Ar
ti
cle
0103 - 5 05 3 $ 6 .00+ 0.00
T h is p ap er is dedicated to our f riend Prof essor I caro M oreira in memoriam
* e-mail: ademir@ q mc.uf sc.b r
Catalytic Promiscuity: Catecholase-like Activity and Hydrolytic DNA Cleavage
Promoted by a Mixed-Valence F e
I I IF e
I IComp lex
Ademir Neves,*,a Adailton J. Bortoluzzi,a Rafael Jovito, a Rosely A. Peralta,a
Bernardo de Souza,a Bruno Szpoganicz,a Antônio C . Joussef,a H ernán T erenzi,b
Patricia C . Severino,b F ranciele L . F isch er,b G erh ard Sch enk ,c M ark J. Riley,c
Sarah J. Smithc and L aw rence R. G ah anc
aLABINC, Departamento de Química and bCentro de Biologia M olecular E s trutural,
Departamento de Bioq uímica, U niv ers idade F ederal de Santa Catarina, 8 8 0 4 0 - 9 0 0 F lorianópolis - SC, Brazil
cSchool of Chemis try and M olecular Bios ciences , T he U niv ers ity of Queens land,
Bris bane 4 0 7 2 , Aus tralia
O s estudos sob re enzimas têm mostrado q ue ex iste uma relação clara entre a ev olução enzimática e a p rop riedade de p romiscuidade catalítica q ue elas p odem ap resentar. C om isto, o desenv olv imento de sistemas b iomiméticos cap azes de atuar de modo p romíscuo, de f orma similar a alg umas enzimas, mostrou-se um nov o desaio p ara os q uímicos b ioinorg ânicos. Neste trab alh o, rep ortamos a estrutura de raios X e os estudos em solução e de p romiscuidade catalítica do comp lex o [ ( b p b p mp ) F eI I I(µ-O A c)
2F e I I] ( C lO
4) , (1) , contendo o lig ante b inucleante não-simétrico
2-{ [ ( 2-h idrox ib enzil) ( 2-p iridilmetil) aminometil] -4 -metil-6 -[ b is( 2-p iridilmetil) aminometil] } f enol ( H2b p b p mp ) . E studos de titulações p otenciométrica e esp ectrof otométrica e cinéticos mostraram q ue esse comp osto de coordenação g era esp écies ativ as cap azes de cliv ar o dsD NA ( D NA de ita dup la) h idroliticamente, com um aumento de 1,9 ×108 na tax a de reação em comp aração com a reação
não catalisada, e cap azes de ox idar o 3,5 -di-terc-b utilcatecol ( 3,5 -dtb c) , com kcat = 1,16 × 10-2 s-1
e KM = 7,1×10-4 mol L-1. A ssim, o comp lex o 1 ap resenta ativ idades de h idrolase e ox idoredutase,
sendo um interessante modelo sintético p ara estudos de p romiscuidade catalítica.
C ataly tic p romiscuity h as emerg ed as an imp ortant prop erty of many enzy mes since th e relationsh ip of th is p rop erty to enzy matic ev olution b ecame clear. Simultaneously , th e dev elop ment of suitab le b iomimetic cataly tic sy stems cap ab le of mimick ing th e p romiscuous cataly tic p rop erties
of such enzy mes rep resents a new ch alleng e f or b ioinorg anic ch emists. I n th is p ap er w e rep ort on th e X -ray structure, th e solution studies and th e promiscuous cataly tic activ ity of th e mix ed-v alence
comp lex [ ( b p b p mp ) F eI I I(µ-O A c) 2F e
I I] ( C lO
4) , (1) , containing th e unsy mmetrical dinucleating
lig and 2-{ [ ( 2-h y drox y b enzy l) ( 2-p y ridy lmeth y l) aminometh y l] -4 -meth y l-6 -[ b is( 2-p y ridy lmeth y l) aminometh y l] } p h enol ( H2b p b p mp ) . Potentiometric and sp ectrop h otometric titrations and k inetics
studies sh ow ed th at th is coordination comp ound g enerates activ e sp ecies th at p romote h y droly tic cleav ag e of doub le strand D NA ( dsD NA ) , w ith a rate enh ancement of 1.9 ×108 ov er th e non-cataly zed
reaction, as w ell as p romote ox idation of 3,5 -di-tert-b uty lcatech ol ( 3,5 -dtb c) , w ith kcat = 1.16 × 10-2 s-1
and KM = 7.1×10-4 mol L-1. T h us, comp lex 1 sh ow s b oth h y drolase and ox idoreductase activ ities
and can b e reg arded as a man-made model f or study ing cataly tic p romiscuity .
K eyw ords: mix ed-v alence F eI I IF eI I comp lex , unsy mmetrical lig and, cataly tic p romiscuity ,
diester and D NA cleav ag e, catech olase activ ity
I ntroduction
C ataly tic p romiscuity means, in th e b roadest sense, th e ab ility of a g iv en natural or non-natural active site
to cataly ze more th an one ch emical transf ormation. I t
constitutes a v ery imp ortant p rop erty of many enzy mes, h av ing a natural role in ev olution and, occasionally , in th e b iosy nth esis of secondary metab olites.1-7 F or ex amp le,
it h as b een demonstrated th at v anadium-dep endent
v anadate is not p resent, indicating th at its activ e site is v ery similar to th at of th e acid p h osp h atases.8 O n th e oth er
h and, it w as f ound th at an acid p h osp h atase f rom p lants ( in contrast to th e k idney b ean p urp le acid p h osp h atase ( PA P) )
uniq uely ex h ib its ch lorop erox idase activ ity w ith loss of p h osp h atase activ ity w h en orth ov anadate is added to th e ap o
f orm of th e enzy me.9 A lth oug h th ese activ ities constitute
v ery dif f erent ov erall reactions, th ey inv olv e enzyme-b ound sp ecies th at are structurally related. F urth ermore, it h as b een demonstrated th at th e PA P isolated f rom tomato may act as a b if unctional enzy me th at op erates in th e h y droly sis of p h osp h ate-esters and also disp lay s sig niicant alk aline p erox idase activ ity w ith th e inv olv ement of reactive ox y g en sp ecies.10 I ndeed, sev eral p lant PA Ps h av e b een rep orted
to b e ex p ressed in resp onse to p ath og en attack or elicitor treatment.11,12 Nev erth eless, no catech olase activ ity f or any
of th e w ell ch aracterized PA Ps h as as y et b een describ ed. W h ile sev eral enzy mes disp lay ing cataly tic p romiscuity h av e b een th e sub j ect of recent related inv estig ations,1-7
th ere are f ew ex amp les of sy nth etic analog ues w h ich ex h ib it such multif unctional activ ities.13-18
I n recent y ears, considerab le attention h as b een dev oted to th e sy nth esis, X -ray structural analy sis and p h y sicoch emical ch aracterization of structural and f unctional dinuclear F eI I IMI I mimetics f or th e activ e site
of metalloh y drolases such as p urp le acid p h osp h atase.19 -21
I n p articular, w e h av e successf ully dev elop ed a g eneral meth od f or th e p rep aration of mix ed-v alence h omo- and h etero-dinuclear MI I IMI I comp lex es, using th e
unsy mmetrical lig and ( H2b p b p mp ) 2-{ [ ( 2-h y drox y b enzy l) ( p y ridy lmeth y l) aminometh y l] -4 -meth y l-6 -[ b is( 2-p y ridy lmeth y l) aminometh y l] } 2-p h enol ( Sch eme 1) .22-32
F or ex amp le, it h as b een estab lish ed th at th e [ ( b p bp mp ) F eI I I(µ-O A c)
2F e
I I] ( C lO 4)
•H
2O comp lex , (1) , is a g ood
model f or th e electroch emical p rop erties of uterof errin, w ith a reduction p otential f or th e F eI I IF eI I I to F eI I IF eI I
coup le of 38 0 mV (v ers us th e normal h y drog en electrode,
NH E ) , w h ich comp ares w ell w ith th e 34 4 mV measured f or uterof errin at p H 4 .1 v ia direct electroch emistry and
36 7 mV at p H 5 v ia microcoulometry .9 , 33,34 I t h as also b een
w ell documented th at comp lex 1 and its corresp onding
ox idized comp lex [ ( b p b p mp ) F eI I I(µ-O A c) 2F e
I I I] ( C lO 4)2,
(2) , are g ood models f or th e resonance R aman p rop erties of mammalian PA Ps in th eir reduced and ox idized f orms, resp ectiv ely .30 I n addition, to understand th e sp ectral
sig natures of th e dinuclear center and to relate these to th e di-iron enzy me uterof errin, sp ectroelectroch emical, sp ectroscop ic, and comp utational studies, and p h osph atase-lik e reactiv ity of th e [ ( b p b p mp ) F eI I I(µ-O A c)
2F e I I]+ ion
comp lex h av e also b een rep orted.31 Very recently , th e
[ ( b p b p mp ) F eI I I(µ-O A c) 2F e
I I]+ comp lex h as b een used as an
electroch emical b iomimetic sensor f or th e determination of p h enolic comp ounds.32
I n th is study , w e describ e th e X -ray structure and solution studies of th e mix ed-v alence comp lex [ ( b p b p mp ) F eI I I(µ-O A c)
2F e
I I] ( C lO
4)•H2O , (1) , w h ich is ab le to cleav e
p h osp h odiester b onds of th e activ ated sub strate b is( 2,4 -dinitrop h eny l) p h osp h ate ( 2,4 -b dnp p )35 and of D NA itself ,
b ut also sh ow s sig niicant cataly tic activ ity in th e ox idation of th e model sub strate 3,5 -di-tert-b uty lcatech ol ( 3,5 -dtb c)
and can th us b e considered as a p romiscuous man-made cataly st.
E xp erimental
M aterials and meas urements
A ll reag ents of h ig h p urity g rade w ere p urch ased f rom commercial sources and used as receiv ed. I n the
ch aracterization of H2b p b p mp and its comp lex es, and in th e k inetic studies, sp ectroscop ic g rade solv ents f rom M erck , dried using molecular siev es, w ere used.
T h e p otentiometric studies w ere carried out w ith a M icronal 375 p H meter itted w ith b lue-g lass and calomel ref erence electrodes calib rated to read –log [ H+] directly ,
desig nated as p H . Bidistilled w ater in th e p resence of K M nO4 w as used to p rep are C H3C N ( 1: 1 v / v ) solutions. T h e electrode w as calib rated using th e data ob tained f rom a p otentiometric titration of a k now n v olume of a standard 0.0100 mol L-1 H C l solution w ith standard 0.100 mol L-1
K O H . T h e temp erature w as k ep t at 25 .00 ± 0.05 °C , and th e ionic streng th w as adj usted to 0.100 mol L-1 b y
addition of K C l. T h e ex p erimental solutions w ere titrated w ith 0.100 mol L-1 standard C O
2-f ree K O H . E q uilib rium
measurements w ere tak en using solutions containing 0.05 mmol of comp lex . T h e ex p eriments w ere p erf ormed on 5 0.00 mL of th e C H3C N: H2O ( 1: 1 v / v ) solution in a th ermostated cell p urg ed w ith arg on, using 0.100 mol L-1
K O H solutions. T h e ex p eriments w ere carried out in trip licate. C omp utations of th e trip licates w ere all carried out w ith th e BE ST 7 p rog ram, and th e sp ecies diag rams w ere ob tained w ith SPE and SPE PL O T p rog rams.36
C omp lex es 1 and 2 w ere also titrated sp ectrop h
oto-metrically . A ll titrations w ere carried out under an arg on atmosp h ere at 25 ºC in 1: 1 ( v / v ) C H3C N: H2O . T h e p H w as irst adj usted w ith H C l and th e comp lex titrated w ith successiv e additions of C O2 f ree K O H 0.124 mol L-1. A t each measured
p H , an aliq uot w as remov ed to record th e U V-Vis sp ectrum. A dark red cry stal w as selected f rom th e samp le of comp lex 1 and ix ed at th e end of a g lass ib er f or X -ray
analy sis. C ry stallog rap h ic measurements of comp lex 1 w ere
carried out w ith an E nraf -Nonius C A D 4 dif f ractometer w ith g rap h ite-monoch romated M o-Kα radiation, at room
temp erature. C ell p arameters w ere determined f rom 25 caref ully centered relections using a standard p rocedure.37
A ll data w ere corrected f or L orentz and p olarization ef f ects.38 A semi-emp irical ab sorp tion correction b ased on
th e azimuth al scans of sev en ap p rop riate relections w as also ap p lied to th e collected intensities w ith th e PL A T O N p rog ram,39 ,4 0 w ith max imum and minimum transmission
f actors of 0.8 73 and 0.79 1, resp ectiv ely . T h e structure w as solv ed b y direct meth ods and reined b y f ull-matrix least-sq uares meth ods using SI R 9 74 1 and SH E L X L 9 74 2 p rog rams,
resp ectiv ely . A ll non-h y drog en atoms w ere ref ined anisotrop ically . H atoms attach ed to C atoms w ere placed at
th eir idealized p ositions, w ith C -H distances and Ueq v alues
tak en f rom th e def ault setting s of th e reinement p rog ram. T h e w ater molecule H atoms w ere f ound f rom a F ourier dif f erence map and treated w ith th e riding model. Tw o ox y g en atoms of th e p erch lorate counterion are disordered ov er tw o alternativ e p ositions. T h e site occup ancies of
0.5 5 5 ( 14 ) and 0.4 4 5 ( 14 ) f or th e disordered atoms w ere reined and th e g eometric p arameters around th e ch lorine atom w ere constrained. F urth er selected cry stallog rap h ic data are summarized in T ab le 1.
Sy nthes es
T h e unsy mmetric dinucleating lig and H2b p b p mp w as p rep ared according ly to p rocedures p rev iously describ ed in th e literature.23
[ ( bpbpmp) F eIII(µ- O Ac)
2F e
II] ( ClO
4)•H2O , (1)
0.5 mmol of H2b p b p mp w ere dissolv ed in 10 mL of C H3C N under arg on. T o th is solution w ere added, resp ectiv ely , 1 mmol of F e( C lO4)2•6 H
2O and 2 mmol of
NaO A c in M eO H ( 10 mL ) also under arg on. T h e resulting solution b ecame deep p urp le. F inally , 5 mL of diisop rop y l eth er in th e p resence of air w ere added to th e p urple
solution, w h ich w as k ep t in th e ref rig erator. A f ter 24 h cry stals suitab le f or X -ray cry stallog rap h y w ere f ormed, w h ich w ere collected b y iltration ( 0.32 g , 70 % ) . Elemental
A naly sis f or C38H4 1C lF e2N5O11 ( 8 9 0.9 1 g mol-1) : F ound C ,
5 1.5 6 ; H , 4 .6 0; N, 7.78 . C alculated C , 5 1.23; H , 4 .6 4 ; N, 7.8 6 % . T h e comp ound is stab le in air in th e solid state.
(C aution! As w ith all perchlorate s alts , s amples s hould be handled w ith care.)
[ ( bpbpmp) F eIII(µ- O Ac)
2F e
III] ( ClO
4)2•3 CH3O H•3 H2O ( 2)
T o a solution of F e( C lO4)3•9 H
2O ( 2.0 mmol) in 20 mL
of meth anol, sodium acetate trih y drate ( 2.5 mmol) and
H2b p b p mp ( 1.07 mmol) w ere added. T h e clear dark b lue solution w as h eated to 4 0 °C and stirred f or 10 min at
amb ient atmosp h ere. A f ter cooling of th e solution to room temp erature, a b lue microcry stalline p recip itate
w as f ormed, w h ich w as f iltered of f and w ash ed w ith 2-p rop anol and eth er ( 0.73 g , 6 5 % ) . E lemental A naly sis f or C4 1H5 7C l2F e2N5O20 ( 1121.17 g mol-1) : F ound C , 4 4 .3;
H , 4 .5 8 ; N, 6 .24 . C alculated C , 4 3.8 7; H , 5 .12; N, 6 .24 % .
K inetics
C atech olase-lik e activ ity tow ard th e ox idation of th e model sub strate 3,5 -di-tertb uty lcatech ol ( 3,5
-dtb c) w as inv estig ated. T h e ex p eriments w ere f ollowed sp ectrop h otometrically on a Varian C ary 5 0 Bio sp ectrop h otometer connected to a comp uter, b y monitoring th e increase in th e 3,5 -di-tert-b uty l-q uinone ( 3,5 -dtb q )
ch aracteristic ab sorp tion b and at 4 00 nm ov er time. T h e conv ersion of th e reaction rate units w as carried out using
ε = 176 0 L mol-1 cm-1 ( measured in th e reaction conditions)
f or 3,5 -di-tert-b uty lq uinone, and th e initial concentration
of comp lex .
T h e ex p eriments w ere carried as f ollow s: a stock solution of th e comp lex w as p rep ared in meth anol ( 16 h earlier) , saturated w ith arg on ( 4 .4 ×10-4 mol L-1) . A stock solution of
th e sub strate w as also p rep ared in meth anol ( [ 3.5 -dtb c] = 8 .26 ×10-2 mol L-1) . T o a 4 mL cuv ette, 100 µL of arg
on-saturated b uf f er and 100 µL of 1 w ere added. Sub seq uently ,
O2-saturated meth anol, deionized w ater and sub strate
( 20-200 µL ) w ere added to comp lete 3.0 mL ( g iv ing th e p rop ortion 1.8 mL meth anol : 1.2 mL w ater, v / v ) .
T h e initial rate w as ob tained f rom th e slop e of th e ab sorb ance v s. time p lot ov er th e irst 10 min of reaction.
E ach ex p eriment w as p erf ormed at least th ree times, and th e ob tained p lots w ere analy zed b y th e initial rate meth od.
A k inetic treatment using nonlinear M ich aelis-Mn ente ap p roach w as ap p lied.
T h e inluence of th e p H on th e cataly zed ox idation of th e 3,5 -di-tert-b uty lcatech ol w as determined ov er th e
C H3O H / H2O , 3: 2 v / v , [1] = 1.39 ×10-5mol L-1, [ 3,5 -dtb c] =
2.77×10-3 mol L-1, [ b uf f er] = 3.3×10-2 mol L-1 ( Buf f er =
M E S, 2-[N-morp h oline] eth anesulf onic acid, or T R I S,
tris( h y drox y meth y l) aminometh ane) . D ep endence of th e reaction rates on [ 3,5 -dtb c] w as determined at p H
6 .0, in a meth anol: w ater ( 3: 2, v / v ) solution. C onditions: [1] = 1.4 6 ×10-5 mol L-1; [ 3,5 -dtb c] = 0.5 -5 .5 ×10-3 mol L-1,
[ b uf f er] = 3.3×10-2 mol L-1 ( b uf f er = M E S) .
T o determine w h ich reduced ox y g en sp ecies ( h y drog en p erox ide or w ater) w as f ormed during th e reaction, mix tures
w ere p rep ared as in th e k inetics ex p eriments. A f ter 1 h of reaction an eq ual v olume of H2SO4 ( 5 ×10-3 mol L-1)
w as added. T h e q uinone w as w ash ed tw ice w ith dich lorometh ane ( 2×3 mL ) . T o a 2 mL aliq uot of th e aq ueous lay er a K I solution ( 1 mL , 0.30 mol L-1) w as added.
F inally , ammonium moly b date ( 0.1 mol L-1) solution w as
added in cataly tic amounts to sp eciically accelerate th e f ormation of I3– in th e p resence of H
2O2. T h e increase in
th e I3−ab sorp tion b and at 35 3 nm (ε = 26 ,000 L mol-1 cm-1)
w as monitored b y U V-Vis sp ectroscop y .4 3-4 5 T h e reaction
w ith out ox y g en w as p rep ared under th e k inetic conditions and th e ab sorb ance increase at 4 00 nm due to q uinone
release w as not ob serv ed.
DNA ex traction
Plasmid D NA ( p BSK I I ) ( Stratag ene) w as p roduced in E s cherichia coli and p uriied w ith H iSp eed M ax ip rep K it ( Qiag en) f ollow ing th e p rotocol describ ed b y the manuf acturer.
DNA cleav age under anaerobic and aerobic conditions
C omp lex 1 solutions w ere diluted in acetonitrile
in an arg on-f illed g lov e b ag to av oid F e ox idation. A tw o-step p rocedure w as used to ob tain deox y g enated w ater, p ip erazine-1,4 -b is( 2-eth anesulf onic acid) ( PI PE S) b uf f er and eth y lenediaminetetraacetic acid ( edta) .
A p arallel ex p eriment w ith th e ox idativ e cleav ag e sy stem [ F e( edta) ]2/ D T T ( dith ioth reitol) w as used to demonstrate th e deox y g enation p rocedure ef iciency .4 6 ,4 7 D NA cleav ag e
ex p eriments w ere p erf ormed in a inal v olume of 20 µL containing 35 µmol L-1 b p ( D NA b ase p airs) p BSK I I
p lasmid D NA , comp lex 1 [ 4 0, 8 0 and 16 0 µmol L-1] , 25 %
acetonitrile ( v / v ) and 25 mmol L-1 PI PE S b uf f er, p H 6 .0.
R eaction mix tures w ere incub ated at 5 0 oC f or 2 h in a
sealed arg on-illed v acuum desiccator.
I dentical comp lex 1 solutions as p rep ared in arg on-illed
b ag s w ere used f or th e reactions under aerob ic conditions. A ll oth er conditions and p rocedures w ere essentially th e same as th ose describ ed f or anaerob ic reactions.
R eactions w ere stop p ed b y ch illing tub es on ice and adding 4 µL of ice-cold loading -b uf f er ( 5 0% g ly cerol, 0.2 mol L-1 edta, 0.05 % b romop h enol b lue) . Samp les w ere
sub mitted to ag arose g el electrop h oresis and stained w ith eth idium b romide. R esulting g els w ere dig italized w ith a p h oto-documentation sy stem ( U VP, C A , U SA ) and D NA b ands w ere q uantiied using L ab W ork s sof tw are v ersion 4 .0 ( U VP) .
K inetics DNA as s ay s
D NA cleav ag e reactions w ere p erf ormed at 5 0 oC in
25 mmol L-1 PI PE S ( p H 6 .0) , using 35 µmol L-1 b p ( D NA
b ase p airs) p BSK I I p lasmid D NA at concentrations of comp lex 1 rang ing f rom 0 to 16 0 µmol L-1, in a inal v olume
of 16 0 µL . A liq uots of 20 µL w ere w ith draw n at dif f erent time interv als ( 0-6 0 min) and w ere analy zed as describ ed
f or th e D NA cleav ag e.22, 4 8
R eaction mechanis m
C leav ag e ex p eriments to understand th e reaction mech anism w ere carried out w ith p otential inh ib iting ag ents
such as a h y drox y l radical scav eng er dimeth y l sulf ox ide ( dmso) , a sing let ox y g en scav eng er ( sodium azide, NaN3) and a minor g roov e b inder ( distamy cin) .7,4 9 -5 1
R eactions in th e p resence of th e ab ov e comp ounds w ere carried out in 20 µL inal v olume, containing : 35 µmol L-1
b p ( p lasmid D NA ) , in 25 mmol L-1 PI PE S, p H 6 .0, f or
1 h at 5 0 oC ( oth er conditions are describ ed in th e F ig ure
leg ends) . R eactions w ere stop p ed b y ch illing tub es on ice and adding 4 µL of ice-cold loading -b uf f er. Samp les w ere analy zed in 0.8 % ag arose g el.
R esults and Discussion
Sy nthes is of 1 and 2 and cry s tal s tructure of 1
R eaction of one eq uiv alent of th e unsy mmetrical lig and H2b p b p mp w ith tw o eq uiv alents of [ F e( O H2)6] ( C lO4)2 in th e p resence of NaO A c in a 1: 1 mix ture of C H3C N and meth anol results in th e f ormation of th e title comp lex [ ( b p b p mp ) F eI I I(µ-O A c)
2F e
I I] ( C lO 4)•H2O
(1) . T h e p rop erties determined b y elemental, U V-Vis and electroch emical analy sis are in ag reement w ith th e comp osition p rev iously p rop osed f or 1.29 C ry stals of 1
suitab le f or X -ray cry stallog rap h y w ere ob tained b y slow dif f usion of diisop rop y l eth er in a meth anolic solution of
of ex cess p erch lorate, th e ox idized dinuclear comp lex [ ( b p b p mp ) F eI I I( µ-O A c)
2F e
I I I] ( C lO 4)
•3C H
3C O H
•3H
2O (2)
w as ob tained. T h is comp lex w as ch aracterized b y elemental analy sis, electronic sp ectroscop y , and electroch emistry / sp ectroelectroch emistry . A ll attemp ts to ob tain suitab le cry stals of 2 f or X -ray analy sis w ere unsuccessf ul.
T h e structure of comp lex 1 consists of discrete dinuclear
[ F eI I I( b p b p mp ) (µ-O A c) 2F e
I I]+ cations, uncoordinated
p erch lorate anions and one molecule of w ater in th e
cry stallog rap h ic asy mmetric unit. A n O R T E P draw ing of 1 w ith its atom-lab eling sch eme is sh ow n in F ig ure 1. Selected b ond distances and ang les are g iv en in T ab le 2.
T h e structure of th e cation [ F eI I I( b p b p mp ) (µ-O A c) 2F e
I I]+
in 1 is similar to th at determined f or th e corresp onding BF4− salt.31 Briely , in th e structure of [ F eI I I( b p b p mp ) (µ-O A c)
2F e I I]+
th e metal centers are b ridg ed b y th e p h enolate ox y gen atom of th e b p b p mp2− lig and and b y tw o ex og enous acetate lig ands, as h as b een ob serv ed in numerous isostructural comp lex es containing th e same lig and.22-29 A s ex p ected
f or a dinuclear mix ed-v alence comp lex containing the unsy mmetrical b p b p mp2−lig and, th e coordination sp h ere of th e F eI I I is comp leted b y tw o nitrog en atoms f rom th e tertiary
amine ( N1) and p y ridy l ( N22) g roup s, and b y a terminal p h enolate ox y g en ( O 10) , w h ile th e F eI I is on th e sof ter side
of b p b p mp2−, coordinated b y th ree nitrog en atoms, p y ridy l N32 and N4 2 and tertiary amine N2.
T h e b ridg ing Op h enolate is coordinated unsy mmetrically to th e tw o metal centers, w ith th e b ond distances F eI I I−O = 2.005 ( 3) and F eI I−O = 2.102( 3) Å b eing
slig h tly long er th an th ose ob serv ed in th e mix ed-v alence comp lex [ F eI I IF eI I( b p mp ) ( O Pr)
2]
2+ ( F eI I I−O = 1.9 4 3( 2)
and F eI I−O 1 = 2.09 0( 2) Å) containing th e sy mmetric
H b p mp ( 2,6 b is[ ( b is( 2p y ridy lmeth y l) amino) meth y l] -4 -meth y lp h enol) lig and.29 A s in th e analog ous F eI I IMI I
comp lex es ( MI I = Z n, C u, Ni, M n) , th e terminal p h enolate
coordinated to th e F eI I I is trans to th e b ridg ing p h enolate.
T h e metal-metal distance ( 3.5 04 1( 13) Å) as w ell as th e F eI I I-O -F eI I ang le ( 117.14 ( 13)°) in th e F eI I IF eI I comp lex
are similar to th ose ob serv ed in all of th e isostructural F eI I IMI I comp lex es ( 3.4 70-3.5 10 Å and 115 -119 °) .22-29
A dditionally , th e terminal p h enox o–F eI I I-O
p h enolate b ond
leng th ( 1.9 02( 3) Å) in 1 is slig h tly long er th an th e
corresp onding b ond in th e F eI I IZ nI I comp lex ( 1.8 9 0 Å) .5 2
Solution s tudies of 1 and 2
Potentiometric titration studies of th e comp lex [ F eI I I( b p b p mp ) (µ-O A c)
2F e I I] C lO
4 (1) in 1: 1 acetonitrile/
b uf f er h av e b een describ ed p rev iously and sh ow ed the p resence of f our p rotonation eq uilib ria, tw o of w h ich ( pKa2 = 4 .13 and pKa4 = 7.5 3) are cataly tically relev ant f or th e h y droly sis of p h osp h ate diester b onds.31 R esults
are summarized in T ab le 3, w ith th e k inetic pKas of 1 f or comp arison.
C onsidering th at th e ox idized f orm ofcomp lex 2 in
aq ueous solution is th e orig in of th e cataly tically activ e
T able 1 . C ry stal data and structure reinement f or comp lex 1
E mp irical f ormula 38HC4 1C lF e2N5O11
F ormula w eig h t 8 9 0.9 1
T emp erature 29 3( 2) K
W av eleng th 0.71073 Å, M o-Kα
C ry stal sy stem, sp ace g roup monoclinic, P21/n
U nit cell dimensions a = 12.74 6 ( 3) Å b = 18 .309 ( 4 ) Å c = 17.077( 3) Å
β = 9 3.8 7( 3)o
Volume 39 76 .0( 14 ) Å3
Z 4
C alculated density 1.4 8 8 M g / m3
µ 0.8 6 3 mm-1
F ( 000) 18 4 4
C ry stal size 0.4 7 × 0.20 × 0.16 mm
T h eta rang e f or data collection o2.06 to 25 .00o
I ndex rang es ≤0 h ≤ 15 , 0 ≤ k ≤ 21, –20 ≤ l ≤ 20
R elections collected / uniq ue 7331 / 6 9 9 6 ( Rint = 0.034 6 )
R einement meth od F ull-matrix least-sq uares on F2
D ata / restraints / p arameters 6 9 9 6 / 14 0 / 5 33
G oodness of it ( F2) 0.9 74
R indices [ I > 2σ( I ) ] 1 = 0.04 31, w RR 2 = 0.1024
R indices ( all data) 1 = 0.1275 , w RR 2 = 0.114 5
L arg est dif f . p eak and h ole 0.36 4 and −0.375 e. Å-3
F igure 1 . O R T E P p roj ection of th e molecular structure of th e cation of comp lex 1. E llip soids are sh ow n at th e 4 0% p rob ab ility lev el. H atoms
sp ecies f or th e ox idation of catech ols (v ide inf ra) , w e also carried out p otentiometric titration of th is comp lex under ex p erimental conditions identical to th ose emp loy ed f or comp lex 1. A s can b e ob serv ed in F ig ure 2, f our mol
of K O H w ere neutralized in th e p H rang e of 2.4 -10.9. F itting th e data w ith th e BE ST p rog ram results in th e f ollow ing dep rotonation constants: pKa2 = 3.14 , pKa3 = 4 .22,
pKa4 = 6 .4 5 and pKa5 = 9 .6 5 . I t is w ell k now n th at in related
MI I I(µ-O A c) 2M
I I dinuclear comp lex es, th e b ridg ing
carb ox y late g roup s dissociate resulting in th e f ormation of F eI I I-O H / O H
2 sp ecies w h en in aq ueous solution. T h us,
th e f our-dep rotonation/ p rotonation eq uilib rium step s f or comp lex 2 are p rop osed in Sch eme 2 w ith th e corresp onding
sp ecies distrib ution g iv en in F ig ure 2. I t is assumed th at w h en 2 is dissolv ed in aq ueous solution at p H 2.4 , tw o
acetate g roup s are released, leading to th e [ ( O H2) F eI I I(µ-O H )
F eI I I( O H
2) ] sp ecies ( structure A in Sch eme 2) .
D ep rotonation of th e w ater molecule ( pKa2 = 3.14 ) coordinated to th e F eI I I center containing th e sof ter terminal
tridentate g roup ( one amine and tw o p y ridine) leads to th e f ormation of th e [ ( O H2) F eI I I(µ-O H ) F eI I I( O H ) ] sp ecies
( structure B in Sch eme 2) . T h is ob serv ation is b ased mainly on th e f act th at th e F eI I I center b ound b y th ree nitrog ens
of b p b p mp2− is a strong er L ew is acid th an th e F eI I I center
coordinated b y th e h arder N2O -donor p endant arm containing
T able 2 . Selected b ond leng th s ( Å) and ang les ( deg ree) f or comp lex 1
F e1-O 10 1.9 02( 3) F e2-O 5 2 2.032( 3)
F e1-O 6 1 1.9 6 3( 3) F e2-O 1 2.102( 3)
F e1-O 1 2.005 ( 3) F e2-O 6 2 2.127( 3)
F e1-O 5 1 2.022( 3) F e2-N4 2 2.14 3( 3)
F e1-N22 2.19 2( 3) F e2-N32 2.217( 3)
F e1-N1 2.223( 4 ) F e2-N2 2.219 ( 3)
F e1-F e2 3.5 04 1( 13)
O 10-F e1-O 6 1 9 0.20( 13) O 5 2-F e2-O 6 2 9 5 .75 ( 13)
O 10-F e1-O 1 175 .6 0( 12) O 1-F e2-O 6 2 8 8 .35 ( 12)
O 6 1-F e1-O 1 9 4 .17( 12) O 5 2-F e2-N4 2 100.75 ( 13)
O 10-F e1-O 5 1 8 9 .9 1( 13) O 1-F e2-N4 2 16 3.8 3( 13)
O 6 1-F e1-O 5 1 100.12( 13) O 6 2-F e2-N4 2 8 4 .4 1( 13)
O 1-F e1-O 5 1 8 9 .8 0( 12) O 5 2-F e2-N32 9 3.25 ( 13)
O 10-F e1-N22 9 5 .36 ( 13) O 1-F e2-N32 9 0.16 ( 12)
O 6 1-F e1-N22 9 2.31( 13) O 6 2-F e2-N32 170.9 6 ( 13)
O 1-F e1-N22 8 3.9 9 ( 12) N4 2-F e2-N32 9 4 .73( 13)
O 5 1-F e1-N22 16 6 .4 9 ( 13) O 5 2-F e2-N2 170.4 6 ( 13)
O 10-F e1-N1 8 7.5 3( 13) O 1-F e2-N2 8 9 .4 1( 12)
O 6 1-F e1-N1 16 8 .00( 13) O 6 2-F e2-N2 9 3.12( 13)
O 1-F e1-N1 8 8 .09 ( 12) N4 2-F e2-N2 76 .6 1( 13)
O 5 1-F e1-N1 9 1.6 7( 13) N32-F e2-N2 77.9 5 ( 13)
N22-F e1-N1 76 .19 ( 13) F e1-O 1-F e2 117.14 ( 13)
O 5 2-F e2-O 1 9 4 .33( 11)
S cheme 2. Prop osed eq uilib ria f or th e comp lex [ F eI I I( b p b p mp ) (µ-O A c) 2F e
I I I]2+in C H
3C N: H2O 1: 1.
F igure 2. Sp ecies diag ram f or th e p otentiometric titration of th e comp lex [ F eI I I( b p b p mp ) (µ-O A c)
2F e I I I] ( C lO
4)2•H2Oin C H3C N: H2O 1: 1. A : [ ( O H2)
F eI I I(µ-O H ) F eI I I( O H
2) ] ; B: [ ( O H ) F e
I I I(µ-O H ) F eI I I( O H
2) ] ; C : [ ( O H )
F eI I I(µ-O ) F eI I I( O H
2) ] ; D : [ ( O H ) F e
I I I(µ-O ) F eI I I( O H ) ] ; E : [ ( O H ) F eI I I(µ-O )
th e terminal p h enolate g roup . T h e pKa3 of 4 .22 is attrib uted to dissociation of th e b ridg ing µ-O H g roup w h ich results in th e f ormation of [ ( O H2) F eI I I(µ-O ) F eI I I( O H ) ] ( structure C in
Sch eme 2) as one of th e cataly tically activ e sp ecies in th e ox idation of catech ols (v ide inf ra) . T h is pKa v alue is around 1.5 p H units low er th an th e corresp onding pKa determined in th e mix ed-v alence F eI I IF eI I comp lex 1 and can b e ex p lained
in terms of a h ig h er L ew is acidity ex erted b y tw o FeI I I centers
on th e b ridg ing µ-O H g roup in comp lex 2. D ep rotonation of
th e w ater molecule ( pKa4 = 6 .4 5 ) terminally coordinated to th e F eI I I center w h ich contains th e h arder tridentate N
2O -donor
g roup ( one amine, one p y ridine and one p h enolate) leads to th e f ormation of th e [ ( O H ) F eI I I(µ-O ) F eI I I( O H ) ] sp ecies
( structure D in Sch eme 2) w h ich is th e most activ e cataly st in th e ox idation of catech ols. A g ain, as ex p ected, th is pKa4 = 6 .4 5
is ap p rox imately one p H unit low er w h en comp ared to th e p rotonation/ dep rotonation eq uilib rium f ound f or th e corresp onding F eI I−O H
2 b ond in th e mix ed-v alence
comp lex 1 . M oreov er, it is also imp ortant to note th at th is pKa4
v alue is in g ood ag reement w ith th e pKa of 7.0 determined f or th e dissociation of th e second F eI I I-b ound w ater molecule in
a dinuclear F eI I I comp lex w h ich contains a similar NO -donor
set as th at p rop osed f or th e [ ( O H2) F eI I I(µ-O ) F eI I I( O H ) ]
sp ecies.5 3 F inally , a f ourth dep rotonation/ p rotonation
eq uilib rium at pKa5 = 9 .6 5 w as ob serv ed, desp ite th e f act th at in th e [ ( O H ) F eI I I(µ-O ) F eI I I( O H ) ] sp ecies th ere ap p ears to b e
no ionizab le p rotons under th ese ex p erimental conditions. W e tentativ ely sug g est th e sub stitution of one p y ridine g roup b ound to one of th e F eI I I centers b y one w ater molecule w ith
sub seq uent dep rotonation of th is w ater molecule as b eing resp onsib le f or th is eq uilib rium.5 3
U V-v isib le sp ectroscop y is anoth er tool emp loy ed to identif y sp ecies in eq uilib rium and can b e v ery usef ul to assig n th e cataly tically activ e sp ecies in solution. M oreov er, it sh ould b e noted th at comp lex es 1 and 2 disp lay lig
and-to-metal ch arg e transf er ( L M C T ) p rocesses w h ich are v ery sensitiv e to any electronic ch ang e (e.g. L ew is acidity ) in
th e metal centers. T h us, w e used electronic sp ectroscop y to f urth er conirm th e cataly tically activ e sp ecies of 1 and 2 under k inetic conditions.
Sp ectrop h ometric titration of th e mix ed-v alence comp lex
1 rev eals f our p rotonation/ dep rotonation constants wh ich
are comp arab le to th ose ob tained f rom th e p otentiometric ex p eriments ( T ab le 3, F ig ure S1) .31 I n g eneral, h y p soch romic
sh if ts are detected ( F ig ure 3) w h en th e p H of th e solution rises, sug g esting th at th e rep lacement of F eI I I-O H
2 b y
F eI I I-O H , or of F eI I I(µ-O H ) F eI I I b y F eI I I(µ-O ) F eI I I in th e
sy stems in eq uilib rium does af f ect th e energ y of the L M C T p rocesses. T h is is p rob ab ly due to a low er order of L ew is acidity of th e F eI I I-centers w h en th e strong er donor h y drox o
or ox o g roup s are coordinated, so th at th e t2 g orb itals in th ese sp ecies are at h ig h er energ y th an th e t2 g orb itals in th e F eI I I-O H
2 comp lex es. Similarly , sp ectrop h otometric titration
of th e ox idized f orm of comp lex 2 also demonstrates th at
th e λmax of th e L M C T is strong ly inluenced b y th e p H of th e solution ( F ig ure S2, F ig ure S3) . H ow ev er, in th is case only
th ree reliab le pKa v alues ( T ab le 3) could b e ob tained in th e p H rang e of 2.4 -10.9 , w h ich are in g ood ag reement with th ose calculated in th e p otentiometry studies.
T able 3 . Summary of pKa v alues in C H3C N: H2O 1: 1 at 25 ºC
C omp lex 1 . . . . C omp lex 2 k inetic ( h y droly sis)a
k inetic ( ox idation)b
p otentiometrica sp ectrop h otometricb p otentiometric sp ectrop h otometric
3.02 3.4 7 * * -
-4 .13 4 .4 4 3.14 nd 4 .74
-5 .76 6 .4 7 4 .22 3.9 5 - 5 .0
7.5 3 7.9 7 6 .4 5 6 .5 6 7.5 4 6 .7
- - 9 .6 5 9 .22 -
-aD ata f rom ref erence 11; bT h is w ork ; * T h ere is no eq uiv alent pK
a f or th is comp lex ; nd: Not determined.
F igure 3. Sp ectral ch ang es of th e comp lex [ F eI I I( b p b p mp ) (µ-O A c) 2F e
I I]
C lO4 during sp ectrop h otometric titration. T h e sp ectra w ere recorded in
C H3C N: H2O 1: 1 at successiv e p H . [ comp lex ] = 3×10
K inetics
I n recent y ears, it h as b een demonstrated th at many enzy mes are ab le to cataly ze distinctly more th an one ch emical reaction. T h is ab ility , called cataly tic promiscuity , may b e related to dif f erent k inds of b ond f ormation or cleav ag e or dif f erent cataly tic p ath w ay s or ev en b oth .1,5 4
Biomimetic models can b e more suscep tib le to cataly tic p romiscuity , since th ey do not h av e a comp lex structure as do enzy mes. I n th is reg ard, b iomimetic models are ex p ected to b e much more accessib le to dif f erent sub strates and more dep endent on th e env ironmental conditions.
T h e cataly tic p romiscuity of th e comp lex [ F eI I I( b p b p mp )
(µ-O A c)2F eI I] C lO
4, (1) , w as ev aluated b y testing its
h y drolase-lik e activ ity in th e cleav ag e of D NA and its catech olase-lik e activ ity tow ard th e ox idation of th e model sub strate 3,5 -di-tert-b uty lcatech ol ( 3,5 -dtb c) .31 T h e
p h osp h atase-lik e activ ity of 1 in th e h y droly sis of th e model
sub strate b is-2,4 -dinitrop h eny lp h osp h ate ( 2,4 -b dnp p ) h as b een demonstrated v ery recently .31
T h e catech olase activ ity of comp lex es 1 and 2 w as
determined using th e sub strate 3,5 -di-tert-b uty lcatech ol
b y f ollow ing sp ectrop h otometrically th e ab sorb ance increase due to th e f ormation of 3,5 -di-tert-b uty lq uinone
(λmax = 4 00 nm) , under conditions of ex cess sub strate. It is imp ortant to note th at th ere is no reaction ( not even one cy cle) using 1 as th e cataly st during a p eriod of th ree h ours in th e
ab sence of ox y g en. H ow ev er, af ter th is time, if th e cuv ettes w ere op ened to allow th e entrance of ox y g en, th e reaction b eg ins immediately . T h eref ore, th e k inetics data obtained sug g est th e f ormation of an ox idized f orm of F eI I IF eI I I as th e
cataly st, w h ich can b e ob tained th roug h ox idation of 1 in th e p resence of air or th roug h ch emical ox idation of 1 w ith H2O2 or f rom th e dissolution of th e comp lex 2 in k inetic conditions.
I ndeed, th e sp ectrum of 1 under k inetic conditions w ith out
th e sub strate sh ow s th e disap p earance of th e intervalence b and centered at 1100 nm, indicating th at th e sp ecies [ ( O H2) F eI I I(µ-O ) F eI I( O H ) ] is ox idized to comp lex [ ( O H
2) F e I I I(µ-O )
F eI I I( O H ) ] in th e p resence of air ( F ig ure S4 ) .
T h e p H -dep endence of th e ox idation reaction of 3,5 -dtb c w as ev aluated f rom p H 4 .4 to 9 .0 and, as can b e ob serv ed in F ig ure 4 , th ere are tw o distinct k inetic b eh av iors indicating th e p resence of dif f erent cataly sts. I nitially , th e reaction rate increases only slig h tly and reach es a p lateau in th e p H rang e 6 .0-6 .5 . A f urth er increase in p H results in an almost uninterrup ted increase in th e initial rate V0, sug g esting th at a second sp ecies is acting as th e cataly st. H ow ev er, f rom th e inlection p oints in th e p lot g iv en in F ig ure 4 , th e pKas at 5 .0 and 6 .7 are only tentativ ely attrib uted g iv en th e small increase in reactiv ity in th e p H rang e 4 .3-6 .5 and th e p oor deinition of th e p lateau at p H 7.0. E v en so, th ese
pKa v alues are in relativ ely g ood ag reement w ith th ose ob tained f rom th e p otentiometric and sp ectrop h otometric ex p eriments ( 4 .2 and 6 .5 ) , th us sug g esting th at th e [ ( O H2) F eI I I(µ-O ) F eI I I( O H ) ] ( C ) and [ ( O H ) F eI I I(µ-O ) F eI I I( O H ) ]
( D ) in Sch eme 2 are most p rob ab ly th e cataly tically activ e sp ecies in th e ox idation of 3,5 -dtb c. I ndeed, th e ob serv ed p H dep endence of th e reaction may b e due to th e successiv e
dep rotonation of F eI I I-coordinated w ater molecules, and
p rob ab ly th e F eI I I-O H g roup s are ab le to interact w ith
catech ol p romoting th e dep rotonation of th e sub strate and concomitant coordination to th e metal centers.
T h e dep endence of th e initial rates on th e concentration of 3,5 -dtb c using comp lex 1 as th e cataly st w as inv estig ated
at 25 ºC at p H 6 .0 and rev ealed saturation k inetics w ith M ich aelis-M enten-lik e b eh av ior ( F ig ure 5 ) .
F igure 4. p H dep endence f or th e ox idation of 3,5 -dtb c cataly zed b y 1.
C onditions: 3: 2 C H3O H : H2O , [1] = 1.39 ×10
-5 mol L-1, [ 3,5 -dtb c] =
2.77×10-3 mol L-1, [ b uf f er] = 3.3×10-2 mol L-1 ( b uf f er = M E S or T R I S) .
F igure 5. O x idation of 3,5 -dtb c cataly zed b y 1(), 2() and ox idized comp lex g enerated in s itu (1 w ith one eq uiv alent of H2O2) () :
D ep endence of th e reaction rates on [ 3,5 -dtb c] at p H 6 .0, in a meth anol/ w ater ( 3: 2) solution. C onditions: [1] = 1.5 ×10-5 mol L-1; [ 3,5 -dtb c] =
T h e k inetic p arameters ( Vmax= 1.6 9 ×10-7 mol L-1 s-1,
KM= 7.1×10-4 mol L-1 and k
cat= 11.6 ×10
-3 s-1) , w h ere K M and
kcat are th e M ich aelis-M enten and th e cataly tic constants, resp ectiv ely , w ere ob tained f rom M ich aelis-M enten nonlinear treatment ( V0 v s . [ 3,5 -dtb c] ) . T h e k inetic p arameters ob tained f or th e reaction cataly zed b y 2 are in th e
same order as th ose f ound f or comp lex 1 in th e p resence of
ox y g en or H2O2 ( T ab le 4 ) , th us sup p orting th e mech anistic p rop osal th at th e F eI I IF eI I I sp ecies must b e p resent to initiate
th e cataly tic cy cle. M oreov er, accumulation of H2O2 during
turnov er w as conf irmed b y means of th e moly b date-accelerated I3− assay , w h ich indicates th at reox idation of th e F eI IF eI I sp ecies b ack to th e activ e F eI I IF eI I I sp ecies occurs w ith
a 1: 1 ( O2: 3,5 -dtb c) stoich iometry and concomitant f ormation of h y drog en p erox ide.
F inally , p ap ers f ound in th e literature related to catech ol artiicial b iomimetics g enerally ref er to b inuclear C uI I
comp lex es.4 3-4 5 ,5 5 I t is interesting to note th at th e k inetic
p arameters ob tained in th is study f or comp lex 1 or
comp lex 2 are of th e same order as th ose f ound f or C uI IC uI I
comp lex es w ith similar lig ands, b ut w ith 1 or 2 b eing activ e
at low er p H v alues ( 6 .0 v s . 8 -9 ) ,4 3-4 5 w h ich is a conseq uence
of th e h ig h er L ew is acidity of F eI I I w h en comp ared to C uI I.
C onsidering all of th e ex p erimental data ob tained, th e mech anism sh ow n in Sch eme 3 is p rop osed.
DNA cleav age
T o v erif y th e ab ility of comp lex 1 to cleav e D NA
p h osp h odiester b onds, w e tested its activ ity in th e ab sence and in th e p resence of ox y g en. D ata f rom th e ex p eriments under aerob ic and anaerob ic conditions ( F ig ure 6 ) sug g est th at a h y droly tic mech anism is resp onsib le f or th e D NA cleav ag e, since th e reaction is not af f ected b y th e ab sence of ox y g en. T h e control ex p eriment w ith F e( edta)2–/
dith ioth reitol demonstrated th at th ere is no residual
ox y g en b ecause D NA cleav ag e w as totally inh ib ited under anaerob ic conditions.
T able 4 . K inetic p arameters of th e ox idation reaction of th e sub strate 3,5 -dtb c
1 2 + H12O2 ( 1: 1)
Vmax /( mol L
-1 s-1) 1.6 9 ×10-7 1.04 ×10-7 1.4 8 ×10-7
kcat / ( s-1) 11.6 ×10-3 6 .34 ×10-3 9 .6 3×10-3 KM / ( mol L
-1) 7.1×10-4 5 .3×10-4 4 .7×10-4
(kcat / KM) / ( L mol-1 s-1) 16 .33 11.9 6 20.4 8
S cheme 3. Prop osed mech anism f or ox idation of 3,5 -dtb c b y 1.
F igure 6 . Plasmid D NA cleav ag e b y comp lex 1 in anaerob ic ( lanes 1-5 )
and aerob ic ( lanes 6 -10) conditions. L anes 1 and 6 control D NA ( p BSK -I I 35 µmol L-1 b p ) ; lanes 2 and 7: D NA + comp lex 1, 4 0 µmol L-1; lanes 3
and 8 : D NA + comp lex 1, 8 0 µmol L-1; lanes 4 and 9 : D NA + comp lex 1,
16 0 µmol L-1; lanes 5 and 10: D NA + F e( edta)2− 25 mmol L-1 + D T T
2.5 mmol L-1. Samp les w ere incub ated f or 2 h at 5 0 °C , 25 mmol L-1
I t is also clear f rom F ig ure 6 th at 1 w as ab le to cleav e
p lasmid D NA in a concentration-dep endent manner. A n increase in circular ( nick ed, f orm I I ) p lasmid D NA , and a p rop ortional decrease in sup ercoiled D NA ( f orm I ) w as ob serv ed w ith increasing comp lex concentration.
Pseudo-M ich aelis-M enten conditions w ere used in th e k inetic studies of th e D NA cleav ag e b y 1.4 8 ,5 6 ,5 7 W e
ob serv ed th at th e decrease in sup ercoiled D NA itted a sing le ex p onential decay curv e ( F ig ure 7) . C leav ag e rates at v arious concentrations of 1 ( 4 0-16 0 µmol L-1) and a
constant D NA concentration w ere calculated and a p lot of
kob s v ersus th e concentration of 1 sh ow s saturation b eh av ior.
L inew eav er-Burk linearization of th ese data ( inset F ig ure 7) g iv es th e f ollow ing k inetics p arameters: kcat 1.9 1×10-3 s-1;
KM 7.9 6 ×10-4 mol L-1; k
cat/KM 2.4 0 mol s
-1 L-1, p rov iding a
rate enh ancement of 1.9 1×108 ov er th e uncataly zed doub
le-strand D NA cleav ag e.
F ig ure 8 p resents th e results of D NA cleav ag e b y 1 in th e
p resence of p otential inh ib iting ag ents: a h y drox y l radical scav eng er ( dmso) , a sing let ox y g en scav eng er ( sodium azide, NaN3) and a D NA minor g roov e b inder ( distamy cin) . Neith er dmso nor sodium azide interf ered w ith th e cleav ag e
activ ity of th e comp lex . T h ese results are in ag reement w ith th ose f ound under anaerob ic conditions, demonstrating th at
1 cleav es D NA h y droly tically and th at ox y g en radicals are
not inv olv ed in th e reaction. A lso in F ig ure 8 , th e activ ity of
1 is not inh ib ited b y distamy cin, sug g esting th at 1 interacts
w ith doub le strand D NA th roug h th e maj or g rov e.
Conclusions
T h e comp lex es [ ( b p b p mp ) F eI I I(µ-O A c) 2F e
I I] ( C lO 4) (1)
and [ ( b p b p mp ) F eI I I(µ-O A c) 2F e
I I I] ( C lO
4)2 (2) containing
th e unsy mmetrical dinucleating lig and H2b p b p mp ,
2{ [ ( 2h y drox y b enzy l) ( 2p y ridy lmeth y l) aminometh y l] -4 -meth y l-6 -[ b is( 2-p y ridy lmeth y l) aminometh y l] } p h enol, w ere sy nth esized and th eir p rop erties and cataly tic
f eatures ex p lored. T h e X -ray structure of 1 rev eals an
{ F eI I I(µ-O
p h enolate) (µ-O A c)2F e
I I} core in w h ich th e F eI I I and
F eI I metal centers are, resp ectiv ely , b ound b y th e h ard
and sof t side of th e b p b p mp2− lig and. K inetic studies on th e cleav ag e of doub le strand D NA ( dsD NA ) b y 1 at th e
op timum p H ( 6 .0) and 5 0 oC sh ow M ich aelis-M enten
b eh av ior w ith a rate enh ancement of 1.9 ×108 ov er th e
uncataly zed reaction. D ata f rom th e ex p eriments under aerob ic and anaerob ic conditions along w ith k inetic studies on a model diester sub strate sug g est a h y droly tic mech anism in w h ich a terminal F eI I I-b ound h y drox ide is th e
initiating nucleop h ile in th e cataly tic reaction. On th e oth er h and, w h en th e [ ( b p b p mp ) ( H O ) F eI I I(µ-O H ) F eI I( H
2O ) ] +
sp ecies is ex p osed to air, th e ox idized F eI I IF eI I I comp lex
is f ormed. T h is comp lex sh ow s sig niicant catech olase activ ity in th e ox idation of 3,5 -di-tert-b uty lcatech ol
( meth anol/ w ater b uf f er, 6 0: 4 0, p H = 5 .8 ) and th e reaction f ollow s M ich aelis-M enten b eh av ior w ith kcat = 1.16 ×10-2 s-1
and KM = 7.1×10-4 mol L-1. T h e comp lex is th us rev ealed
to b e an imp ortant ex amp le of a sy nth esized p romiscuous cataly st, w ith activ ity in b oth th e ox idation of catech ols and th e h y droly sis of p h osp h odiester b onds.
S up p lementary I nf ormation
F ig ures S1-S4 are av ailab le f ree of ch arg e at
h ttp : / / j b cs.sb q .org .b r, as a PD F ile. C ry stallog rap h ic data ( ex cluding structural f actors) f or th e structures in th is p ap er
h av e b een dep osited w ith th e C amb ridg e C ry stallog rap h ic D ata C entre as sup p lementary p ub lication numb er C C D C 74 76 19 . C op ies of th e data can b e ob tained f ree of ch arg e v ia w w w .ccdc.cam.ac.uk / conts/ retriev ing .h tml ( or f rom th e
C amb ridg e C ry stallog rap h ic D ata C entre, C C D C , 12 U nion R oad, C amb ridg e C B2 1E Z , U K ; f ax : + 4 4 1223 336 033 ; or e-mail: dep osit@ ccdc.cam.ac.uk ) .
F igure 7. K inetic analy sis of D NA cleav ag e b y 1. F inal comp lex
concentration v aried f rom 4 0 to 16 0 µmol L-1. R eactions w ere carried
out in 25 mmol L-1 PI PE S p H 6 .0 at 5 0 oC and k
ob s w as calculated f or
each concentration.
F igure 8. D NA cleav ag e in th e p resence of radical scav eng ers ( dmso and NaN3) and D NA minor g rov e b inder ( distamy cin) . F inal concentration in 20 µL reaction of dmso, NaN3 and distamy cin are indicated in th e ig ure.
C ontrol samp les f or p lasmid D NA w ith acetonitrile (lane 1) , dmso ( lane 3) ,
Acknow ledgments
T h e auth ors th ank C NPq , C A PE S, F A PE SC and I NC T -C atálise f or inancial sup p ort. G . Sch enk and L.
G ah an ack now ledg e f unds aw arded b y th e A ustralian R esearch C ouncil th roug h g rants D P09 8 6 29 2, D P09 8 6 6 13, D P06 6 4 039 and D P05 5 8 6 5 2.
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0103 - 5053 $6.00+0.00
This paper is dedicated to our friend Professor Icaro Moreira in memoriam *e-mail: ademir@qmc.ufsc.br
Catalytic Promiscuity: Catecholase-like Activity and Hydrolytic DNA Cleavage
Promoted by a Mixed-Valence F e
I I IF e
I IComp lex
Ademir Neves,*,a Adailton J. Bortoluzzi,a Rafael Jovito, a Rosely A. Peralta,a
Bernardo de Souza,a Bruno Szpoganicz,a Antônio C . Joussef,a H ernán T erenzi,b
Patricia C . Severino,b F ranciele L . F isch er,b G erh ard Sch enk ,c M ark J. Riley,c
Sarah J. Smithc and L aw rence R. G ah anc
aLABINC, Departamento de Química and bCentro de Biologia M olecular E s trutural,
Departamento de Bioq uímica, U niv ers idade F ederal de Santa Catarina, 8 8 0 4 0 - 9 0 0 F lorianópolis - SC, Brazil
cSchool of Chemis try and M olecular Bios ciences , T he U niv ers ity of Queens land,
Bris bane 4 0 7 2 , Aus tralia
F igure S1. Species diagram for the spectrophotometric titration of complex [FeIII(bpbpmp)(µ-OAc) 2Fe
II]+ in CH
3CN:H2O 1:1. µ-H2O: [(OH2)Fe III(OH
2)
FeII(OH
2)]; A: [(OH2)Fe
III(µ-OH)FeII(OH
2)]; B: [(OH)Fe
III(µ-OH)FeII(OH
2)]; C: [(OH)Fe
III(µ-O)FeII(OH
2)]; D: [(OH)Fe
Figure S2. Species diagram for the spectrophotometric titration of complex [FeIII(bpbpmp)(µ-OAc) 2Fe
III]2+ in CH
3CN:H2O 1:1. B: [(OH)Fe III(µ-OH)
FeIII(OH
2)]; C: [(OH)Fe
III(µ-O)FeIII(OH
2)]; D: [(OH)Fe
III(µ-O)FeIII(OH)]; E: [(OH)FeIII(µ-O)FeII(OH) 2].
Figure S3. Spectral changes of the complex [FeIII(bpbpmp)(µ-OAc) 2Fe
III]2+ during titration. The spectra were recorded on CH
3CN:H2O 1:1 at successive
Figure S4. Spectral change of the intervalence band for the mixed-valence complex [FeIII(bpbpmp)(µ-OAc) 2Fe
II]+ over time under kinetic conditions: 3:2
![Figure S2. Species diagram for the spectrophotometric titration of complex [Fe III (bpbpmp)(µ-OAc) 2 Fe III ] 2+ in CH 3 CN:H 2 O 1:1](https://thumb-eu.123doks.com/thumbv2/123dok_br/18994129.461651/14.892.153.719.115.541/figure-species-diagram-spectrophotometric-titration-complex-iii-bpbpmp.webp)
![Figure S4. Spectral change of the intervalence band for the mixed-valence complex [Fe III (bpbpmp)(µ-OAc) 2 Fe II ] + over time under kinetic conditions: 3:2 CH 3 OH:H 2 O at pH 7.0](https://thumb-eu.123doks.com/thumbv2/123dok_br/18994129.461651/15.892.184.730.109.545/figure-spectral-change-intervalence-valence-complex-kinetic-conditions.webp)
