Structural analysis of compound I in hemoproteins: Study on Proteus mirabilis catalase

Biochimi¢ l! 997~ 79, 667--671 (0 Socidld fran~'aise tic biochhnie el biologic moldculairc i Else\ icr. Paris

Structural ana|ysis of conlpound I in hemoproteins: Study on Proteus mirabi{is catalase HM Jouve", P Andreoletti", P Gouet b, J Hajdw: J Gagnon" ah:st.itut d.e Nio!ogi~' Smt~Turah' .h,an-Pierre-Ebel, CEA/CNRS, 41. rue des Martyrs, 3h'027 Gremd~le ccde.~ I. Fram'c: Laboralorv o.f M,~h'cular BioldO'.~ics am/O.~ford ('enter for Molecular S~icm'e.s. University olO~.ford, South Park Road. O~/'ord OXI 3QU, UK; CDeparlnle,t of Biochcmisll 3. Uppstda Univ~ rsiO, Box 576, S-751 23, Ul~p,s'ala, Swede.

I Received 2 June I t)97; accepted 27 Oclobcr 1997~ S|ln|mary ~ Ferryl calalysis has ailracled considerable hlleresi, because a diverse variely of enzyme.,, use ferryl inlermediales to perform difficull chemis|ry. The slructure of the re.'lclionai inlennediale compound ! of Prote,s mirabilis catalase (PMC) has been sol~ed using lime-resolved X-ray diffraction techniques and single cryslal nficrospeclropht~lonlelry, l:ormation of compound i is characlerized by signilicant changes in file absorbance spectrum, and the creation of an oxoferryi ~roup on the distal side of lhe home. This group is clearl.~ visible in Ihe X-ray elecmm densily maps. An unidenlified electron density, likely Io be an anion because of 1he nature of its en~ironmem, appears during the teat|ion, in a nile dlM;|ill frolll 1he henle. The slruclure of conlpound ! in PMC in compared wilh Ihal of compotmd ! in cvtochromc c peroxidase (CCP~.

catalase compound I / time-resolved cryslallography / X-ray slrueture Introduction

Occurrence of compound I in ea~alases

Calahlse is an ubiquitous enzyme present in aerobic organ-

('oml)oLmd ! results from the two-electron oxklalion of lhc ferric ¢atalase by one molecule of llydrogen peroxide (fig i) I6, 7, 14 I. This intermediate contains an oxoferryl group and a x-cation radical delocalised on the porphyrinic ring 115 I. Compound ! is selbjecl to a l'asl iwo-elecI|'o|l reduclion by a second molecule of hydrogen peroxide, leading the cnz~ nit back to itsresting form. A single-electron reduction ~I ¢omimtmd i may also occur under reacli~m ~xilh a slcatly Ilo~v of hydrogen peroxide, a,s under physi~dogic condi|iom, 116 I. Hydrogen donors such as phenols, ascorbale, tcrmcyani~lc or superoxide can similarly reduce compound 1 Io Ibis form named comi_~ound Ii 14, 131. Willl 1111eXCeSS el" hydrogen peroxide, compound !i in replaced by an hyperoxidized species named COml'~ound ill, which in inactive on hydrogen peroxide 16, 7, 13 I. Thus, the pathway via compounds 11 and ill leads to a decreasing aclivily of catalase on hydrogen peroxide 16, 7, 13 I. h., rive, this process can he responsible Ibr an accumuh|tion of hydrogen peroxide, causing dele° terious consequences such as illfJamlllalioll, iiltltaliOilS. aging or cancer 1171. Celt,us '" " catalases use NADPH to pre° vent the formation of compound I! 118-211. However, NADPH does not reacl rapidly neither with compound ! nor with compound I1, and its mechanism in not clearly understood 122, 231. It has been proposed that NADPH could reduce a transient protein radical appearing, during the decay of compound i to compound II. !1 appears thai compound ! plays an important role ill the calalylic cycle of ca|;dascs, by posilioning the enzyme to-

isms, which achieves disproporlionalion of hydrogen peroxide Io molecular oxygen and waler. It has an imporlanl role in Ihe del~nse of aerobic organisms ;.IgaillSl oxidants Ii I. Catalase was firsl crystallized before the Second World War l2 I. Fifty years ago, Chance discovered an intermediate ill Ihe reac|ion of C;llalase wilh hydrt~gell peroxide, by stopped-flow spcctroi~hotonlelric measurements 131. 'Mils transient f¢ffin WaS lalcr called conlpound I. Calahlse coin,poulld I has a very high rate o1' Ibrmation and decomposi o lion, d e p e n d e n t on lhe inilial h y d r o g e n p e r o x i d e concenlralion, it reacts wilh acceplors or inhibilors in tile same way as the so-called complex pemxidase-hydrogen peroxide 13, 41. it has a green color, while the ferric enzyme is normally brown and exhibits a characteristic electronic absorption spectrum 15]. Other properties have been reviewed 16-81. Early experiments revealed thai acyiperox-

ides, such as peroxoaeetic acid, could be used to give a more stable intermediate 191. Thin property Ires recently been used to obtain cryslals of stable compound i in the catalase of the bacteria Proteus mirabilis I101, This review emphasizes on the importance of compound i in catalases and on the methods tmlized to delermine ils crystallographic structure in PMC. A parallel is made with compound I in cytochmnle c peroxidase, which was Ihe firsl structure of an oxoferryl hemoprolein intermediate to be delermined by lime-resolved studies IIII.

J

~8 NADPH

NADI~ + H÷

H20

? Compound |

FOlil

o2=

I ~-:

o•

o2+x2o

fA.

xoox

Ferrocyanide Ascorbate Phenols

o[ X,,~ A •

lc,=po- ,ul

NADPH

I H20

HOOH

wards a two or one electron reduction pathway I131. It is present as a reactional intermediate in other heine proteins ~uch as peroxidases, ¢ata!ase-peroxidases. cytochrome P450 and myoglobin 124. 251.

Tiai~e.~soived crystallography mi compound i The stability of the intermediate during data collection is

th~ main problem arising in timc,.rcsolvcdcrystallographic studies 1261,This stabilitycan be monitored with a singlecrystal nficrospectrophotometer, if the protein carries a chromophot~ 127, 28 I. The catalytic reaction can be started, by supplying a solution of substrate through the open-ended flow cell to the crystal. The cell can be adapted on a goniometer head (fig 2) and spectral changes recorded simultaneously with X-ray data, Compound I in catalase is characterized by an important decrease of the Sorer band and the apparition of a typical charge-transfer band at about 660 nm 151, The extinction coefficients are low enough (< 40 mM--I cm-I) to authorize absorbance measurements in crystals with a high concentration in protein. Stabilization el" comlxmnd I in PMC crystals is I~rformed using ~ta~xoacetic as a pseudosubstrate ill tile absence of N ADPH I9, 291, Figu~ 3 shows typical spectral changes recorded on a crystal I lOf At wavelengths inferior to 450 ran, the crystal is black to incident light, The spectrum of the ferric

I

Fig |. Possible redox transferrallions in |he catalytic cycle of catalase. Compounds !, 11 and ill are intermediates in fiwmal oxidation ~lalcs Fe IVk Fc ~lV~ and Fe ~Vll, respeclively: AH is a oneocquivalent eicclron donor. Ferrocyanide, ascorbate, phenols and supemxide are one-electron donors speeding up the transition of compound I to compound !!: phenols speed up the return of compound I| to the native enzyme. Question marks indicate that some reactions are not clearly proved (inspired from Schonbaum and Chance 171, Goner et al 1121, Lardinois 1131).

enzyme shows maxima at 507.540 and 630 nm. Ill compound I, maxima at 507 and 540 m!mdisappeaL while the 63{) nm peak moves to 660 nm. This spectrum remains filirly constant during 30 mill at 16 °C. Pelventage of compound I in the crystal is about 90%. It is wortli noting thai compound i obtained with pelx~xoacolic acid is less stable in solution 130L X-ray lima can be collected fi~r tillie~resolved experimt~vts using either a Laue method 131 ] or a fast collection methi)d with nlonochnm|atic X-rays 126]. For tile first nlethod, one shot wit!l l~flychromatic X-rays call be sufficient to solve a 3-D structure: however, the processing of Laue data is frequently altel~d by spatial overhtpping, leading to a lack of completeness at low resolution and a poor quality of the calculated electron density map [26]. For tile second approach, as for PMC comlxmnd I, synchrotron data were measured using a Weissenberg camera in Photon Factory, Japan 1321. Using this method, IO frames of Weissenberg data ate collected within 30 mill leading to tin excellent completeness after data pr(~essing due to the high symnletry of tile PMC crystals (hexagonal space group P6222 J331).

Structural results on PMC compound ! The structure of PMC compound ! was solved at 2.7 /~ resolution without bound NADPH I lO]. The results reveal no major structural rearrangements relative to the native

6{59 I I lOI. Superoxidc which ma> ha,,c a ro~c in the formation of compound II !131 fits the extra electron density the bcq. CAPILLARY

Comparison of compound I structures in PMC and CCP

X4~,Y$

au~wR

GONIOMETER I DETECTOR

MOTOnlZED

SVnINGE DEWCE

~

..................

l .......................

/\ y\. WAVE LENGHI

SPEGTROPNOTOMETER

Fig 2. Schematic representation of the device used to de|ermine the structure of compound 1 of Prole,s mirabili.v catalase by lime-resolved X-ray crystallography I101: tile calalase crystal (C) is wedged in a tapered quartz capillary adapted on a goniometer head: the buffer is flowed around the crystal by a motorized syringe device; the absorbahce spectrum of the crystal is recorded during X-ray diffraction measurements.

enzyme. However, dilTcrence Fourier maps show without ambiguily Ihe oxoferryl group of compound ! interacting with a waler molecule present in all catalase struclures [34 ]. Formation of tt:c oxoterryl group is accompanied by small movements of the heine and of the proxinlal ty~osine too wards the distal histidine. A strong electron density is observed more than 10 A away fi'om the heine site, in one of the larges! cavities of the enzyme 112 I. This density is likely to acconnl I'or the ,'eplacement of a water molecule by a stronger scatterer in compound !. Considering the residues lining the new site (R342, It 349, H 42(Q)), the unknown molecule is likely to be anionic. Interestingly, H42(Q) belongs to the N-terminal arm of a related subunit in the cataiase tetramer [121. The binding of the unknown anion is reversible as checked by the disappearance of the extra density in the ferric structure I I 0 f A few anions are contained in the solution bathing compound 1 crystals: chloride, acetate, sulfate, and probably superoxide produced by auloxidation of peroxoacetic acid. The binding of chloride to catalase has been described at low pH (4.4) and can produce the inhibition of the enzyme activity 1351. However, at the pH used for PMC crystals (pit 5.9), ! to 501) mM chloride involved no change on the kinetic parameters of compotmd

At the present time, only the structure of compound I of cytochrome c peroxidase (CCP) has been solved using methodologies similar to that used on PMC compound | [ l 1]. This structure, determined using a Laue method, confirms but also improves that obtained by Edwards et a11361, using conventional monochromatic X-rays on crystals soaked in hydrogen peroxide and then frozen. Structural changes are only observed around the peroxide binding site and movements of the heine iron are very similar to those observed in PMC. However, |brmation of compound I in CCP causes the removal of fllree water molecules on tl~e distal side and displacement of an arginine residue toward tile oxoferryl group. Moreover, it is known that tl~e flu'malion of compound ! in CCP inwflves the appearance of an organic radical on a proximal tryptophan (Trp 191)!37]. This radical is essential for the electron transfer bdween CCP and cyctochrome c 137, 381. The results obtained by Ffil/Sp et ai [ I i ] show ~,hat no obviously detectable structural changes are seen in the vicinity of Trp 191. contrary to previous results [361, and have been interpreted as file formation of a neutral radical at Trp 191. The fo|'mation of a tryptophan radical is not observed in the cornpound I form of ascorbate peroxidase, which heminic site is nearly identical to the one of CCP. But the binding of a potassium cation at about 8 A from the Iryptophan observed in ascorbate peroxidase may destabilize the formation of such a radical in this enzyme 139, 40]. A tyro° sine radical has been observed by EPR measuremenls in the well studied bovine li~¢r catalase after reaction with perox~

2.2[

~=

1.8 '

"',,,, 1.4

,,, \

1.0 0.6 0.2

Native (3i

,

"-.

,

' •

, - , , , .....

450 490 530 570 610 650 690 730"770 Wavelength

~

....

810

Fig 3, Changes observed in tile absorbance spectrmn of PMC cryslals, when compound ! is t'orl ed by slowly (0. 3 mL/h) tlowinp i.6 mM peroxoacetic acid m 0.1 M Tris maleat¢ buffer. 3,7 M ammonium sulfate, final pH 5.9. The wavelength is expressed in nm {adapled from Gouel ct al I IOI).

d•..•Wat4 tl

oxygen

matte reaction. This method has been used in d~e determimaion of the structure of compound I in PMC catalase. The presence of an unexpected anion binding site in PMC compound 1 requires further experiments to elucidate its real biological significance. Structural changes observed in the formation of compounds 1 in oxidoreductases like CCP and PMC are of little amplitude, but sufficient to explain differences in their respective activity.

Acknowledgment

H34

We ~hank Dr J GallIard for fruitful discus.,,ions and I'~u" help in writin:.

H349 References

H42~ H350 Fig 4. Difference Fourier map,~ showing tl~e structural changes of PMC ¢Oml~und I near the active site. Positive electronic densities ealerl.'tled from F~b~¢r~vd,t3mqaumd ! ~ F.h.¢~¢d, t~,!ixc m~p~ l~pfes¢!ll lhe binding of the oxygen atom in the distill site altd tile binding of u~ u~lidentified anion away from the proxinta! ',itc (adapted I'rom G~)ue( el al I I01),

~c~'ti~ i~¢id t~ I'~rm cumt~mmd i 14! l, Tim, radical might be Ih¢ intermediate i~slulated by Hillar and Nicholls to exo, plain the reaction of catalas¢ with NADPH 19">, 23!, The EPR spectrum of PMC compound 11421 does not reveal a similar t:ymsine ', ,1'~cal as in bovine liver catalase, but re, ta. sembles that t'oulld t'or Micmcoccus Ivsodeikticus catalase, which has a typical t~wphyrin n-cati~u radical associated with its oxoferryl group 1431, Binding of an anion in PMC compound I c('~uld have an electrostalic efi~c! on the protein radical fi~rmation and stability as seen in ascorbate ~roxidase !4t)1, Conclusion Single-crystal microspectroplmlometry i~.,rtnits ~o follow enzymatic reactions in a crystal made up of proteins carrying a chromophore, This technique in association with time-~solved crystallography allows to correlate electronic transitions to structural changes in the course of the euzy-

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671 I~) Jouvc HM, Pehnom J, (;aiihud J ~ 1~)~6~ h~erac~i~n be~xveen t~'~idine adcufinc tlinuc]colidcs and bl~\~ine lixcr ealala,,e: a ch[omahL,eraphic mid spectral sludy, :l~x'h ]t~oc/wm i~/ophv~" 24g, 7 I-7~ 20 Kh°kman t|N, (;aliano S, (;aelm~i t;F'I I~)g71Tht' ltlllCliOli ol Calalttsebollnd NAI)Ptl, ,t B/ot (Twin _(',_, ~ ~' 6~0-¢~60 21 .iouvc HM, Beammmi t ( l.{'gcr I, l:ora)J, [Pehnonl ,I t It~St))"l'i~htly bound NADPtt ill t)~i~lc~r.~mh'idfi/is calalase. B#ochi,m ('ell fliol (~7, 271-.7. 22 Hillar A. Nicholls P ~1992) A mechanism for NADPH hlhibilion ~1 calalase compound il fonnaiion. FEBS Lett 314, ! 79- i 82 23 Hillar A, Nicholls P, S~'ilala L, i.owen PC ( 1¢)941 NADPH binding and con|rol ol'ca~alase compound I! fonnaihm: comparison of bovine, l;" ,.I,,,,." ):casl, and ,,s . . . . . ~ hm coil enzymes. BioHwm J 3lill 531-539 24 Weiss R, Mandon [), Woiler T, Trauiw..'in AX, Miither M, Bill E, Gold A, Jayanti K, Terser ,! (lt~gt~) Delocalizalion over the henle and the axial ligand ligands tit one of the two oxidizing equivalents stored above the t~rric stale in the peroxidase and calalase colnpound-! in|¢i'mediales: hldh'ect participation of the proximal axial ligand of iron ill lilt oxidalion i'eaetii!lln eal;li'v'ted by hclllt'-ba.,~cd peloxid;.ises ;iild ca|ala,ses? ,I Iliol hlo/'g ('hiqtt l, 377-383 25 P~lihls TI. I I!)t}{~1 The role of lilt' prllxiiilal li~2and ill lilt' Ileillt' CII/.~Illt.'S. ,I l j h j l #nor~ ('h~'/ll l, .15(1- ~15i) 26 Hajdu J, Andersson ! 11993i Fasl X-ray crystallography and tithe-resolved sti'uctures. Amlu Roy Biophvs Riomol Strm't 22,467-4qI'; 27 Hadfield A, Hajdu J (It)931 A fasl and ptlrtablc iniero.,,pc¢lrophotometer for protein crys/allography. J App# ('rvst. 26, 830-842 28 Moz#arclli A, Rossi GI, ( 191J61 Protein l'tlnciiOll in the crystal..,t#mu Rev Bhlllh.vs l'Jlolllo/,~lFiit'l 25, 3 4 3 - 3 0 5 29 Caltani L, Fcrri A. (!q94) The lilnction of NADPH bound to calalase. J Blot Res Boll Sol" it Biol Sper 70, 75-82 3(.) SIoddard BL, Farbcr GK 119951 Direct nwa,,,urcnlt'nl of rea¢livil 3 in the prolein crystal by sleat, ly-slale kinetic studies. Strumlure 3. t)i)lt}96 31 Farber GK (1995) Laue cryslalhlgraphy, it's show tilnc. Curr lli,II 5, 1088-10q0 32 Sakabe N (1983) A focusing Weisscnbcrg camera with multi-layerline screens for Inacronlole¢iil;ir crysl;ilh)graph)..1 AID/i/ ('rv.~l 16. 542-5,17 ,

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t'r\',tal~i/ali~mand c~)slal packin~ ~I Pr~wu~ mtr~d~U~ ~'R ~,~.L~c. ,/.4hd Hiol ._"'~[. 1075-. 1¢0T7 t~ravo J, Fila I. (;ouel P..h~u~c [|.N.I. 5/lclik-..\dain~ati ~k ~',lu~,,hu.?.,~ ( ;,N t let)7 ) Sllth.:lUie ~,)1c;.t[a~;.ly,cx. /H. ()Aid~Hiv¢" ,~'ttt'~s ~Hht lilt' ~,'/~/t' ~ u/ar /h,dotLv ,~1 Anrt~,.~td~mt I)Cti,n.~cs t Scandalios JG. cd) (~)ld Spring tiarbor i.aborator,, Pre,,s. Nex~-x(~rl,. 4~17 p Lilchfield WJ t Iq771 |nacti~,ation of caialasc b.~ chhuidc. It:B.'~ l.¢ll 83.281-284 Edwards Sl.. Xuong Nh. Hamlm RC. Krau! J 119871Cr~,,tal Structure of cviochrome, c peroxidase Compound I. Bio~'In'mi.~trv _6. "~ 15(t3-1511 Sivaraja M. Goodin DB, Smhh M. Hoffman BM (1¢)~9t ldentilicalitm by F.NDOR of Trp ~'~i us the free-radical site in c~,toct~romc, pen~xidane compound ES S,icm c ,~4 . . 733-740 Huyell JE. Doan PE. Gurbiel R. Houseman ALE Si~araja M. Go,)din DB. Hofl'man BM (I qq5) ('onq~ound ES t)l c%iochlOllle ¢" peroxidas¢ COlll;.lins a "rrp ~ - c a t i o n radical: cha|;.lclerizalioll b~, ('\~,' and pulsed Q-band ENI)()R spectroscopy.'..I Am ('lwm S,,= II 7. ql133-qi)41 Bon;,eura CA. Stmdaramoor0~v M. Pappa itS. Paller,,on ~,R. Poulo,, TI. (19961 An engineerin~ cation site in cytochromc , pcroxid,t,,c ahern tile reaclivh.v of the redox actixc Ir) pttq~h,ul, t~,iotl,,mi~t/'v 35. 0107-6115 Pappa li, P;ltterstHI ~,VI,L Potlhi,, ! i . ( It,~ql~i | h e honlohlgl~tl,, try ptt,phan critical Ior c3 h~cllriune , pcroxidase I'tlllt.'litlll in Iltll c,,semi:d lot ascorb,fl¢ pcroxidase aclixi!~..IIH(" I. 61-(~(~ I,~;.lncit:hA, Jouve ltM, Gaillard J (1990) i'.l'R cxidem.'c 1~, d Lx~,~,xl radical illlernlediale ill bovine li~,cr u'alalane..1 ,,~m L'/Icm .'~',~t I,q,~. 12~52- ! 2,~53 lvancich A. Jouvu IIM. Satfor il, (;;tillard J ( It)971 EPR inxe~ti~ation of conlpt)tllld I ill Proteu.s mirahili.x alld ho~ ine lixer catalases: loNnalion of porphyrin ;ind t.s,ros>l radical inlcrllletlial¢,,, leto~hcmi~lr~ 16. t) 351~-9304

43 Bcneck~ MJ. Frev,' .IE. Scoxvcn N. Jose,, P. I|offman BM (It)tL~,) lilq~ and I~NI)()I ,~, dcleclion of conlpound I I1o111/llltrt,~l,s I~w,dcd,,,,~ catzllasc. Biot'hcmi~trv 32. IIt,12q-IIq33