896
BIOCHEMICAL SOCIETY TRANSACTlONS
3. Moody, A. J. & Rich, P. R. ( 1 988) EBEC'Rep. 5 , 6 9
4. Rich, P. R., West, I. C. & Mitchell, P. ( 1 988) FEBS Lett. 233,
25-30
5. George, P. & Tsou, C. L. ( 1952) Biochem. J. 50,440-448
6. Artzatbanov, V. Yu.,Konstantinov, A. A. & Skulachev, V. P.
( 1978) FEBS Lett. 87, 180-1 85
7. Wikstrom, M. ( 1 989) A n n . N . Y. Acad. Sci. 550, 199-220
8. Goodman, G. ( 1984) J. Biol. Chem. 259,15094- 15099
9. Wikstrom, M., Krab, K. & Saraste, M. (1981) Cytochrome
Oxidase: A Synthesis, Academic Press, London
10. Blair, D. F., Ellis, W. R., Wang, H., Gray, H. B. & Chan, S. I.
(1986)J.Biol. Chem. 261,11524-11537
11. Nicholls, P. & Wrigglesworth, J. M. (1989) Ann. N. Y. Acad.
Sci. 5 50, 5 9-6 7
12. Moody, A. J. & Rich, P. K. ( 1989) Hiochern. Soc. Trcrns. 17,
896-897
Received 28 March 1989
Redox titration of haem a in cyanide-ligandedcytochrome-c oxidase: simulation studies on
interacting, pH-dependent, redox centres
A. JOHN MOODY and PETER R. RICH
Glynn Research Institute, Bodmin, Cornwall PL30 4A U, U.K.
We have investigated a model system, containing two '12 = 1'
redox components, which can exist in two states that are
interconverted by (de)protonationof a single group. The two
states are characterized by differing redox midpoint potentials for one or more of the redox couples. Fig. l(6, inset)
shows an 'arrow diagram' for this system; the arrows point in
the direction of electronation or deprotonation. Therefore
the ratios are defined as appropriate, e.g. R , = red./ox. =
1()I'
l/W at 20"C, where Em is the midpoint potential and
E,, is the redox potential, and R,,,,= deprot./prot. = 10'pH-pKI.
The minimum number of arrows required to 'connect in' all
the substates is used. This is the number of equilibria needed
t o define the whole system; the choice of equilibria is
arbitrary provided that there is only one route between any
two substrates.
Three midpoint potentials (equilibria)can define a singlestate two-component system (bold arrows). At a given pH,
the two-state system is analogous to the single-state system
and can be described using three apparent midpoint potentials. The relationship between pH and the apparent interaction between the components, 1', is given in eqn ( l ) ,where
R; and R', are analogous to RY and RY in the single state
system.
5.6
pKol 6.8
6.3
PH
(b)
3601.
340
-
320
-
300 '
6
v
4280
Eqn ( 1 ) can be used to simulate the effect of pH on 1'.
However, it is simpler to construct a Pourbaix diagram [ 11 for
one of the components. There are two couples per component, i.e. oxidoreduction while the other component is
oxidized and oxidoreduction while the other is reduced, The
difference between the idealized EJpH profiles is a semiquantitative measure of 1'. There are three general cases.
(1) If both couples for either (or both) component(s) are
pH independent then I' is pH independent.
( 2 ) If one couple for either (or both) component(s) is pH
independent then I' changes with pH in a similar way to the
midpoints (Fig. 1b).
( 3 ) If all couples are pH dependent then there is an additional interaction superimposed on a change in I' which is
identical to that in case (2).This interaction is co-operative if
the net effect of pH on the midpoints for each component is
in the same direction (Fig. la); anticooperative if the effect is
in the opposite direction.
-
260 r
240 I
6
7
I
PK, 8
7.8
I.
1
pKlO9 P K , ~ 10
8.9
PK,,
9.4
1
PH
Fig. 1 . Examples of the effect of pH on the apparent midpoint
potentials of the redox couples involving component I (00- 10
and 01 11)for the model shown in the insert
-
(a) E:,, = E:.z = 367 mV; E:,3 = 280 mV; Em = Em,:= 328
mV; Em,,=2S1 mV; pK,,,,=5.6. (b) E:,,=Ei,,=328 mV;
E:,3 = 25 1 mV; Ern,'= 263 mV; Em->
= 235 mV; En,%?
= 223
mV; pK,,,,= 7.75. The dashed lines are Pourbaix diagrams for
the same values. Note that the ratios R:' and I?,refer to the
couples 01"- l l H and 01-11, respectively, which are not
labelled with arrows in the insert.
1989
897
630th MEETING, ABERYSTWYTH
The two-state, two-component model cannot account for
the effect of pH on the titration of haem a in cyanideliganded cytochrome-c oxidase over all of the range studied
(6.2-9.2, see [2]). However, the parameters used for the
simulations shown in Fig. 1 were chosen to illustrate how it
can account for the effect over two limited ranges: ( a )6.2-7
and ( b )7 5 9 . 2 (component 1, haem a; component 2, Cu,).
Combination of these two partial models into a three-state
system involving sequential (de)protonation of two groups
yields a model that fits well over the whole pH range (see
curves in [ 2 ] ) .
Wikstriim et al. [3] have shown that a model involving
sequential (de)protonationof two groups, with pK values like
those used here, can explain the pH dependency of haem a in
unliganded oxidase (though at that time the interaction
observed at haem a was entirely attributed to haem a3).The
three-state model can also explain the results for COliganded oxidase. Component 2 is now an ‘n = 2’ component,
comprising Cu, and haem a3,whose midpoint potential with
oxidized interactant (Em,o)
is more than 6 0 mV more positive than
of haem a [4]. In this case, haem a titrates
essentially as an ’ n = 1’ component with Em= Em,R(midpoint
potential with reduced interactant) and the slight pH dependency [S]can easily be simulated, again using similar pK
values to those used here.
We conclude that the interaction between two components
at a given pH for a system with two or more pH-dependent
states is composed of two parts: ( a ) direct interaction, e.g.
electrostatic, and/or ( b), indirect interaction, occurring via
the linkage between the oxidoreduction of the components
and the (de)protonation of one or more common acid/base
groups.
We propose a minimal model for cyanide-liganded cytochrome-c oxidase in which the oxidoreduction of both haem
a and Cu,, but not Cu,, are linked to the (de)protonation of
two common acid/base groups with well-separated pK
values.
This work was supported by a grant from the S.E.R.C. (No. GR/
F/176OS).
I . Bockris. O M , & Reddy. A. K. N. (1973) Modern Elecirochemistry, p. 1 123, Plenum Press, London
2. Moody, A. J.. Mitchell, R. & Rich, P. R. ( 1 989) Riochern. Soc.
Trans. 17,895-896
3. Wikstrom, M., Krab, K. & Saraste, M. (198 I ) Cyrochrome
Oxidase: A Synthesis, Academic Press, London
4. Lindsay, J. G., Owen, C. S. & Wilson. D. F. (1975) Arch.
Biochem. Biophys. 169,492-505
5. Ellis, W. R., Wang, H., Blair, D. F., Gray, H. B. & Chan, S. 1.
( 1986) Biochemistry 25.16 1 - I67
Received 28 March 1989
Does cytochrome-c oxidase exist in both low- and high-spin pulsed forms?
NIKOLAOS IOANNIDIS and
JOHN M. WRIGGLESWORTH
Division of Biomolecular Sciences, Biochemistry Section,
King’s College London, Camden Hill Road, London
W8 7AH, U.K.
Isolated cytochrome c oxidase (ferrocytochrome-c oxygen
oxidoreductase EC 1.9.3.1) can exist in different forms,
when in its oxidized state. The enzyme is considered to be in
a ‘resting’ form, as obtained by some purifications, when it
exhibits slow intramolecular electron transfer ( t , , z= SO s ) [ 11
and multiphasic cyanide-binding kinetics [2].In contrast, the
‘pulsed’ form of the enzyme shows fast intramolecular
electron transfer [3] and monophasic cyanide-binding
kinetics [2].The pulsed form was originally defined in kinetic
terms from measurements of the oxidation rate of ferrocytochrome c when the reduced enzyme was exposed to an
oxygen pulse [4].The species generated was characterized by
a substantially higher turnover number, when compared to
that of the ‘resting’ form 131. The optical properties of the two
forms are different: it is well accepted that the ‘resting’
enzyme has a Soret maximum centred around 4 18 nm, while
the pulsed form, immediately following a redox cycle,
absorbs maximally at 428 nm. However, the latter absorption
band seems to shift to lower values with time, a process
strongly dependent on experimental conditions [ 51. In the
present work, some of the properties of the decaying pulsed
species produced by the reoxidation of the fully reduced
100
426
80
60
40
20
n
”
0
50
100
Time (min)
150
200
50
100
150
200
0
Time (s)
Fig. 1. Spectral changes (a) arid rates of iritramolecular electron transfer (b) of the
pulsed form of cytochrome-C oxidase at various rimes follo wing preparation
( a )Time-dependent changes of the Soret maximum. ( b )Rate of reduction of haems u
( 0 ) and a , ( w , A , A ) following the addition of sodium dithionite ( 2 5 mM) to the
‘resting’ form ( m ) and to the pulsed form 20 min (A ) and 120 min ( A ) after preparation. The enzyme (3-5 ,UM)was diluted in 0.1 M-potassium phosphate buffer, at pH
7.0, containing 0.5% (v/v) Tween 80. The rates of haem u reduction for all samples
are shown ( 0 ) .
Vol. 17