J. Biol. Chem.-1980-Poulos-8199-205

8/12/2019 J. Biol. Chem.-1980-Poulos-8199-205

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T H E O U R N A L F BIOLOGICALH EMIS TRYVal 255 No. 7. Insue o September IO pp. 8199-8205, 19W1nntrd tn 1 S A

The Stereochemistry of Peroxidase Catalysis*(Received for publication, Janua ry 14, 1980, and in revised form, May 2, 1980)

Thomas L. Poulos and Joseph KrautFrom the Department of Ch em isto , University of California, San D iego, L a Jolla, California 92093

A stereochemical mechanism is proposed for the per-oxidase-cata lyzed heterolytic cleavage of the RO-OHbond. It is based on the 2.5 A struc ture of cytochromec peroxidase, model building experiments, and a n ex-tensive body of literature on peroxidase biochemistry.The essential featuresof this mechanism are acid-basecatalysis by an invariant distal histidine (His-52 incytochrome c peroxidase), charge stabilization by aninvariant arginine residue (Arg-48), and stabilizationof higher oxidation states of the heme iron atom bymeans of interaction between an invariant proximalhistidine (His-174) and a nearbyburied glutamine-glu-tamate pair (Gln-239, Glu-187).The indole ring of Trp-01 on the distal side of theheme ring i s probably the site of radical formation incompound I of cytochrome c peroxidase.The essential role of the catalytic arginine in theproposed mechanism offers an explanation for whyoxygen-binding globins are poor peroxidases. Quitesimply, the globins possess no homolog to the peroxi-dase catalytic arginine and thus re unable to promoteheterolysis of peroxides by stabilizing a developingnegative charge on the leaving group of the substratein the transition state.Struc tural differences in the vicinity of the proximalhistidine also explain why the midpoint potential ofcytochromec peroxidase - 194mV) is much lower thanthat of myoglobin (+50 mV). In myoglobin the proximalhistidine is hydrogen bonded to a backbone carbonyloxygen atom, while in cytochrome c peroxidase theproximal histidine contactsand probably hydrogenbonds with the side chain of Gln-239, which in turninteracts with the buried carboxylate group of Glu-187.This latter set of interactions in cytochrome c peroxi-dase may impart sufficient anionic chara cter to theproximal histidine to stabilize higher oxidation statesof the heme iron during the eroxidase cataly tic cycle.

Peroxidases are widely distri buted throughout the biologi-cal world. They are found in many plants, in some animaltissues, and n microorganisms (Saunders et al., 1964) andtheycarryout a variety of biosyntheticanddegradativefunctions calling for the use of peroxides, especially H202,asan oxidant. In so doing the peroxidases also protect the cellagainstaccumulation of these dangerously reactive com-pounds. Indeed he enzyme catalase, he sole function ofwhich appears to be just that, the destructionf excess H202,is also considered to be a specialized peroxidase which uses

* This work was supported by Grant PCM 77-08554 from theNational Science Foundation and Grants RR-00757 and GM 10928from the National Insti tutes of Health. T he costs of publication ofthis article were defrayed in part by the payment of page charges.This article must therefore be hereby marked aduertisement inaccordance with 18U.S.C. Section 1734 solely to indicate this fact.

H202 s both oxidant and reductant and thereby converts itinto H20 and 02Some recent reviews of peroxidase biochemistry are t o befound in the following references: Williams et al. 1977; Mor-rison and Schonbaum, 1976; Yonetani, 1976; Schonbaum andChance, 1976.The overall net reactioncatalyzed by many peroxidasesmay be written most simply as

ROOH + 2D + 2H -+ ROH + Hz0 + 2D+where ROOH is e ither hydrogen peroxide or an organic hy-droperoxide and D the electron donor. The oxidized speciesD is often a highly reactive free radicalwhich can enter intoa variety of further nonenzymic reactions, including reactionwith itself. Additionally, certain two-electron donors can alsobe utilized, and the overall peroxidase reaction is sometimeswritten

ROOH + HzD - ROH 20The prosthetic group in the well studied plant and yeast

peroxidases is noncovalently bound heme. In this respect theyresemble the globins, the b type cytochromes, and the P-450cytochromes. Moreover, as in t he globins, the fifth coordina-tion position of the heme iron in the peroxidases is occupiedby a proximal histidine side chain and the sixth coordinationposition by an aquoigand. In contras t to thelobins, however,E,, for th e peroxidases is about -200 mV instead of +50 mV,and correspondingly the normaloxidation state of peroxidaseis Fe(1II) instead of Fe(I1). In addit ion, the amino acid sidechains forming the ligand binding pocket on the distalide ofthe heme aredifferent in cytochrome c peroxidase and myo-globin. Although both prote ins conta in a similarly situateddistal histidine, in myoglobin a valine and a phenylalaninecontact the distal surfacef the hemewhile the correspondingresidues in cytochrome c peroxidase are tryptophan and ar-ginine (Poulos et al., 1980). A very fundamental question tobe addressed by structural investigations, then, is how suchdifferences in the surrounding prote in can confer peroxidaseactivity in one case and reversible oxygen binding capabilityin the other.As a contribution towardanswering this question, the mainpurpose of the present communication is to describe thosefeatures of the cytochrome c peroxidase structure tha t webelieve are responsible or itscharacteristic eaction withperoxides, and to propose a detailed stereochemical mecha-nism based on that structure.The peroxidase reaction is a two-electron oxidation-reduc-tion, but the reaction ormally occurs (excluding catalase) inthree distinct steps:

P + ROOH P 1) + ROH + H20P 1) + D + H P(II) + D

P(I1)+ D + H P + D8199


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82 Peroxidase Catalytic MechanismIn this ormalism P represents the nativeeroxidase molecule,P( I) is the so-called compound , which is wo oxidationequivalents above the native enzyme, and P(I1) s compound11,which is one oxidation equivalent above the native nzyme.Th e electron donor substrate D may in general be a widevariety of organic and inorganic compounds,but in the specialcase of cytochrome c peroxidase with which we shall beconcerned th e enzyme exhibitsa high degree of specificity forthe reduced cytochrome c molecule.In his paper we shall consideronly the stereochemicalbasis for catalysis of the first of the foregoing reaction steps,the formation of compound I. Clearly, however, now th at th ecrystal struc ture of cytochrome c peroxidase is known, weshould soon be in a position to il luminate the long standingquestion of how an elect ron s transferred between hemes, theprocess represented by the second and third of these reactionsteps.

Yonetani and his colleagues, in a painstaking and sophisti-cated investigation extendingovermanyyears Yonetani,1976) have thoroughly characterized the cytochrome c per-oxidase reaction sequence. They have shown that ompoundI (compound ES in their terminology) is rapidly formed byreaction between cytochrome c peroxidase and hydroperox-ides, with a rate constant k 1of 4.5 X 10 M s- , and containstwo oxidation equivalents more than the native Fe(II1) en-zyme (Loo and Erman,1975). From this nformation, togetherwith the fact that ROH s released, it appears likely tha t thereaction involves initial heterolytic cleavage of the RO-OHbond to give RO- as a leaving group andOH , or some peciesin the equivalent oxidation sta te, bound to the enzyme ncompound I (Schonbaum and Lo, 1972).Spectral methods have been the principle tools employedin elucidating the nature of compound 1. Low temperaturemagnetic susceptibility measurements (Iizuka et al. 1971) andMossbauer spectroscopic data (Langet al., 1976) suggest t ha tthe heme iron in compound I is in the Fe(IV)oxidation state,and electron spin esonance spectroscopy reveals he presencein compound I of a stable free radical at a spin concentrationof 1 mol/mol of cytochrome c peroxidase (Yonetani et al.,1966). The obvious nference is that one of th e two extraoxidation equivalents in cytochrome c peroxidase compoundI resides on the iron atom and the other on ome amino acidside chain. As will be seen below, we can now identify thatsidechain as he indole ring of a tryptophan residue nintimate contactwith both the heme roup and the hydroper-oxide substrate molecule.

THE M O D E LIn apreviouspublication we have described the overall

structure of cytochrome c peroxidase and the arrangementof

certain amino cid side hains close to the heme as determinedfrom a 2.5 A electron density map (Poulos t al., 1980). Sincethen the primary sequence of cytochrome c peroxidase hasbeen established (Takio et al., 1980 and we can now refer toresidues in the struc ture by their location within the primarysequence. Thus, the proximal histidine occupying the fifthcoordination positionis His-174 and the dista l histidines His-52. The exact geometry of these and other important aminoacid side chains interacting with the heme group is depictedin Fig. 1.A small sphere represents the aquoigand occupyingthe sixth coordinationposition. NE2 of His-52 is approxi-mately 3.2 A from the aquo ligand and is therefore withinhydrogen bonding distance. However, th e hydrogen bondinggeometry is not ood, as can be seen by inspection of Fig. 1.

Such an arrangementf a proximal istid ine at coordinationposition 5, water molecule at position 6, and a distal histidinewithin hydrogen bonding distance of the latt er s highly rem-iniscent of the metmyoglobin and methemoglobin s tructu resand indeed a close resemblance between the globin and per-oxidase heme coordination has been expected for a ong time.It is strongly suggested by the similarities in their spectralproperties especially of the oxygenated ferrous derivatives(Wittenberg et al., 1967) and the ability of t he ferric proteinsto bind various anions like CN-, Nn-, and F- (Yonetani, 1976).In addition, Yonetani et al., (1972) predicted on the basis ofelectron spin resonance tudies of peroxidase and globin nitricoxide complexes that peroxidases also contain aproximalhistidine heme ligand as was later substantiated byx-raycrystallography (Pouloset al., 1980). However, there is reasonto believe the water ligand a t position 6 in peroxidases is lessstrongly boundand in a omewhat different environment thanit is in metmyoglobin. For example, results from both elec-tronic absorption (Tamura t al., 1980) and nuclear magneticresonance spectroscopy (Lanir and Schej ter, 1975) indicatethat in horseradish peroxidase the axial aquo ligand is eitherabsent or oosely bound. This difference may be related to thefact tha t the dis tal imidazole in metmyoglobin is normallyhydrogenbonded to heboundwater molecule while, asmentioned above, His-52 in cytochrome c peroxidase is notproperly oriented for that task.Also close to the sixth oordination site are wo amino acid

side chains, those of Arg-48 and Trp-51,which we believe areintimately involved in the function of cytochrome c peroxi-dase.

Th e side chain of Arg-48 extends over the distalurfaces ofpyrroles I11 and 1V with its partially uried guanidinium group3.6 8 above and nearly parallel to t he heme . buried chargegroup of this kind situated in the active site always suggestssome sort of involvement in the enzymic mechanism. Inter-estingly, the guanidinium is about 3.5 A from the buried

5 2

4 8

f

FIG. 1. Theheme group in cyto-chrome c peroxidase and some sur-rounding amino acid side chains. Asmall sphere is used to epresent heaquo ligand at he sixthcoordinationposition. This figure as well as Fig. 2 wasplotted directly from an Evans and Suth-erland Picture System display.

\ \G L N 239 G L N 2 3 9


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Peroxidase Catalytic Mechanism 8201carboxylate group of pyrrole IV, but the local geometry indi-cates a charge-charge interac tion rather thanydrogen bond-ing.Th e indole ring of Trp-51 is also about 3.6 A above andapproximately parallel to the distal surfacef the heme. It issituated above pyrrole 11, well within the heme crevice. Th eindole ring NE1 is about 2.7 A from and appears tohydrogenbond with the water igand at coordination position 6.

PEROXIDE BINDING SITEInorde r o explore the possible binding geometry of a

hypothetical substrate peroxide molecule, the heme and thedistal residues described above were displayed on an Evansand Sutherland Picture System simultaneously ith a repre-senta tion of the 0 0 group of a peroxide. The la tte r onsistedof two small spheres igidly connected at an 0 0 distance of1.48 A. The peroxide group was first positioned withoneoxygen atom, identified as 0 2 in subsequent discussion, an-chored at the sit e of the wa ter ligand and then the 01-02bond of the peroxide model was moved through all possibleorientations. It quickly became obvious tha t the re is littleroom for maneuvering, and in fact just one learly preferableorientation for the peroxide group stands out. This is shownin Fig. 2, where it will be seen tha t the 01 atom is neatlypositioned to accepthydrogen bonds from both NE and NH2of Arg-48, with dis tances of 2.7 and 2.9 A , respectively. Noticealso tha t NE2 of His-52 and NE1 of Trp-51 remain, respec-tively, 3.2 and 2.7 A away from the peroxide 0 2 atom, as ofcourse they must since 0 2 still occupies the siteof the heme-boundwater molecule. Th e Fe-02-01 angle is approxi-mately 135 as might be expected for an oxygen ligand.

Let usnow go back and consider the geometry of distal His-52 in relat ion to the model peroxide. As mentioned earlier,NE2 of His-52 is within hydrogen bonding distance 3.2 A ) ofthe water molecule coordinated to the heme iron at position6, but thehydrogen bonding geometry is poor. Bearing that nmind, we now find upon examining our peroxide-containingmodel that in fact His-52 appears to be bet ter oriented tomake a hydrogen bond to th e 01 atom of our hypotheticalperoxide substrate than to 02. The distanceetween NE2 ofHis-52 and 01 is 2.7 A and the geometrys essentially normal.We shall return to this important observationn th e followingsection.

As mentioned in the introduction, cytochromeperoxidaseand other eroxidases also utilize organic hydroperoxides,andin particular Yonetani (1976) has shown that eth yl alcohol isreleased in he reaction between cytochromeperoxidase andethyl hydroperoxide. We are lead therefore to ask whetherthe geometry justescribed can accommodate the ethyl group

4 8

H I

TR P 5 1

or other R groups of an alkyl hydroperoxide, which in con-formity with th e oxygen-numbering convention we have beenusing above, must be representedas R-01-02-H. Inspec-tion of the model on the Pic ture System howed th at indeedit can, with thelkyl carbon chainextending out over pyrroleIV and thence into the surrounding solvent. Moreover, thespatial arrangement of t he four orbitals surrounding 01 isvery nearly etrahedral. Moreprecisely, the direc tions efinedby the 01-02 bond, th e R-01 bond, the 01-His-52 hydro-gen bond and he remaining one pairorbital involved inhydrogen bonding to theguanidinium group of Arg-48 are allfour tetrahedrally arranged around oxygen atom 01 of theperoxide molecule. This is exactly the geometry one wouldhave anticipated, but we should emphasize that it was notput into themodel, so to speak,at the beginning.

PROPOSED MECHANISMHaving just described the geometry of cytochrome c per-

oxidase in the vicinity of our hypothetical peroxide bindingsite, we now present a probable mechanism for th e reactionbetween a peroxide molecule and cytochrome c peroxidaseleading to compound I. This mechanism is of course based ona large body of biochemical, kinetic, and spectroscopic evi-dence in addition to our wn structural data, but or the sakeof clarity we shallpresent he mechanism first and deferdiscussion of some of th at evidence unt il the ollowing section.

Fig. 3 depicts the roposed mechanism in aighly schematicform. The resting cytochrome peroxidase moleculeis shownin Fig. 3a, with the side chain of Trp-51 represented as TRPHfor late r convenience. T he side chains of Arg-48 and distalHis-52 are also included, together with the heme , proximalHis-174, and a water molecule bound a t he hemes 6thcoordination site. The distal histidine s shown as making nohydrogen bonds in the nativeenzyme, in accordance with thediscussion above.

In the irst step a peroxide subst rate R-01-02-H entersthe heme crevice, displaces the bound water molecule, andforms a short-lived intermediate (Fig. 3b). The most probablestructure for this intermediate contains a singly ionized per-oxide molecuie covalently bound to the heme ron atom withthe hydrogen ion transferred to His-52.

In he next step, he enzyme.peroxide complex passesthrough the activated transition state (Fig. 3c) on its way tocompound I. In the transition state theovalent bond betweenperoxide atoms 01 and 0 2 is undergoing heterolytic cleavageto give a leaving group R-01- and a single oxygen atom, 0 2 ,covalently bound to the hem e ron, while the proton isbeingtransferred to the leaving group from His-52. Here, His-52serves as ancid-base catalyst by facilitating the transferof a

-I-,4 a

FIG.2. A closer view of the distalstde of the heme with Arg-48, Trp-51, and His-52. A model peroxide 0 1 -0 2 is represented by a pair of smallspheres separated by 1.48 A . The proxi-mal histidine His-174) is also visible be-low the heme plane.


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8202 Peroxidase Catalyt ic Mechanism

FIG. 3. Schematic representation of the cytochrome c per-oxidase-catalyzed heterolytic cleavage of the R01-02H bond.a, the restingenzymewith a watermoleculebound at the sixthcoordination position; b, a hypothetical enzyme .substrate complexwith the substrate bound as a hydroperoxyl anion, the 0 2 protonhaving transferred to the distal histidine; c, the transition state. Thehydroperoxide 0 0 bond is undergoing heterolytic cleavage and aproton from 0 2 to 01. One might at first expect His-52 toform hydrogen bonds with oth oxygen atoms of the subs tra te.In fact, the hydrogen bonding geometry and distance (3.2Abetween His-52 and 0 2 is poorwhile both he hydrogenbonding distance (2.7 A and geometry between His-52 andthe leaving group, ROl-, are quiteood. Such an arrangementactually favors proton transfer from 02 t o 1 and is reminis-cent of ano ther well studied class of enzymes, theserineproteases. In that case the active site histidine formsbetterhydrogen bond with the subst rates eaving group which is tobe protonated than with the proton donatingOG atom of thecatalytic serine residue (Matthews etal., 1977).

Other than theacid-base catalytic role of His-52, the moststriking feature of this mechanism, and certainly the one orwhich the evidence so far comesexclusively from the presentcrystal struct ure, has to do with th e role of Arg-48. Noticethat the transition state depictedn Fig. 3c necessarily involvesa developing negative charge on 01, and recall that 01 isprecisely positioned to accept a pair of hydrogen bonds fromNE and NH2 of Arg-48. In short, the buried and positivelycharged side chain of Arg-48 must function in a simple andobvious way to stabilize the ransit ionstate equired forheterolytic cleavage of th e peroxide group. Considering th atthe coordination sphere of the heme ron atom in cytochromec peroxidase on the one hand and in metmyoglobin or met-hemoglobin on the other are almost identical, it may not be

dlnegative charge developing on the leaving group, ROI- is stabilizedby hydrogen bonding with Arg-48 and by the transfer of a protonfrom the distal histidine.d to f possible structuresof compound I. dand e, two esonance forms of the oxene ntermediate; f thestructure of compoundI after the oxene oxygen atomas abstracteda hydrogen atom from the indole ring of Trp-51.

going too far to uess that thepresence of such a strategicallysituated positively charged group is an essential stru cturalfeature thatdistinguishes a peroxidase from n oxygen-carry-ing globin.

We come next to Fig. 3d. His-52 delivers i t s proton to R-01- which the n dissociates from th e enzyme as R-01-H.Left behind is a single oxygen atom, an oxene (Hamilton,1974), attach ed to a heme tha t was originally in the Fe(II1)state . Formally, therefore, the iron is now at th e Fe(V) oxi-dation level, that is, two oxidizing equivalentsabove heground state. However the weight of spectroscopic evidencefavors an electronic description of compound I with th e ironatom in the Fe(1V) state, and so in Fig. 3e we include thatpresumably predominating canonical resonance struc ture.

As depicted in Fig. 3f, the indole ring of Trp-51 is oxidizedto an aromaticadical. Th e edge of the Trp -51 ing is only 2.7A from 0 2 and is the only readily oxidizable amino acid sidechain contacting the substrate. Thus, the reactive oxene in-termediate should be able easily to abstract a hydrogen atomfrom the ndole ring leaving an indole-free radical. Th e pres-ence of such a radical is consistent with Yonetanis inte rpre-tation of the cytochrome c peroxidase compound I electronspin resonance spectrum (Yonetani etal., 1966). However, onthe basis of cytochrome c peroxidase compound I elect ronspin resonance and electron nuclear double resonance spectraobtained at 1.4K, Hoffman et al. 1979) concluded th at th e


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Peroxidaseatalyticechanism 8203free radical in compound I resides on the sulfur atom of amethionine residue. Of the five methionine residues in cyto-chrome c peroxidase, he closest to the hydrope roxideindingsite is Met-171with its sulfur atom approximately .6A belowthe proximal surface of the heme and A from the heme ironatom. Thus, it is difficult to see how the oxene intermediatecould preferentially oxidize Met-171 rat her tha n Trp-51. Un-fortunately, we cannot for the present resolve this apparen tinconsistency, although tshould be emphasized that hemagnetic resonance spectral results ere interpreted withoutfull benefit of the three-dimensional structure.Finally, two one-electron reduction steps bring compoundI back to the nat ive enzyme to begin the catalytic cycle allover again. As stated at t he outset, we will not attempt todiscuss this part of the mechanism in the present communi-cation.

DISCUSSIONModel Reactions-It is a matter of both reason and faith

that any redible enzymic eaction mechanismwill have somesort of counterpart in the realm of classical organic or inor-ganic chemistry. We are thu s mpelled to ask whether or notthe preceding peroxidase mechanism is consistent with wellestablished peroxide chemistry. More specifically, one mightwish to know if there isany convincing evidence ha t peroxidesare capable of heterolytic cleavage in the presence of Fe3+ oproduce an intermedia te complex of oxygen with iron in ahigher oxidation sta te. The question is easier to ask than toanswer, unfortunately, but the attempt does shed some lighton the matter.

As a generality, t is a well known characteri stic of peroxidesthat they eadily undergo homolytic cleavage under the nflu-ence of heat or radia tion ith production of RO- free adicals.This property is the consequence of a relatively low 0 0bond energy, 34 kcal/mol, to be compared with, forxample,an S S bond energy of 63 kcal/mol or anO=O bond energyof 119 kcal/mol (Purcell andKotz, 1977). On the other hand,several well characterized reactions f peroxides are known toproceed y nonradicalmechanisms involving heterolyticcleavage of the 0 0 bond to yield an oxidized product plusOH- or H20. Examples are the oxidationbyperoxides ofhalide ions to oxyhalides and of various organic sulfides tosulfoxides (Edwards, 1964). Th e problem with trying to findmodelperoxidase reactionsamong his class of reactions,however, is that they all involve attack on the peroxide byreadilypolarizablenucleophiles, and normally theFe(II1)center of the heme group is not considered to be n thatcategory.

In partbecause of their intrinsicbiochemical relevance, thechemistry of several ironperoxide systems have been studiedin some detail, but apparently there is still considerable con-troversy concerning mechanisms. T he prototypica l system isthe Fenton reaction (Walling, 1975), in which catalytic con-centrations of Fe2 ion promote the xidation of a wide varietyof organic compounds by H202 .Although the Fenton reactionhas been known for ome 86 years (Fenton,1894), it is not yetgenerally agreed whether he primary oxidant s he HO.radical (Walling, 1975) or an iron. xygen complex, the fer rylion [Fe(IV)=O] (Groves andVan Der Puy, 1976). From thevantage point of the nonexpert , however, it seems that thedetailed mechanism mightwell vary with reaction conditions,such as solvent, Fez+ igation, etc.

Of perhaps greaterelevance for he peroxidase mechanism,since peroxidases inheir active state containe3+ ather thanFe, is the catalysis of peroxide decomposition by Fe ion.Additionally, the Fe3+-H202 system also can oxidize organicsubst rates in peroxidase and catalase like reactions. Again,

two reaction schemes have been proposed. One involves in-termediacy of the HO. radical (Barb et al., 1951) and th eother a higher valence ironoxygen complex analogous to th atproposed in our peroxidase mechanism (Fig.3d) (Kremer andStein, 1959). However, later work by Walling and Goosen(1973) favors the radical mechanism.

An interesting extension of t he Fe3+-H202 react ion hasbeen studied by Hamilton as a model for catalase and cyto-chrome P-450 (Hamilton, 1974). In Hamiltons system aro-matic compounds are hydroxylated by Hz02 in the presenceof catalytic amounts of Fe3 and catechol. A detailed kineticand product studyed to the onclusion th at th e ydroxylatingagent is a ternary complex of Fe(II I), catechol, and a singleoxygen atom derived by heterolysis of H202 The similaritybetween such an intermediate and the structureroposed forcompound I of cytochrome c peroxidase is evident.

Hamilton has also focused attention on the resemblancebetween reactions of carbenes and nitrenes on the one handand both enzymic and model nonenzymic hydroxylation re-action on he other, and refers o he atter as oxenoidmechanisms. In particular, Hamilton points out that a loneoxygen atom coordinated to an Fe3+ center is isoelectronicwith carbene CH2 or nitrene NH, and might herefore beexpected to participate in imilar C-H bond insertions, dou-blebondadditions, and hydrogen abstractions(Hamilton,1974). This is the rationale behind our labeling the speciesrepresented inFig. 3d an oxene intermediate. Hamilton andothers believe a species of this kind may be active in heme-containingoxygenases ike cytochrome P-450. Presumablycytochrome c peroxidase and othe r peroxidases are not oxy-genases because of restricted access to the sit e of oxeneformation and rapidonversion of the latt er to theerryl stateplus a radical.

Finally, th e presence of the higher, F e(IV), oxidation statesis not without precedent n porphyrin chemistry. Felton etal.have demonstrated that one elect ron can be removed fromthe iron atom in femc tetraphenyl and octaethyl porphyrinsby cyclic voltammetry to generate Fe(1V)porphyrin (Feltonet al. 1971).

The Role of Arg-48Regardless of what mechanism sultimately agreed upon for these potential model reactions,there is considerable direct evidence or the existence ncompound I of an iron.oxygen atom complex. As mentionedearlier, the presence of two additional oxidation equivalentsin cytochrome c peroxidase compound I (Coulson et al., 1971)and the elease of ROH whenROOH is the peroxide substrateforhorseradish peroxidase (Schonbaumand Lo, 1972) orcytochrome c peroxidase (Yonetani, 1976) certainly argues orbinding of a single oxygen atom. In the case of chloroperoxi-dase, experiments with *O-labeled subs tra tes i ndicated tha tone substrate-derived oxygen atom is retained in compoundI of that enzyme (Hager et al., 1973). Additionally, Coulsonand Yonetani (1975) found th at various hydroxylamine deriv-atives of the type RONH2 react with cytochromeperoxidaseto form compound I only if the RO moiety is a good leavinggroup. These results are compatibleonly with heterolysis ofthe RO-OH bond.

In view of the foregoing, it is satisfying to find an arrange-ment of amino acid side chainsat the distal side f the hemein cytochrome c peroxidase which is ideally suited to promoteheterolysis of a peroxide molecule, namely Arg-48 which sta-bilizes a developing chargeeparation andHis-52 which servesas an acid-base catalyst by facilitating proton transfer to theleaving group.

The need for some kind of enzymic machinery to promotecharge separation and proton transfer n the catalase mecha-nism was recognized by JonesandSuggett (1968). They


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8204 Peroxidase Catalytic Mechanismsuggested that an arginine side chain and a heme propionateside chain would meet this requirement,which, assuming thecatalase active site turns out to resemble cytochrome c per-oxidase, may be at least half right.

The importanceof Arg-48 for peroxidase catalysis is under-scored by comparing cytochrome c peroxidase with metmy-oglobin. Even though the heme environmentf metmyoglobinis superficially similar to that of cytochrome c peroxidase,metmyoglobin onIy weakly promotes cleavage of H 202(George and Irvine, 1956; King and Winfield, 1963). We pro-pose that the chief reason for this difference between met-myoglobin and the peroxidases is that no analog of Arg-48exists n myoglobin, where the only site that even roughlycorresponds is occupied by phenylalanine CDl instead (Pou-10s et al., 1980).

The charge stabilizing influence of Arg-48 is also reflectedin another property of peroxidases. By suitab le reatmenthorseradish peroxidase can be prepared in the Fe(I1) sta te, asthe so-called compound I11 (Yamazaki, 1974).Thus compound111should resemble myoglobin in its active Fe(1I) state, andindeed compound 111 does contain bound molecular oxygenand exhibits an oxymyoglobin-like spectrum (George, 1953).In fact, it would appear from qualitative observations that2is bound exceptionally strongly in compound 111. Now it isthought that 2 binding to transition metal centers involvesa major resonance contribution from the' 1 -02- canon-ical structure (McLendon and Martell, 1976), and th e sameview has come to prevail regarding oxygen binding to hemeproteins (Peisach,1975). Thus it s reasonable to suppose thatcharge stabilization by Arg-48 (or itshomolog in horseradishperoxidase) would strengthen the oxygen binding capabilityof Fe(I1) peroxidase. By the same mechanism we would alsoexpect ready reaction between Fe(II) peroxidase and 2 toproduce Fe(II1)peroxidase and superoxide anion, OZ-.

Comparison with Other Peroxidases-A rather obviousquestion that should be dealt with before proceeding is theextent to which t he molecular machinery we have been de-scribing is preserved in peroxidases other than cytochromecperoxidase. S tructurally the sequence of residues 48 through52 in cytochrome peroxidase, consisting f Arg-Leu-Ala-Trp-His, forms themiddle of helix B with the carbonyl oxygen ofArg-48 hydrogen bonded to the backbone amidoroup of His-52. As one can see from Table I the homologs of Arg-48, Leu-49, and His-52 are present in all six peroxidases for whichpartial or complete sequence data arevailable. Preservationof Arg-48 and His-52 is expected, of course, and implies thatthe mechanism we have proposed here for cytochrome cperoxidase is also applicable to other peroxidases. Howeverthe function of Leu-49 is not obvious. Probably it serves tomainta in the overall positioning and orientationof helix 3.

Apparently, then, cytochrome c peroxidase and the plantperoxidases catalyze the reduction of peroxides by virtuallyidentical mechanisms. Nevertheless, cytochrome c peroxidaseand horseradish peroxidase compounds I exhibit two mark-edly different spectral properties: (1)cytochrome c peroxidasecompound I generatesan easily discernable elect ronspinresonance signal while horseradish peroxidase compound Idoes not, and 2) cytochrome c peroxidase compound I is redwhile horseradish peroxidase compound I is green. Becausecompound I in both enzymes contains a ferry1 iron, thesespectral differences are most ikely associated with the sourceof the secondreducingequivalent. As discussed above, incytochrome c peroxidase the second equivalent is probablyremoved from Trp-51, while examination of Table I showsthat the correspondingresidue in he plant peroxidases isphenylalanine. Since phenylalanine is more resistant to oxi-dation than tryptophan, the econd equivalent in horseradish

TABLEAmino acid sequencespreceding the distal histidine in

cytochrome c peroxidase CCP) Takio et al., 1980); horseradishisoperoxidase c HRP c) Welinder, 1976 ;and four isoenzymes ofturnip peroxidase PI ,Pz a, and P7 Welinder and Mazza,1977 .The sequence numbersrefer to cytochrome c peroxidase.

48 49 50 51 52CCPHRP c Arg

Leu Ala Trp HisArg Leu His Phe HisPI Arg Leu His Phe His

P Arg Leu His Phe HisPS Arg Leu His Phe HisP7 Arg Leu Phe Phe His

peroxidase compound I must be abstracted from some otherneighboring group, most probably th e heme ring itself. Thepresence of a heme radical in horseradish peroxidase com-pound I is consistent with both itsgreen color (Dolphin et al.,1971) and a ecentlydetailedanalysis of itselectronspinresonance spectra (Schulz et al., 1979).

Role of the Proximal Histidine-Thus far we have dis-cussed only the function of the dista l residues in cytochromec peroxidase catalysis, but obviously the proximal histidine,His-174, and its surroundings mustlso be considered.Peisach(1975) and others (Nappa et al., 1977) have suggested thatinteraction between t he proximal histidine and neighboringgroups in the prote in molecule will regula te the electronicproperties and therefore the reactivity of the heme iron. If,for example, this interaction imparts enhanced anionic char-acter to the roximal histidine, higher oxidation stat es will bestabilized and O will be lowered. Indeed, Mincey and Traylor(1979) have found that model heme compounds containinganionic axial ligands are more readily oxidized than hosecontaining neutral ligands.

In the case of cytochrome c peroxidase, the oxidation-re-ductionpotential of the ferrous /femc couple is -194 mV(Conroy et al., 1978) which is tobe compared with myoglobinat +50 mV (Cassa tt et l., 1975). Mechanistically this require-ment for a lowered oxidation-reduction potential in cyto-chrome c peroxidase is understandable, since the Fe5+ formaloxidation state (Fig. 3d) must bestabilizedsufficiently topermit it to beroduced at least transientlyn a two-electronoxidation of Fe3+ by peroxide.

An intriguing question, then, is how can the structure ofcytochrome c peroxidase explain its low oxidation-reductionpotential in comparison with myoglobin. We had expected tofind the proximal histidine, His-174, hydrogen bonded to anegativelychargedcarboxylate ide chain. Now that heamino acid sequence of cytochrome c peroxidase is in hand,however, we find instead hat His-174 is withinhydrogenbonding distance of the side chain of Gln-239. Specifically,ND1 of His-174 is about 2.9 A from OE2 of Gln-239. In turn,Gln-239 is hydrogen bonded to theside chain of Glu-187 (seeFig. 1 .All three side chains (His-174, Gln-239, and Glu-187)are buried and inaccessible to solvent. A preliminary equencealignment of cytochrome c peroxidase and horseradish per-oxidase is so far inconclusive concerning the possible conser-vation of this structural feature. However, i t is noteworthythat both Gln-239 and cytochrome c peroxidase and Gh-245in horseradish peroxidase are located in identical Leu-Ile-Glnsequences.By way of comparison, t he proximal histidine of myoglobin,His-8F, is hydrogen bonded to the ackbone carbonyl of Leu89.Presumably this arrangement of a buried negative chargeand hydrogen bonding o the roximal histidine in cytochromec peroxidase is responsible for regulating its oxidation-reduc-


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Peroxidaseatalyticechanism 8205tion potential. However, it isnot entirely clear i there wouldbe sufficiently large inductive effect at the heme iron due toa negative charge on Glu-187 to explain the observed oxida-tion-reduction potential difference between cytochromeper-oxidase and myoglobin, but for he moment that seems to bethe best working hypothesis. Moreover, the hydrogen bondingnetwork ncytochrome c peroxidase is considerablymoreflexible than the carbonyl oxygen-His interaction in myoglo-bin. Therefore, it is intriguing to consider the possibility thatheme reactivity is modulated by the dynamics of the His-174-Gln-239-Glu-187 interaction during the catalytic cycle.The recent observation Hayashi and Yamazaki, 1979) thattheoxidation-reductionpotentialof the twocouples, com-pound I/compound I1 and compound II/native peroxidase inhorseradish peroxidase,s nearly the same suggests that somesuch mechanism may be at work.

Acknowledgments-We wish to acknowledge the Chemistry De-partment Computing Facility and especially the expertise of StephenDempsey for his continued development of the Pic ture System.REFERENCES

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