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.
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