Plant Oxygenases, Peroxidases and Oxidases
hlontc4ano. 1’. It. (1000) Arch. Biochciii. Hiophys.
280,2 17-223 1.5. Marquez, I,.‘ Wariishi, H.,I)unford, 11. 1%. & Gold, hl. tl. (1988)J. 1 h l . Clieni. 263, 10540-10552 10. 1 I;irvey, 1’. J.. I’almer, J. M.. Schoemalter, I I. E.? Ikkker, I I. I,. Kr Wever, Ii. (lO80)13iochini. I%iophys. Acta 994, SO-03 17. Cai, 1). 8r Tien, M.(1001) J. 13iol. Cheni. 266, 1440-114460 18. Wariishi, 1 I., 1 luang, Dunford, 11. 1%.& Gold, M. 11. ( IW 1) J. Hiol. Chem. 266, 20004-20000 19. I Iarvey. 1’. J.. Schoeniakcr. I I. 1.: K: I’almcr. J. hl. (1087) I,es C o l l o g ~ ede~ 1,’INKA 40, 145-150 ’0. Wariishi. I I. & (;old, M. I I. (1080) FEBS I,c,tt. 243. 10s-I08 21. Wariishi, I I., Marquez, I,.. Ihnford. € 1. 1%. Kr Cold, M. tl. (1000)J. l h l . Chem. 265. 1 1 1.37-1 1 1-12 7 7 Ciii, I). Kr ‘rim, M. (1080) I3iocheni. 13iophys. K c x --. Con1Iiiun. 162. 464-469 ‘3. Wariishi, t 1. & (;old, hl. 11. (l(JO0)J. Hiol. Chem. 265, J.?
2070-2077 ’4. Stiinxida, hl.. Nakatsubo. F.%Kirk. T. K. Kr I liguchi, 7‘.( 1 0 X l ) Arch. Microbiol. 129. 321-324 2.5. Kawai, S., Ilmeza\va. T. & 1 liguchi. T. (108h)Wood lies. 73. 18-2 1 20. ‘I‘ien, M. & Kirk. T. K. (1084) I’roc. Natl. Acad. Sci. k1.S.A. 81, 2280-2284 27. I I;trvcy, 1’. J.. Schocmakcr, I I. 13. & I’almer, J. M.
(1086) FEHS I x t t . 195, 242-240 28. ‘I’ien, M., Kirk, T. K., l3ull. C. Kr I;ee. J. A. (1080) J. Hiol. Chem. 261, 1087-1603 29. Valli. K.. Wariishi, tl. & Cold, M. 11. (1000) I h chemistry 29, 835-839 30. tlaemmerli. S. I)., Schoeniaker, €1. E., Schmidt, k1, W. H. & 1,eisola. M. S.A. (1087) FERS I,ett. 220. 140-154 31. Schmidt, € 1. W. [I.. I laeninierli, S. I)., Schoemaker, €1, E. & I,eisola, M. S. A. (1080) Biochemistry 28, 1770- 1783 32. Palmer. J. M.. I larvey. I). J. & Schoeniakcr. €1. E. (1987) Phil. Trans K. Soc. Imnd. A 321, 405-505 33. Kerstcn. 1’. J., ‘I’ien, M.. Kalyanar;iman, 1%.Kr Kirk, T, K. (1985)J. Hiol. Chem. 260,2000-2612 .34. (;ilardi, G.? Harvey. I). J., Chss, A. E. G. Kr I’almer, J. M. ( 1000) Hiochim. Ihphys. Acta 104 1, 120- 132 35. Keference deleted 30. I’aszczynski, A. & Cra\vford. l i . I,. (1001) f%iochcm. Biophys. Kes. Conmiin. 178, 1050- 1 O h 3 37. Keference deleted 38. I,cisola, M. S. A., Haemnierli. S. I).. Waldner. K.. Schoeniakcr, 11. I;., Schmidt, 11. W. 11. Kr l;iechter, A. ( 1088) Cellulose Chcm. ’I’cctinol.22, 207-277 30. Schoemaker, H. E. (IOOO) Keel. ‘I’rav. Chini. I’aysHas 109,255-272
Received 23 Ilecember 1 0 0 1
A comparison of peroxidase and cytochrome P-450 Paul M. Wood Department of Biochemistry, University of Bristol, School of Medical Sciences, Bristol BS8 I TD, U.K.
Summary A peroxidase has 1 -electron oxidation as its characteristic activity, while that of cytochrome P-450 is hydroxylation. Both catalytic cycles involve similar high valency states of iron. However, a peroxidase can only accept electrons at the haem edge, while the SiibStrate for cytochrome 1’-450 is bound in a precise orientation before the active state is created.
Introduction A peroxidase is defined as an oxidoreductase acting with hydrogen peroxide as acceptor (EC I . 1 1). This discussion will be restricted to haem proteins such ;is horseradish peroxidase o r the ligninase of whiterot fungi, in their characteristic action as 1-electron oxidizing agents:
I I,0,
+ 2x + 21 1’
-
2IILO+ 2X.’ (Scheme 1 )
Cytochrome 1’-450 is the collective name for haem proteins that give a n Fe(I1)-CO complex with a yband near 450 nm, a property that correlates with a
cysteine anion (thiolate) as axial ligand to the haem 11 1. T h e cytochromes P 4 5 0 typically act as monooxygenases; the covalent bond of dioxygen is broken, one 0 - a t o m being incorporated into an organic substrate, while the other is reduced to water:
MI + OL+21 I + + 2 e -
-
KO11 + I I L O (Scheme 2)
T h e 0 - a t o m incorporation is often followed by an elimination or rearrangement [ 21; such secondary reactions are outside the scope of this discussion.
Redox states of oxygen T h e redox chemistry of oxygen underlies the catalytic cycles of these enzymes. Successive I -electron reductions of dioxygen yield superoxide, hydrogen peroxide, the hydroxyl radical and water, with 1-electron potentials (Eb) in volts as shown below
[S]: OL
01.
HLOi HZO+ HO. 2HL0 +0.89 +0.38 + 2.32
~
-0.33
I992
349
Biochemical Society Transactions
The 2-electron couples linking dioxygen, hydrogen peroxide and water have redox potentials that are the mean of the corresponding 1-electron values, since 2 nE must be the same for different pathways 350
131: I I@?
0 1
+0.28
21 I10
+ 1.35
For the 4-electron reduction, E~,(0,/214,0)= + 0.815 V, the mean of the 2-electron values. Two points to note are the low potential for 1-electron reduction of dioxygen to superoxide and the high value for hydrogen peroxide reduction to water. The 1-electron reduction of hydrogen peroxide has an unremarkable potential (E(,= +0.38 V), but generates a very potent oxidizing agent - the hydroxyl radical. With Fe(I1) as the reductant, this is the Fenton reaction. Ilepending on how the iron is complexed, the hydroxyl radical may remain associated in an iron-oxo complex, or be released in the free state 141:
Fez++11101 [Fe(IV)=O]” +H1O +
or
-
F e ” ++ H O . + H O
The Fe(IV) valence state is known as ferryl. The 0-atom in [Fe(IV)= O f + can be regarded as a water molecule deprotonated by the 4 + positive charge, as in permanganate or chromate. The catalytic cycles or peroxidases and cytochrome P-450 involve Fe(1V) (or higher) states of iron, as will be explained. The ferryl iron of the Fenton reaction can be almost as reactive as a free hydroxyl radical [ 51. Peroxidase and cytochrome P-450 have both succeeded in taming the reactivity of Fe(IV), so that the enzyme does not become inactivated, and the substrate is modified in a specific way.
The catalytic cycles In the peroxidase catalytic cycle, peroxide reduction to water is coupled to 2-electron oxidation of the enzyme [6].This generates a strong oxidant, Compound I, which is reduced in two 1-electron steps, with Compound I1 as intermediate. The electron distribution in Compound I will be discussed below. The cytochrome P-450 catalytic cycle is more complex. The standard scheme [ l , 71 is based partly on mammalian studies, but primarily on work with the soluble camphor-oxidizing cytochrome P-450 from Pseudomonas putida. The resting enzyme has haem in the Fe(II1) state. The
Volume 20
obligatory first step is binding of substrate. One electron is then supplied, from an electron transport chain with NADPH as reductant, and the haem is reduced to Fe(I1). The Fe(I1) state binds O,, as for myoglobin. A second electron is supplied. A shortlived active state is created, and products are formed [see scheme (2)]. With care, the two electrons and dioxygen can be replaced by a peroxide or another 0-atom donor such as iodosylbenzene:
K H + [O]
-
ROfI
(Scheme 3)
The term ‘peroxygenase’ is used for a monooxygenase acting with a peroxide as 0-atom donor. At first sight these catalytic cycles seem very different. I Iowever, consider chloroperoxidase from Caldariomyces fumago, discussed in 181. Chloroperoxidase is unusual among peroxidases in having a cysteine sulphur as axial ligand to the haem, making it formally a cytochrome P-450. Its name is based on chlorination reactions, not relevant to the present discussion. With hydrogen peroxide as oxidant, its activities include a conventional peroxidase catalytic cycle [ Scheme (l)]. I Iowever, in addition, chloroperoxidase is capable of peroxygenase reactions [Scheme ( 3 ) ] ,such as epoxidation of alkenes.
Explanation for differing activities The lack of specificity of chloroperoxidase raises two questions: what stops most peroxidases from catalysing oxotransfer reactions, like a monooxygenase? And, what stops a typical cytochrome P-450 from catalysing 1-electron oxidations. like a peroxidase? An answer to the first question is provided by the manner of horseradish peroxidase inactivation by mechanism-based inhibitors such as alkylhydrazines 19, 101. It was found that alkylation had a high specificity for the 8-meso position on the haem. N-alkylation of the haem (common for cytochrome P-450) was not observed, even with agents as small as methylhydrazine. Such studies (discussed in [8])indicate that the haem edge is the site of electron transfer. The hydrogen peroxide has access to the iron, but steric factors prevent substrates for oxidation from getting close enough for oxotransfer to occur [XI. The 0-atom of the oxohaem state is converted into water, with His-42 of horseradish peroxidase supplying the first proton [ h I. What stops cytochrome P-450 from acting as a peroxidase? T h e crucial difference is that the organic substrate for cytochrome P-450 must bind
Plant Oxygenases, Peroxidases and Oxidases
first. When the reactive state is formed, the organic substrate is already in place. In the resting state the iron of cytochrome P-450 has two axial ligands, the cysteine sulphur and an oxygen donor, which may be water [I]. Physical studies indicate that the organic substrate displaces the oxygen donor [ 1, 1 I]. The substrate does not bind directly to the iron, but rather promotes formation of a state with only the cysteine as axial ligand. This causes a change from low to high spin, as in other haem proteins [ 121. So why does reduction of Fe(II1) only occur after substrate has bound? For some cytochromes P-450 this is easily explained - in the resting state the redox potential of the haem is far lower than that of its electron donor, but once substrate has bound the two potentials become similar. Thus for the camphor-hydroxylating cytochrome P-450 of Ps. putzdu the redox potentials for haem, haem-plussubstrate and donor were -0.340, -0.170 and - 0.106 V respectively [ 131, while for cytochrome 1'-450 from bovine adrenal mitochondria the corresponding values were - 0.4 12, - 0.305 and - 0.290 V [ 141. The change in potential correlates with the stronger binding of water to Fe(II1) than to Fe(l1). I Iowever. for many cytochromes P-450, substrate binding makes little difference to the redox potential. For example, Guengerich [ 151 tested eight rat liver cytochromes P-450 and found the increase in redox potential on substrate binding never exceeded 33 mV, and in many cases was too small to measure. Two reasons have been proposed for reduction only occurring after substrate is bound: (i) the reaction may be spin-forbidden when the haem is in a low-spin state 111, or (ii) substrate binding may cause conformational changes that promote binding of the electron donor [ 111. For some cytochromes P-450 there are further complications, such as the haem being not totally in a high-spin state in the presence of substrate, and burst kinetics for electron transfer [ 11. Hefore hydroxylation takes place, the haem of cytochrome P-450 must receive a second electron. The potential for the second 1-electron reduction is more difficult to measure, but has been estimated as + 0.05 V [ I S ] . A higher value than for the first 1 -electron reduction explains why cytochrome b; in some reductases, El,near +0.02 V, supplies only the second electron [2, 151. In the peroxygenase activity of cytochrome P-450, the two 1-electron reductions and O2 are replaced by a peroxide or other 0-atom donor. Most papers that discuss these reactions say little about the order of binding. An ordered sequence, with the substrate binding first, was
reported in [16J. There is no obvious reason why the peroxide should not bind before the substrate for hydroxylation, but if it does, there is a strong risk of the active state becoming suicidal. Indeed, there are reports of the haem being rapidly destroyed [ 21.
The active state In peroxidases there is agreement that the iron of Compound I is promoted to Fe(IV), as opposed to Fe(V), while the second electron is taken from elsewhere [6]. For horseradish peroxidase there are many lines of evidence for a porphyrin cation radical [6], although a recent study finds delocalization that includes the proximal histidine [ 171. Cytochrome c peroxidase (yeast) has recently been shown to have the radical located on a tryptophan, Trp-191 [lX]. For cytochrome P-450, the replacement of NADPH by HLOLor other 0-atom donors often has little effect on the product distribution. This implies that cleavage of the 0-0 bond of 0 , forms an intermediate with a single 0-atom, similar to Compound I. The electron distribution is still unresolved, although alternatives have been proposed [ 11. This intermediate is commonly written (FeO)'+, as for Fe(V). In the normal catalytic cycle its lifetime is very short. The mechanism of 0-atom insertion into the substrate has been probed with strained cycloalkanes, in which the radical state undergoes rearrangement at a knoun rate (radical clocks). Such studies indicate a radical intermediate that collapses with a lifetime of 1 0 - y - l O ~ " s IlO]. IJydrogen-atom abstraction from the substrate generates a transient free-radical, and oxygen rebound creates the hydroxylated product: (FeO)'+ + C-H
+
(Fe01 l)'+
+ C.
+
Fe'+ +C-011 The multiplicity of pathways for oxidation of a nbonded system are discussed in [7].
Comparison of oxidizing activity For both enzymes the 2-electron reduction of I IIOL can serve as the activating reaction, E;,(H201/ 2II,O)= + 1.35 V. This standard value refers to 1 \I-1 120Z,whereas the concentration in practice will be lower. The potential is also pH dependent; a common assay for ligninase uses a pH of 3, at which E ( H L 0 2 / 2 H L 0will ) be 0.24 V higher than at pH 7. For horseradish peroxidase, 1-electron redox potentials Em,(, (Compound I/Compound 11)=
I992
35 I
Biochemical Society Transactions
352
+ 0.04 V and B,,,,, (Compound WFerric) = + 0.96 V have been measured by titration with iridium complexes [20]. These relative values are surprising, since the first I-electron reduction would be expected to have the higher potential. In yeast cytochrome c peroxidase the potential of the tryptophan radical may be modified by the protein. However, the potential has recently been measured in solution as E:,(Trp.+/Trp)= + 1.05 V [21]. One or more tyrosines in cytochrome c peroxidase may be important in electron transfer [ 1XI, EI,(Tyr.+/Tyr) being + 0.94 V in solution [21]. Most aliphatic compounds cannot be oxidized by a peroxidase, because their redox potentials for 1-electron oxidation are too high. For some peroxidases the electron is taken from a transition metal, as in cytochrome c peroxidase or the manganese peroxidase of white rot fungi. For aromatic compounds (which provide the substrate for inany peroxidases), the redox potential for 1-electron oxidation will be below + 1.0 V if there are electron donating substituents; compare + 0.95 V for phenol at pH 7 with + 0.73 V for p-methoxyphenol [ 221. For cytochrome 1’-450 the instability of the active state prevents direct measurement of redox potential. A study of rates of oxidation of substituted N,N-din~ethylanilines led to an estimated apparent potential of 1.0-2.2 V (quoted as 1.7-2.0 V versus saturated calomel) 12.11. The initial (I;e = O)’+state is electrically neutral, because the haem has a protoporphyrin dianion and cysteine anions as ligands. The rate-limiting step was considered to be transfer of an electron from the substrate, creating an ion pair: [(f;eO)’+.P-.S-]+ R’N
+
[ ( F e O ) ’ + . P - . SI + R , N . + The apparent potential u a s interpreted as an intrinsic potential for the (I;e = O);+ state and a coulombic fiactor caused by charge separation. The coulombic fictor would be inversely related to the local dielectric constant, and was proposed to be about 1 V. Thus the intrinsic potential may be similar to that for Compound VCompound If of horseradish peroxidase, but the coulombic factor creates an effective potential almost as high as for 1 -electron reduction of the hydroxyl radical. Whether a radical cation is always an intermediate is still debatable, but it is evident that a cytochrome
Volume 20
P-450 is capable of hydroxylating almost any organic molecule, provided the protein provides an appropriate active site. I . Llawson, J. H. 8r Eble, K. S. (10x0) Adv. Inorg. Hioinorg. Mech. 4, 1-64 2. Ortiz de Montellano, 1’. K. (1086) Cytochromcs f’450: Structure, Mechanism and Hiochemistry, Plenum, New York
3. Wood, 1’. M. (1088) Hiocheni. J. 253,287-289 4. Halliwell, H. 8r Gutteridge, J. M. C. (IOXO) I;rcc Radicals in Hiology and Medicine. 2nd cbdn., Clarendon, Oxford .5. Sutton, H. C. & Winterbourn, C. C. (10x0)1:rce k i d . Hiol. Med. 6, 53-60 0. Dunford, H. H. (1001) in I’eroxidases in Cheinistry and Biology (Everse, J., Everse, K. 17. 8r (;rishain, M. H., eds.). vol. 2. pp. 1-137. CHC I’rcss. 1joc;i Katon. L I S A .
7. Guengerich, F.1’. 8r Macdonald, 7’. I ,. ( 1000) I;ASI
hlontc4ano. 1’. It. (1000) Arch. Biochciii. Hiophys.
280,2 17-223 1.5. Marquez, I,.‘ Wariishi, H.,I)unford, 11. 1%. & Gold, hl. tl. (1988)J. 1 h l . Clieni. 263, 10540-10552 10. 1 I;irvey, 1’. J.. I’almer, J. M.. Schoemalter, I I. E.? Ikkker, I I. I,. Kr Wever, Ii. (lO80)13iochini. I%iophys. Acta 994, SO-03 17. Cai, 1). 8r Tien, M.(1001) J. 13iol. Cheni. 266, 1440-114460 18. Wariishi, 1 I., 1 luang, Dunford, 11. 1%.& Gold, M. 11. ( IW 1) J. Hiol. Chem. 266, 20004-20000 19. I Iarvey. 1’. J.. Schoeniakcr. I I. 1.: K: I’almcr. J. hl. (1087) I,es C o l l o g ~ ede~ 1,’INKA 40, 145-150 ’0. Wariishi. I I. & (;old, M. I I. (1080) FEBS I,c,tt. 243. 10s-I08 21. Wariishi, I I., Marquez, I,.. Ihnford. € 1. 1%. Kr Cold, M. tl. (1000)J. l h l . Chem. 265. 1 1 1.37-1 1 1-12 7 7 Ciii, I). Kr ‘rim, M. (1080) I3iocheni. 13iophys. K c x --. Con1Iiiun. 162. 464-469 ‘3. Wariishi, t 1. & (;old, hl. 11. (l(JO0)J. Hiol. Chem. 265, J.?
2070-2077 ’4. Stiinxida, hl.. Nakatsubo. F.%Kirk. T. K. Kr I liguchi, 7‘.( 1 0 X l ) Arch. Microbiol. 129. 321-324 2.5. Kawai, S., Ilmeza\va. T. & 1 liguchi. T. (108h)Wood lies. 73. 18-2 1 20. ‘I‘ien, M. & Kirk. T. K. (1084) I’roc. Natl. Acad. Sci. k1.S.A. 81, 2280-2284 27. I I;trvcy, 1’. J.. Schocmakcr, I I. 13. & I’almer, J. M.
(1086) FEHS I x t t . 195, 242-240 28. ‘I’ien, M., Kirk, T. K., l3ull. C. Kr I;ee. J. A. (1080) J. Hiol. Chem. 261, 1087-1603 29. Valli. K.. Wariishi, tl. & Cold, M. 11. (1000) I h chemistry 29, 835-839 30. tlaemmerli. S. I)., Schoeniaker, €1. E., Schmidt, k1, W. H. & 1,eisola. M. S.A. (1087) FERS I,ett. 220. 140-154 31. Schmidt, € 1. W. [I.. I laeninierli, S. I)., Schoemaker, €1, E. & I,eisola, M. S. A. (1080) Biochemistry 28, 1770- 1783 32. Palmer. J. M.. I larvey. I). J. & Schoeniakcr. €1. E. (1987) Phil. Trans K. Soc. Imnd. A 321, 405-505 33. Kerstcn. 1’. J., ‘I’ien, M.. Kalyanar;iman, 1%.Kr Kirk, T, K. (1985)J. Hiol. Chem. 260,2000-2612 .34. (;ilardi, G.? Harvey. I). J., Chss, A. E. G. Kr I’almer, J. M. ( 1000) Hiochim. Ihphys. Acta 104 1, 120- 132 35. Keference deleted 30. I’aszczynski, A. & Cra\vford. l i . I,. (1001) f%iochcm. Biophys. Kes. Conmiin. 178, 1050- 1 O h 3 37. Keference deleted 38. I,cisola, M. S. A., Haemnierli. S. I).. Waldner. K.. Schoeniakcr, 11. I;., Schmidt, 11. W. 11. Kr l;iechter, A. ( 1088) Cellulose Chcm. ’I’cctinol.22, 207-277 30. Schoemaker, H. E. (IOOO) Keel. ‘I’rav. Chini. I’aysHas 109,255-272
Received 23 Ilecember 1 0 0 1
A comparison of peroxidase and cytochrome P-450 Paul M. Wood Department of Biochemistry, University of Bristol, School of Medical Sciences, Bristol BS8 I TD, U.K.
Summary A peroxidase has 1 -electron oxidation as its characteristic activity, while that of cytochrome P-450 is hydroxylation. Both catalytic cycles involve similar high valency states of iron. However, a peroxidase can only accept electrons at the haem edge, while the SiibStrate for cytochrome 1’-450 is bound in a precise orientation before the active state is created.
Introduction A peroxidase is defined as an oxidoreductase acting with hydrogen peroxide as acceptor (EC I . 1 1). This discussion will be restricted to haem proteins such ;is horseradish peroxidase o r the ligninase of whiterot fungi, in their characteristic action as 1-electron oxidizing agents:
I I,0,
+ 2x + 21 1’
-
2IILO+ 2X.’ (Scheme 1 )
Cytochrome 1’-450 is the collective name for haem proteins that give a n Fe(I1)-CO complex with a yband near 450 nm, a property that correlates with a
cysteine anion (thiolate) as axial ligand to the haem 11 1. T h e cytochromes P 4 5 0 typically act as monooxygenases; the covalent bond of dioxygen is broken, one 0 - a t o m being incorporated into an organic substrate, while the other is reduced to water:
MI + OL+21 I + + 2 e -
-
KO11 + I I L O (Scheme 2)
T h e 0 - a t o m incorporation is often followed by an elimination or rearrangement [ 21; such secondary reactions are outside the scope of this discussion.
Redox states of oxygen T h e redox chemistry of oxygen underlies the catalytic cycles of these enzymes. Successive I -electron reductions of dioxygen yield superoxide, hydrogen peroxide, the hydroxyl radical and water, with 1-electron potentials (Eb) in volts as shown below
[S]: OL
01.
HLOi HZO+ HO. 2HL0 +0.89 +0.38 + 2.32
~
-0.33
I992
349
Biochemical Society Transactions
The 2-electron couples linking dioxygen, hydrogen peroxide and water have redox potentials that are the mean of the corresponding 1-electron values, since 2 nE must be the same for different pathways 350
131: I I@?
0 1
+0.28
21 I10
+ 1.35
For the 4-electron reduction, E~,(0,/214,0)= + 0.815 V, the mean of the 2-electron values. Two points to note are the low potential for 1-electron reduction of dioxygen to superoxide and the high value for hydrogen peroxide reduction to water. The 1-electron reduction of hydrogen peroxide has an unremarkable potential (E(,= +0.38 V), but generates a very potent oxidizing agent - the hydroxyl radical. With Fe(I1) as the reductant, this is the Fenton reaction. Ilepending on how the iron is complexed, the hydroxyl radical may remain associated in an iron-oxo complex, or be released in the free state 141:
Fez++11101 [Fe(IV)=O]” +H1O +
or
-
F e ” ++ H O . + H O
The Fe(IV) valence state is known as ferryl. The 0-atom in [Fe(IV)= O f + can be regarded as a water molecule deprotonated by the 4 + positive charge, as in permanganate or chromate. The catalytic cycles or peroxidases and cytochrome P-450 involve Fe(1V) (or higher) states of iron, as will be explained. The ferryl iron of the Fenton reaction can be almost as reactive as a free hydroxyl radical [ 51. Peroxidase and cytochrome P-450 have both succeeded in taming the reactivity of Fe(IV), so that the enzyme does not become inactivated, and the substrate is modified in a specific way.
The catalytic cycles In the peroxidase catalytic cycle, peroxide reduction to water is coupled to 2-electron oxidation of the enzyme [6].This generates a strong oxidant, Compound I, which is reduced in two 1-electron steps, with Compound I1 as intermediate. The electron distribution in Compound I will be discussed below. The cytochrome P-450 catalytic cycle is more complex. The standard scheme [ l , 71 is based partly on mammalian studies, but primarily on work with the soluble camphor-oxidizing cytochrome P-450 from Pseudomonas putida. The resting enzyme has haem in the Fe(II1) state. The
Volume 20
obligatory first step is binding of substrate. One electron is then supplied, from an electron transport chain with NADPH as reductant, and the haem is reduced to Fe(I1). The Fe(I1) state binds O,, as for myoglobin. A second electron is supplied. A shortlived active state is created, and products are formed [see scheme (2)]. With care, the two electrons and dioxygen can be replaced by a peroxide or another 0-atom donor such as iodosylbenzene:
K H + [O]
-
ROfI
(Scheme 3)
The term ‘peroxygenase’ is used for a monooxygenase acting with a peroxide as 0-atom donor. At first sight these catalytic cycles seem very different. I Iowever, consider chloroperoxidase from Caldariomyces fumago, discussed in 181. Chloroperoxidase is unusual among peroxidases in having a cysteine sulphur as axial ligand to the haem, making it formally a cytochrome P-450. Its name is based on chlorination reactions, not relevant to the present discussion. With hydrogen peroxide as oxidant, its activities include a conventional peroxidase catalytic cycle [ Scheme (l)]. I Iowever, in addition, chloroperoxidase is capable of peroxygenase reactions [Scheme ( 3 ) ] ,such as epoxidation of alkenes.
Explanation for differing activities The lack of specificity of chloroperoxidase raises two questions: what stops most peroxidases from catalysing oxotransfer reactions, like a monooxygenase? And, what stops a typical cytochrome P-450 from catalysing 1-electron oxidations. like a peroxidase? An answer to the first question is provided by the manner of horseradish peroxidase inactivation by mechanism-based inhibitors such as alkylhydrazines 19, 101. It was found that alkylation had a high specificity for the 8-meso position on the haem. N-alkylation of the haem (common for cytochrome P-450) was not observed, even with agents as small as methylhydrazine. Such studies (discussed in [8])indicate that the haem edge is the site of electron transfer. The hydrogen peroxide has access to the iron, but steric factors prevent substrates for oxidation from getting close enough for oxotransfer to occur [XI. The 0-atom of the oxohaem state is converted into water, with His-42 of horseradish peroxidase supplying the first proton [ h I. What stops cytochrome P-450 from acting as a peroxidase? T h e crucial difference is that the organic substrate for cytochrome P-450 must bind
Plant Oxygenases, Peroxidases and Oxidases
first. When the reactive state is formed, the organic substrate is already in place. In the resting state the iron of cytochrome P-450 has two axial ligands, the cysteine sulphur and an oxygen donor, which may be water [I]. Physical studies indicate that the organic substrate displaces the oxygen donor [ 1, 1 I]. The substrate does not bind directly to the iron, but rather promotes formation of a state with only the cysteine as axial ligand. This causes a change from low to high spin, as in other haem proteins [ 121. So why does reduction of Fe(II1) only occur after substrate has bound? For some cytochromes P-450 this is easily explained - in the resting state the redox potential of the haem is far lower than that of its electron donor, but once substrate has bound the two potentials become similar. Thus for the camphor-hydroxylating cytochrome P-450 of Ps. putzdu the redox potentials for haem, haem-plussubstrate and donor were -0.340, -0.170 and - 0.106 V respectively [ 131, while for cytochrome 1'-450 from bovine adrenal mitochondria the corresponding values were - 0.4 12, - 0.305 and - 0.290 V [ 141. The change in potential correlates with the stronger binding of water to Fe(II1) than to Fe(l1). I Iowever. for many cytochromes P-450, substrate binding makes little difference to the redox potential. For example, Guengerich [ 151 tested eight rat liver cytochromes P-450 and found the increase in redox potential on substrate binding never exceeded 33 mV, and in many cases was too small to measure. Two reasons have been proposed for reduction only occurring after substrate is bound: (i) the reaction may be spin-forbidden when the haem is in a low-spin state 111, or (ii) substrate binding may cause conformational changes that promote binding of the electron donor [ 111. For some cytochromes P-450 there are further complications, such as the haem being not totally in a high-spin state in the presence of substrate, and burst kinetics for electron transfer [ 11. Hefore hydroxylation takes place, the haem of cytochrome P-450 must receive a second electron. The potential for the second 1-electron reduction is more difficult to measure, but has been estimated as + 0.05 V [ I S ] . A higher value than for the first 1 -electron reduction explains why cytochrome b; in some reductases, El,near +0.02 V, supplies only the second electron [2, 151. In the peroxygenase activity of cytochrome P-450, the two 1-electron reductions and O2 are replaced by a peroxide or other 0-atom donor. Most papers that discuss these reactions say little about the order of binding. An ordered sequence, with the substrate binding first, was
reported in [16J. There is no obvious reason why the peroxide should not bind before the substrate for hydroxylation, but if it does, there is a strong risk of the active state becoming suicidal. Indeed, there are reports of the haem being rapidly destroyed [ 21.
The active state In peroxidases there is agreement that the iron of Compound I is promoted to Fe(IV), as opposed to Fe(V), while the second electron is taken from elsewhere [6]. For horseradish peroxidase there are many lines of evidence for a porphyrin cation radical [6], although a recent study finds delocalization that includes the proximal histidine [ 171. Cytochrome c peroxidase (yeast) has recently been shown to have the radical located on a tryptophan, Trp-191 [lX]. For cytochrome P-450, the replacement of NADPH by HLOLor other 0-atom donors often has little effect on the product distribution. This implies that cleavage of the 0-0 bond of 0 , forms an intermediate with a single 0-atom, similar to Compound I. The electron distribution is still unresolved, although alternatives have been proposed [ 11. This intermediate is commonly written (FeO)'+, as for Fe(V). In the normal catalytic cycle its lifetime is very short. The mechanism of 0-atom insertion into the substrate has been probed with strained cycloalkanes, in which the radical state undergoes rearrangement at a knoun rate (radical clocks). Such studies indicate a radical intermediate that collapses with a lifetime of 1 0 - y - l O ~ " s IlO]. IJydrogen-atom abstraction from the substrate generates a transient free-radical, and oxygen rebound creates the hydroxylated product: (FeO)'+ + C-H
+
(Fe01 l)'+
+ C.
+
Fe'+ +C-011 The multiplicity of pathways for oxidation of a nbonded system are discussed in [7].
Comparison of oxidizing activity For both enzymes the 2-electron reduction of I IIOL can serve as the activating reaction, E;,(H201/ 2II,O)= + 1.35 V. This standard value refers to 1 \I-1 120Z,whereas the concentration in practice will be lower. The potential is also pH dependent; a common assay for ligninase uses a pH of 3, at which E ( H L 0 2 / 2 H L 0will ) be 0.24 V higher than at pH 7. For horseradish peroxidase, 1-electron redox potentials Em,(, (Compound I/Compound 11)=
I992
35 I
Biochemical Society Transactions
352
+ 0.04 V and B,,,,, (Compound WFerric) = + 0.96 V have been measured by titration with iridium complexes [20]. These relative values are surprising, since the first I-electron reduction would be expected to have the higher potential. In yeast cytochrome c peroxidase the potential of the tryptophan radical may be modified by the protein. However, the potential has recently been measured in solution as E:,(Trp.+/Trp)= + 1.05 V [21]. One or more tyrosines in cytochrome c peroxidase may be important in electron transfer [ 1XI, EI,(Tyr.+/Tyr) being + 0.94 V in solution [21]. Most aliphatic compounds cannot be oxidized by a peroxidase, because their redox potentials for 1-electron oxidation are too high. For some peroxidases the electron is taken from a transition metal, as in cytochrome c peroxidase or the manganese peroxidase of white rot fungi. For aromatic compounds (which provide the substrate for inany peroxidases), the redox potential for 1-electron oxidation will be below + 1.0 V if there are electron donating substituents; compare + 0.95 V for phenol at pH 7 with + 0.73 V for p-methoxyphenol [ 221. For cytochrome 1’-450 the instability of the active state prevents direct measurement of redox potential. A study of rates of oxidation of substituted N,N-din~ethylanilines led to an estimated apparent potential of 1.0-2.2 V (quoted as 1.7-2.0 V versus saturated calomel) 12.11. The initial (I;e = O)’+state is electrically neutral, because the haem has a protoporphyrin dianion and cysteine anions as ligands. The rate-limiting step was considered to be transfer of an electron from the substrate, creating an ion pair: [(f;eO)’+.P-.S-]+ R’N
+
[ ( F e O ) ’ + . P - . SI + R , N . + The apparent potential u a s interpreted as an intrinsic potential for the (I;e = O);+ state and a coulombic fiactor caused by charge separation. The coulombic fictor would be inversely related to the local dielectric constant, and was proposed to be about 1 V. Thus the intrinsic potential may be similar to that for Compound VCompound If of horseradish peroxidase, but the coulombic factor creates an effective potential almost as high as for 1 -electron reduction of the hydroxyl radical. Whether a radical cation is always an intermediate is still debatable, but it is evident that a cytochrome
Volume 20
P-450 is capable of hydroxylating almost any organic molecule, provided the protein provides an appropriate active site. I . Llawson, J. H. 8r Eble, K. S. (10x0) Adv. Inorg. Hioinorg. Mech. 4, 1-64 2. Ortiz de Montellano, 1’. K. (1086) Cytochromcs f’450: Structure, Mechanism and Hiochemistry, Plenum, New York
3. Wood, 1’. M. (1088) Hiocheni. J. 253,287-289 4. Halliwell, H. 8r Gutteridge, J. M. C. (IOXO) I;rcc Radicals in Hiology and Medicine. 2nd cbdn., Clarendon, Oxford .5. Sutton, H. C. & Winterbourn, C. C. (10x0)1:rce k i d . Hiol. Med. 6, 53-60 0. Dunford, H. H. (1001) in I’eroxidases in Cheinistry and Biology (Everse, J., Everse, K. 17. 8r (;rishain, M. H., eds.). vol. 2. pp. 1-137. CHC I’rcss. 1joc;i Katon. L I S A .
7. Guengerich, F.1’. 8r Macdonald, 7’. I ,. ( 1000) I;ASI
