Download The ways in which the enzymes catalase and horseradish

The ways in which the enzymes catalase and horseradish
peroxidase are oxidized by hydrogen peroxide to porphyrin
p-cation radicals are described and compared to the intermediates
involved in the activation o f dioxygen by the enzyme cytochrome
P-450. I n the latter case a similar p-cation radical o f an
iron( I V ) oxo-complex is examined as the probable intermediate
in the oxidation o f unactivated carbon-hydrogen bonds.
The redox chemistry of oxygen is extensive
(Fig.l) and many of the derivatives of oxygen are
highly reactive and toxic to living systems.
Nevertheless, nature encounters and frequently uses
many of the intermediates shown in figure 1. In the
case of the highly reactive species such as singlet1
oxygen 02 and the hydroxyl radical their
"indiscriminant" reactivity is used for the ultimate
destruction of invading organisms and xenobiotics in
the mammalian polymorphonuclear leukocyte (1) where
myeloperoxidase, a heme protein, plays a major role
(2). Similarly, nature uses both heme and non-heme
iron proteins in the manipulation of other oxygen
d e r i v a t i v e s . T h u s t h e f ou r - e 1 e c t r on r e d u c t i o n o f
dioxygen to water is controlled by the heme protein
cytochrome oxidase (3) which in turn receives its
electrons from the respiratory electron transport
c h a i n w h i c h i n c l u d e s inter a l i a t h e h e m e p r o t e i n s
c t o c h r o m e s b a n d c. ( 4 ) .
Hydrogen peroxide is a powerful oxidizing agent
and if left unattended in a living cell would soon
create havoc. Nature has devised at least three ways
to handle this toxic yet necessary molecule. The heme
protein catalase will, at essentially diffusion
controlled rates, decompose H2O2 to oxygen and water
(5). On the other hand, peroxidases use the oxidizing
power of peroxide to bring about selective oneelectron oxidations of organic substrates, usually
phenols and anilines (6). Finally, and as we shall
see below in some detail, hydrogen peroxide can be
generated in situ by cytochrome P-450 by the
coordination of dioxygen to ferrous P-450 followed by
a one-electron reduction.
We originally became interested in the catalases
and peroxidases as a result of studies on the
reversible one-electron electrochemical oxidations of
metalloporphyrins (7). Even when the meta11oporphyrin
c o n t a i n e d a n o n - ox i d i z a b 1 e m e t a l s u c h a s Z n ( I l ) o r
Mg(II) the system still underwent a reversible oneelectron oxidation with the site of oxidation being
the HOMO of the porphyrin (8). Thus a one-electron
oxidation generates the corresponding porphyrin p+.
cation radical (P ). When a redox active transition
metal is coordinated to a porphyrin both the metal and
the porphyrin macrocycle can under go redox chemistry.
Thus in the case of a Co(II) porphyrin the first oneelectron electrochemical oxidation causes the metal to
be oxidized with small perturbations in the optical
spectrum (9). However, a second one-electron
oxidation leaves the Co(III) unchanged and oxidizes
the porphyrin to the p-cation radical. The second
oxidation is associated with dramatic changes in the
optical spectrum (Fig. 2) and since theelectrochemical
oxidations were carried out in the
p r e s e n c e o f t e t r a - R-p r o p y 1 a m m o n i u m p e r c h l o r a t e t h e
oxidized
porphyrin
is
the
perchlorate salt
+. 2+
[CO(III)P ] 2C104 (1).
Similar oxidations can be performed using bromine as
the oxidant and first the metal and then the
porphyrin macrocycle undergo one-electron oxidations
.
to finally give the complex [Co(III)P+ ]2+2Br (2).
While dramatic changes occur in the optical spectra
during this chemical oxidation the spectra of the
final porphyrin p-cation radicals (1 and 2) differ
as a function of their counter anions. That the
spectra are indeed influenced by the counter ion is
readily demonstrated by treating 2 with AgClO4 which
converts it to 1. During this process the optical
s p e c t r u m o f 2 a l s o c h a n g e s t o t h a t o f 1( 9 ) . T h e
question must then be asked as to how the two p
cation radicals of the same metalloporphyrin can have
such different optical spectra?
T h e t w o h i g h e s t - f i l l e d p- m o l e c u l a r o r b i t a l s o f
porphyrins are essentially degenerate and have a1u
and a2u symmetries. Removal of a single electron
from either of these orbitals will generate a free
2
2
radical having either A1u, or A2u ground states.
The choice of which ground state is more stable in a
given situation depends upon subtle criteria such as
the nature of the fifth and sixth ligands coordinated
to the metalloporphyrin (10) as exemplified above
w i t h 1 a n d 2.
Of special interest was our observation that the
o p t i c a l s p e c t r a o f t h e p- c a t i o n r a d i c a l s 1 a n d 2
paralleled those of catalase and horseradish
peroxidase in their highest, enzymaticalIy active,
oxidation states known as compounds I. Compounds I of
both catalase (Cat I) and peroxidases (as exemplified
by horseradish peroxidase(HRP I)) are generated when
the resting ferric heme proteins undergo a twoelectron, hydrogen peroxide mediated, oxidation.
The congruence of the optical spectra of 1 and 2
with Cat I and HRP I (Fig. 2) allowed us to describe
Cat I and HRP I as porphyrin ir-cation radicals(9).
Such that the overall redox chemistry of both classes
of enzymes is as follows:
From their optical spectra Cat I clearly exhibits the
2
2
A1u and HRP I the A2u ground states. Since our
o r i g i n a l d e s c r i p t i o n o f t h e s e h i g h v a l e n t p- c a t i o n
radicals there has been much controversy as to the
accuracy of the descriptions of these electronic
states. Nevertheless, the evidence is overwhelmingly
i n f a v o r o f t h e p- c a t i o n r a d i c a l f o r m a t i o n ( 1 1 ) . I n
addition, the detection using ENDOR techniques by
H o f f m a n ' s g r o u p o f b o t h t h e p o r p h y r i n p- c a t i o n r a d i c a l
(12) and of an oxygen coordinated to the iron(lV) of
HRP I provides (13) conclusive proof for our original
suggestion. At the same time it allows for HRP I (and
by analogy Cat 1. for bovine catalase) to be described
.
as O=Fe(IV)P+ .
Since we know that catalase is coordinated by a
proximal phenoxide (14) and horseradish peroxidase by
a proximal imidazole ligand (15) the differences in
2
2
their two ground states ( A1u and A2u can beattributed
to this difference in proximal ligation.
We have reconstituted horseradish peroxidase
with a variety of synthetic hemes in order to
explore changes in the electronic character of
the compounds I of these reconstituted
proteins as a function of the reconstituing
heme (16). In the case of the deuteroheme (3)
reconstituted protein the highest oxidation
2
state takes on the A1u ground state of
catalase. However, since deuterohemin
horseradish peroxidase possesses no catalase activity (it behaves
like a peroxidase) the structure of the apoprotein is concluded to
play the major role in determining the reactivity of HRP I toward
hydrogen donors (16).
M e s o h e m i n (4) r e c o n s t i t u t e d h o r s e r a d i s h p e r o x i d a s e o n t h e o t h e r
hand exhibits an optical spectrum for its compound I which is half
2
2
way between that typical of the A1u and A2u ground states. Since
the reconstituted protein is homogeneous with respect towards
porphyrin binding (17) we conclude that the electronic ground state
is best described a s a quantum mechanically mixed ground state.
The symmetries of the a1u and a2u ground states allows
for such quantum mechanical mixing (18).
We now turn our attention to the cytochromes P-450. These enzymes
are widely distributed monooxygenases which activate molecular
oxygen (19).Two principal classes of oxidations are controlled by P450 namely the epoxidation of both aromatic and aliphatic double
bonds and the oxidation of unactivated C-H bonds to give the
corresponding
alcohol (C-OH). In both cases the other oxygen atom of the
dioxygen ends up as water.
The catalytic cycle for cytochrome P-450 is shown in figure 3.
When functioning normally the resting ferric hemoprotein binds
substrate (RH) which generally causes a low to high spin conversion
of the ferric iron. The high spin iron is then reduced, at about 0.0V
to ferrous P-450 which like ferrous hemoglobin and myoglobin binds
dioxygen. A further one-electron reduction of oxy P-450 occurs to
Figure 3. Catalytic cycle of cytochrome P-450
give an intermediate which is formally a ferric porphyrin
coordinated to hydrogen peroxide. This formalism has some reality
since the dioxygen and two one-electron reductions can be shunted
and the enzyme made to function by
using peroxidatic oxidants such as alkyl
hydrogenperoxides or peracids. No
enzymatic intermediates have so far been
observed beyond the"ferric-peroxide"
complex, but we shall consider below
some experimentally based speculations
on the nature of the further ntermediates.
But first let us look at what is known
about cytochrome P-450.
Cytochromes P-450 are widely distributed in nature and perform a
great variety of oxidations based upon the general schemes given
above. The majority of mammalian P-450's are membrane bound
w h i c h m a k e s s t u d y i n g t h e m d i f f i c u l t ( 2 0 ) . Fortunately a
microbial enzyme which metabolizes camphor has been isolated and
crystallized (21). The work that we shall describe below was carried
out on this particular enzyme (P-450cam) but the results can be
generalized to the whole class of these enzymes. Cytochrome P-450
is a protoheme-containing e n z y m e l i k e h e m o g l o b i n , m y o g l o b i n ,
b - t y p e cytochromes, peroxidase and catalase. However, unlike most
ferrous-heme proteins which form CO complexes that absorb at ~420
nm, P-450 forms a CO complex which absorbs at 450 nm in i t s
electronic spectrum (hence its name (22)). The major electronic
d i f f e r e n c e s t h a t a CO c o m p l e x o f a p r o t o h e m e containing
enzyme experience can result only from the nature of the proximal
ligand. Using model syst e m s ( F i g . 4 ) w e w e r e a b l e to m i m i c the
l o w energy Soret band at ~450 nm only when a mercaptide ligand
(RS~) was coordina t e d t o a f e r r o u s - C O complex (23) and further
studies confirm that the ferrous state of P-450 is indeed coordinated
b y a c y s t e i n y l a n i o n w h e n b o u n d t o CO. A s s h o w n i n f i g u r e 4 n o t
o n l y d i d a b a n d a t ~ 4 5 0 nm a p p e a r i n o u r m o d e l s y s t e m b u t a s e c o n d
h i g h e n e r g y b a n d a t ~ 3 6 0 n m w a s o b s e r v e d . Since our first o b s e r v a t i o n o f
this split-Soret band the phenomena has been observed in P-450
i t s e l f ( 2 4 ) . T h e s p l i t t i n g o f t h e n o r m a l p-p* transition o c c u r s d u e t o
a n i n t e r a c t i o n b e t w e e n t h e p-p* a n d a s u l f u r p - p * c h a r g e t r a n s f e r
(facilitated by the high charge density at sulfur) which gives rise to
two new transitions. Spectra which exhibit such split-Soret
bands are known as hyper spectra and are characteristic, inter alia,
of iron porphyrins coordinated by ligands of high charge density
(24).
At first sight oxy-P450 resembles oxymyolobin since both exhibit
Soret absorptions at ~418 nm. If oxy-P450 were coordinated by a
thiolate ligand one would anticipate that it should show a hyper
electronic spectrum. Yet its apparent similarity to
oxymyoglobin suggests otherwise. If oxy-P450 is not coordinated by
RS and, as we know, CO-P450 is coordinated by RS then the
conversion of oxy-P450 to its CO complex should invoke the loss of
one proton/mole. We have found that there is no net proton change
in such a process and conclude that like CO-P450 oxy-P450 is
indeed coordinated by a thiolate anion (25). This is confirmed by a
closer inspection of the electronic spectrum of oxy-P450 which does
indeed show a hyper spectrum (Fig. 5). It is merely coincidental
that the low energy partner of the split Soret of oxy-P450 occurs at
the same position as the Soret band of oxymyob1obin.
What happens when oxy-P450 is reduced by one electron? Shunting the enzyme by
peroxide-1ike oxidants suggests that peroxide may indeed be found bound to iron. Addition of an
electron to the ferric superoxide formulation (26) for oxyferrous porphyrins allow the
visualization of such a process.
We have explored this reaction using model systems and find that the one-electron reduction of
oxygenated ferrous octaethylporphyrin does indeed give the corresponding ferric peroxide
complex but that the peroxide is apparently bound sideways in a V –fashion (27) . Whether or
not these structural features prove to obtain for the enzyme must await further experimentation.
Whatever the structure of the "ferric-peroxide complex in the functioning enzyme at some stage
in the enzymatic process the 0-0 bond must break. Presently the evidence is strongly in favor of
0-0 cleavage before substrate oxidation. Such cleavage could be envisaged as double protonation
followed by loss of water, or as suggested by recent observations (28) an initial formation of
an intermediate peracid with subsequent loss of carboxylate to bring about 0-0 bond cleavage. In
either case the breaking of the 0-0 bond will generate a species which is formally a
ferric-oxene complex. The original observation of Ullrich (29) and the subsequent reactions of
ferric porphyrins with iodosyl benzene as detailed by Groves (30) adds support to the concept of
an iron complex bound to a single oxygen atom-containing species as the powerful oxidizing
agent of P-450. The role of the thiolate anion in breaking the 0-0 bond and more
importantly in stabilizing the Fe-O bond of an oxene complex has been discussed by Ullrich in
this monograph. A ferric oxene complex is but one of several extreme resonance structure (Fig.
6) that can be written for the active oxidizing agent of P-450.
A familiar electronic structure that can be written is the iron(IV) porphyrin f-cation radical that
we have seen earlier describes the compounds I of catalase and horseradish peroxidase. Groves
has recently isolated and characterized such an iron(IV) porphyrin ir-cation radical from the
reactions of a ferric porphyrin and iodosyl benzene (31). This appears to complete the circle and
to suggest that those hemeproteins which control and utilize high oxidation states of oxygen have
a common electronic configuration namely that of the iron(IV) porphyrin T-cation radical. That
each of these high valent oxidation states perform different functions must be attributed both to
the proximal coordination and active site environment provided by the protein.
Finally the mechanisms by which the active oxidizing agent oxidizes organic substrate is
considered. We shall limit the discussion here to the oxidation of unactivated C-H bonds since
this whole area is covered in greater depth in the next chapter by Loew. At first sight one might
imagine that possible similarities between an iron-oxene and carbenes or nitrenes would suggest
a direct insertion of the oxygen atom into the C-H bond to give the alcohol. Nevertheless, the
elegant work of Groves (32) has shown that at least in the oxidation of tetradeuteronorbornane a substrate intermediate is an obligatory intermediate.
If the breaking of a substrate C-H bond is a necessary step in the enzymatic reaction then
the lowest energy intermediate is likely to be a sub strate free radical" since the generation of
ionic intermediates* in what must be a strongly hydrophobic active site, would be energetically
unfavorable. Indeed, the isotope effects and regio- and stereoselectivity of P-450 oxidations all
argue for substrate radical intermediates. A further example of this is our own interpretation of
the microsomal metabolism of the insecticide Dieldrin ( 5 ) (33). Formation of the product ( 6 )
from the pentacyclic starting material requires the intervention of an intermediate and the
chemistry outlined in figure 7 is best accommodated by a radical intermediate.
Figure 7. !Proposed mechanism for the P-450 mediated
oxidation o f Dieldrin.
The formation and hydroxylation of such a substrate free radical is outlined in figure 8
where the abstraction of a hydrogen atom from substrate (R-H) by the active oxidizing agent of
P-450 would give the substrate radical and an iron(lV) porphyrin complexed by hydroxide.
Subsequent transfer of an iron bound hydroxy radical to the substrate radical could generate the
oxidized substrate and the resting enzyme (Fe(III)) ready for another catalytic cycle. While these
speculations on the details of the oxidative steps require more experimental support the
intermediacy of both an oxene and hydroxyl radical (each stabilized by coordination to an iron
porphyrin) would complete the picture for the oxygen chemistry outlined in figure 1 since all of
the possible oxygen intermediates shown there would have been utilized by nature and stabilized
in each case by the use of a heme protein.
Acknowledgment
The writing of this review and much of our work described here was supported by the United
States National Institutes of Health (AM 17989).
1. Babior, B.:
1 9 7 8 , N e w E n g l a n d J . M e d . 2.3. 8 . 7 2 1 .
2. Harrison, J.E. and Schultz, J.:
1 9 7 6 , 2. 5 .1 1 3 7 1 .
3. Malstroma B.G.:
1980 in "Metal Ion Activation
of Dioxygen" ed. Spiro, T.G.,
Wiley Interscience, New York, pp. 181-207.
4. Dolphin, D., ed. "The Porphyrins', 1979, Academic
Press, New York, Vol. VII.
5. Schonbaum1 G.R. And Chance, C. In "The Enzymes"
Ed. Boyer P.D.:
1976, Academic Press, New York,
Vol. XII, pp. 363-408.
6. Hewson,
W.D.
and
Hager,
L.P.:
in "The
Porphyrins" ed Dolphin, D. 1979, Academic
Press, New York, Vol. VII, pp.295-332.
7. Fajer, J., Borg, D.C., Forman, A., Dolphin, D.
and Felton.R.H.: 1 97 0, J. Am. Chem. Soc 92, 3451.
8. Dolphin, D., Muljiani, Z., Rousseau, K., Borg,
D.C.,
Fajer,J.
and Felton,
R.H.:1973, Annals
N.Y. Acad. Sci. 206, 177.
9. Dolphin, D., Forman, A., Borg, D.C., Fajer, J.
and Felton, R.H:
1971, Proc Natl. Acad. Sci.
U.S.A. 6,614.
10. Dolphin, D. and Felton, R.H.: 1974, Accounts
Chem. Res. I, 26.
11. Dolphin, D.:
1 9 8 1 , I s r a e l J . C h e m . 2 .1, 6 7 .
12. Roberts, J.E., Hoffman, B.M., Rutter, R. and
Hager,
L.P.:
1 9 8 1 , J . B i o l . C h e m . 25.6.. 2 1 1 8 .
13. Roberts, J.E., Hoffman, B.M., Rutter, R. and
Hager, L.P.: J.Am. Chem. Soc., in press.
14. Rossmann, M., Tanaka, N. and Reid, T. III.: this
voIume.
15. Yonetani, T., Yamamoto, H., Erman, J.E., Leigh,
J.S., Jr. and Reed, G.H.: 1972, J. Biol.
C h e m . 21, 2 4 4 7 .
16. DiNello, R.K. and Dolphin, D.: 1981, J. Biol.
C h e m . 2. 5 . 6 . , 6 9 0 3 .
17. La Mar, G.N., personal communication.
18. Hanson, L.K., personal communication.
19. Coon,
M.J. and White,
R.E.
in "Metal Ion
Activation of Dioxygen" ed. Spiro, T.G., 1980,
Wiley Interscience, New York, pp. 73.-123.
20. Griffin, B.W., Peterson, J.A. and Estabrook, R.W.
in "The Porphyrins" ed. Dolphin, D., 1979,
Academic Press, New York,
Vol. VII, pp. 333-375.
21. Wagner, G. and Gunsalus,I. C: this volume.
22. Omura, T. and Sato, R.:
1964, J. Biol. Chem.,
111, 2370.
23. Chang, C.K. and Dolphin, D.:
197 5, J. Am. Chem.
24.
Hansont L.K., Eaton, W.A., Sligar, S.G., Cunsaius, I.C., Gouterman,
M. and Connelt C.R.:
1 9 7 6 , J . Am. Chem. Soc, 2 6 7 2 . 2 5 . Dolphin, D., James, B.R. and Welborn, H.C.: 1
9 8 0 , J . Mol.Cat., 7 _ , 2 0 1 .
2 6 . Makinen, M.W. in "Biochemical and Clinical Aspects of Oxygen" ed.
Caughey, W.S., 1 9 7 4 , Academic Press, New York, p. 1 4 3 .
2 7 . Welborn, C.H., Dolphin, D. and James, B.R.: 1 9 8 1 , J. Am.Chem. Soc 1 D
3 , 2 869.
2 8 . Sligar, S.G., Kennedy, K. A. and Pearson, D.C.: 1 9 8 0 , Proc.Natl. Acad. Sci.
U.S.A., 7 7 , 1 2 4 0
2 9 . Lichtenberger, F., Nastainczyk, W. and UllrichtV.: 1 9 7 6 ,
Biochem. Biophys.
Res. Commun., I Q . , 9 3 9 .
3 0 . Groves, J.T. in "Metal Ion Activation of D i o x y g e n " e d . S p i r o ,
T.G., 1 9 8 0 , W i l e y Interscience, New York, pp. 1 2 5 - 1 6 2 .
3 1 . Groves, J.T., Haushatter, R.C., Nakamura, M., Nemo, T.E. and Evans, B.J.: 1 9 8 1 ,
J. Am. Chem. Soc., 1 0 3 , 2 8 8 4 .
3 2 . Groves, J.T., McClusky, G.A., White, R.E. and Coon, M.J.: 1 9 7 8 , B*i o c h e m.
Biophys. Res.Commun., 8 _ 1 . 1 5 4 .
3 3 . Dolphin, D., Addison, A.W., Cairns, M., DiNello, R.K., Farrell, N.P., James, B.R., Paulson,
D.R. and Welborn, C.: 1 9 7 9 , Int. J. Quantum Chem. ,XII,3 1 1 .