peisach, J. and Blumberg, W.E. (1974) Arch. Biochem. Biophys. 165, 691-708. Kivelson, D. and Neiman, R. (1961) J. Chem. Phys. 35, 149-155. Wittenberg, B.A., Kampa, L., Wittenberg, J.B., Blumberg, W.E. and Peisach, J. (1968) J. Biol. Chem. 243, 1863-1871. Van Gelder, B.F. and Beinert, H. (1969) Biochim. Biophys. Acta 189, l-24. Vanneste, W.H. (1966) Biochemistry 5, 838-845. Lindsay, J.G., and Wilson, D.F. (1974) FEBS Lett. 48, 45-49. Lindsay, J.G., Owen, C.S., and Wilson, D.F. (1975)Arch. Biochem. Biophys. 169, 492-505. Chance, B., Saronio, C., and Leigh J.S., Jr. (1975) Proc. Nat. Acad. Sci. 72, 1635-1640. Wikstxm, M.K.F., Harmon, H.J., Ingledew, W.J. and Chance, B. (1976) FEBS Lett. 65, 259-277.

765

An EPR signal from the "invisible" copper of cytochrome oxidase.

BIOCHEMICAL Vol. 78, No. 2, 1977 AND BIOPHYSICAL AN EPK SIGNAL FROM THE "IldVISIBLE" COPPER OF CYTOCHROME OXIDASE C.H.A. Seiter, S.G. Angelos, De...
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