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ferredoxin:NADP+ reductase system from Anabaena
Bioc|fimie { 19~)8~ 80, 837-846 () Soci6t6 ~TaWaise de bh~chhnie el bioh~gie mo16ctdairc / E1se~wr, Paris
Protein-protein imeraction in e|ectron transfer reactions: The ferredoxhfffiavodoxirdferredoxin:NADP + reductase system from Anabaena Carlos G6mez-Moreno"*, Marta Martfnez-Jdlvez'L Milagros Medina". John K. Hurley b, Gordon Tollin b "l)¢7ro't,mento de Bioquh,ica y Biologht Molecuhtr v Cehdat: l'~r'ultad de Ciem'ius. U, iversichM de Z~IFtI~OSgl,50009 ZtIFO~OSO. Spat, t'Delmrtmenl r!t'Biochemislry; I/,iversitv ~!/'Ari-mm. Tin'so,. AZ 85721. USA
(Received 3 April 1998: accep|ed I 7 September ! 998) Abslracl - - Electron Iransl'cr reactions involving protcin-I'wolein inlera¢|ions require Ihe I'ornlalion of a Irat]sient complex ~,~.hich brings together Ihe Ivvo redox celllres exchanging elecll'olls. This is Ihe ¢;.lse for Ih¢ l|axoprotein ferredoxin:NADP + Iedu¢iase t I::NR! fl'onl the Cyallohactel'itllll Amd~.em~. an enzyme which inlcracts with l'erredoxill in lhe photosynthetic pathway to receive the electrons requh'¢d for NADP' redu¢lion. The reduclase show.,, a ¢oncaxe cavi|y in ils slructure inlo vduch small proleins such as lcrrcdoxin can lit. Flavodoxm, an FMN-containmg protein Ihal is synthesined ill c),at~obacl¢i'ia ultder iron-delicient conditions, pla3s the Silllle role as ferredoxin in its interaction with FNR in spite of its different strttclure, size and redox cofactor. There are a number of negafi',ely charged mnino acid residues on the surface of IL'rredoxin m~d Ilavodoxin that play a role in the electron lransfer reaction with the reductase. Thus far, in only one case has charge replacemem of one of the acidic residues produced an increase in the rate of electron transl~'r, whereas in several other cases a decrease in the rate is observed. In the most dramatic example, replacement of Glu at position 94 ol'Amlbaem~ fer,'edoxirt results in virlually the cornplele loss of ability to Iranst~r electrons. Charge-reversal of positively charged amino acid residues in the rcductase also produces strong effects on the rate of electron Iiallsft21". Several degrees oI' impairment have been obsel'~;ed, tile nlost signilicant involving a positively charged Lys at position 75 which appears to be essential for tile stabili|y of the cotnplex between the reductase and fcrredoxin. The results presented in this paper provide it clear dernonstratitm of the inlportance of electrostatic interacliotls o n the stability of the transient complex l'ormed during electron transfer b~ the protein,, presently under study. 0) Stlcit~lO l'rant;aise tie hiochimie el biologic mol,~ctdaire 1 Elsevier, Paris
FNR / ferredoxin / Ilavodoxin / protein-protein interae|ion I. intnJductio,i
Eleclron~,lransfer reaclions involving proleinoprolein interactions require the I'orlualion el t~ ti;ansient ¢o|nplex which brings together the two redox cemres exchanging electrons. During oxygenic plr~losytlthesis four electrons are removed l'rom two water molecules, yieldi~Ig molecuo lar oxygen and protons. The two high potential electrons removed fi'om water are used to reduce one NADP + molecule. In the course of the oxidation-reduction teat-
; C'ii:,:eSpi"id i' :e iii'd ,: p':i'iiS AIH~reviations: FNR. ferredoxiil:NADP' reduclase; FNR,,,. FNR in the oxidised Male; FNR,d. FNR in lhc reducc, l s1~lw: FNR ,,. FNR in the semiquinone slale; l'd, l¢rrcdoxin; I d,,~. l'd in the oxidised slale; Fd,d, Fd in the reduced slale~, Fld. flavodoxin; Fld,,~. Fld in lhe oxidised slale; Fld,,~. Fld in lhe reduced slale; Fld~,r Fld in the semiquinone slal¢; llrrG, isopropyl-l:bl)thiogalacloside: dRf. 5-deazariboflavin: dRill, semiquinone form of dRf: el. electron transfer.
lions involved in electron transfer from water Io N A D P ' . enough energy is also lihcrated Io synthesisc one ATP luoiccul¢. Fcrrcduxhl;N ADP ' rcdtl¢ l;,|s¢ { FN R} catal), sc~, the lransl~r of two eleelrons fronl reduced l~rredoxin (Fd) to N A D P + I l l . This enzyme ¢onlaitls a nonocov;d~ntly bound FAD with a midpoint redox potential of ~344 mV (pH 7.0)121. Plant and cyanobacterial ferredo×in.~ atv small globuhn" proteins (!1 kDa). which contain one [2Fe-2SI centre which participates in reactions in which electrons are u'ansferred at low potentials (E+o = =4,20 mV) 13 I. Under iron-delicient conditions+ certain strains of cyanobacteria arid eukaryotic algae synthesise an FMNo containing Ilavoprotein, llavodoxin (Fld), which replaces I'crredoxin in those reactions in whict~ the ironoprotein ix itwolved 13 ]. Its parlicipation in pholosynlhesis is. then. to substitute for Fd in the transfer of one electron from PSI |o FNR. for which it u~c~ the half reaction in which lhc protein oscillates between the semiquinone and hydro° quinone Ik~rms. al a midpoint redox potential o f - 4 3 6 mV (at pH 7)121. The reaction in which these three proteins are involved requires the I'ommtion of a Irarisietll complex between Fd and F:NR. or Fkl and FNR. so that electron
838 transfer between the two proteins can occur. It is now well established that the rate of interprotein electron transfer is modulated by parameters that include: a) a thermodynamic term that takes into consideration the difference in dox ~ e n t i a l s between the prosthetic groups which are i~ting in the reaction: b) the distance between the two centres that exchange electrons; and c) what is called " the it reo~am,' S atton energy,• a term that refe~ to the change in geometry that protein and solvent molecules undergo during the formation of the transition state of the reaction. This implies that the rate of electron transfer would depend on the structural characteristics of the prt~ein-protein interface during the electron transfer step, ;o; well as distance, relative orientation and redox potential differences between the two centres. three dimensional structu~s of,4m~/r+emr FNR 141, Fd 151 and FId 161 have been detemained at high resolution, providing invaluable intbmlation for the study of this inte~sting problem. The three-dimensional structure of Ana~ena FNR re~mbles that reported lbr the spinach enzyme 171,consisting of two domains. One of these binds IAD, and is made up of a scaffold of six antiparallel strands arranged in two perl~ndicu!ar ~-sheets, tile bottom of which is caplx,~l by a short or-helix and a long ltx~p [41. ~ e NADP+ binding domain consists of a core of live parallel ~--strands surrounded by seven ~Jt-helices. This arrangement corresponds to a variant of the Rossman fold typical of dinucleotide binding proteins 181. Mo~over, tile th~e~dimert~ional struc!u~ of this enzyme has ~ e n pro~sed to be the p~rotyl~ of a large thmi!y of tlavind¢~ndent oxido~ductases that fimetion as transducer-, I~tween nicotinamide dhlu¢leotides (two-electron t~l! ' .... ' 1 !ers) and oneoelectron carriers i7, 9, II)[, The FNR molo ecule has a very noticeable concave cavity, comprising FAD and N A D P binding domains, where the Fd {and FId) is ptt~pos~l lo bind !4, 1!, !21, Fhe I ~ D mol~ule i:s k~ated in the centre of this cavity with its dimethyl~n~e~ ring, the part of the c"~~lactor ',.... molecule through w h~ch " the electrons must ~ exchanged, pointing I o w a , s the solvent, Chemical cross-linking o f F N R to
G6mez-Moreno et al. 2. Materials and methods
2. !. Oligonucleoti~h'-(fire,'ted mtttagenesis Mt~tants of recombinant Amd~aem~ PCC 7 ! 20 Fd were made using the TranslbrmerT M site-directed mutagenesis kit from Clontech and a construct of the petF gene, pAn662 I251, cloned into the plB125 vector (International Biotechnologies Inc., New Haven, CF, USA) as template. E, colt strain JM !09 was translbrmed with this plasmid lbr protein expression. Mutants of recombinant A,abaemt PCC 7119 FNR were produced using a construct of the petH gene which had been previously cloned into the expression vector pTrc99a 1261. Amino acid substitutions were also canted out using 1he Translbrmer si|e-dil'ected mutagenesis kit fi'om Clontech (Palo Al|o, CA, USA ~. Tile constructs containing the mutated FNR gene were used to wansfonn the E. (:oil PC 0225 strain 1271. Mutations were verified by DNA sequence analysis.
2.2. Put'~lication ~.i /'~teins Anahaena PCC 7119 wild type and mutant FNR forlns were purified from IPTG induced cultures as previously descried 124, 271. Ataalnlena PCC 7120 wild type and mutant ferredoxins were purified as previously descri~d[19]. Recombinant flavodoxin from Anahaem~ ~ C 7119 was prepared as described 128]. UV-visible ab.~orption spectra and SDS-PAGE electrophorcsis wet~ used as purity criteria. ....~. !limlit~g ~'ott.~la!m, [ o r the I.~1,,: FNRo,, compl¢.~e.~ "
Dissocia|iolt ¢~tll|stallls, binding energies and extinclion c~flicienls of tile complexes helween oxidised FNR species and oxidised !crrcdoxin or oxidised l!avodoxi!) were obtained as previously d~acri~d 1291. Experimental data were fit to a theoretical equation for I:1 stoichiometry by means of non-linear regression.
2.4. lxtser,lhtsh photolysis measurements
F'dll31, c~mical m~ilicationl!2, 14-161 and site, di~x:t~ mutagenesis II7, 181 exl~riments have identilied, in bolh the spinach and the Am~l~ena enzymes, a numi~r of iml~rtant a~inine and iysine t~sidues in the area where the Fd interaction is proposed to occur 14, ! !1, in ~ e n t yea~, a large amount of structural, kinetic and e l e c t r ~ m i c a l data that demonstrate the crucial role that certain amino acid residues in i~rredoxin play in the re~li~n with FNR have ~ e n t~'el~rtCd ~!~.~231, Simihu~ studies a~ now in progress to study the role of residues at t ~ FNR surface 118, 241, The data presented herein summarises our wo~ concerning the role of acidic amino acid residues in Ana~etm Fd, or FId, and basic residues in Anatatetm FNR at the protein-protein docking interfaces.
The puled laser photolysis apparatus used to obtain transient electron transfer kinetics has been described previously 130-321, as has the photochemical reaction which initiates electron transfer133-351. Laser flashinduced kinetic measurements were performed at room temperature, in addition to protein, samples also contained I mM EDTA and 95-1(~) ltM dRf in 4 mM potassium phosphate buffer, pH 7.0. When necessary, the k~nic s|~ngth oi the solution was adjusted to IO0 mM using 5 M NaCi, Samples were made anaerobic by bubbling for I h with H20-saturated Ar gas prior to addition of protein. Binding constants for the transient Fd.,~:FNR,.,, complexes were determined by fitting the laser flash photolysis data to the exact solution of the differential equation describing
Recognition nile in FNR for its protein partners
839
a mhfimal ttwo-siep} mechanism mvol~hlg complex formation followed by electron ti'~,insli{'l 13{>t.
2.5. St~q~ped-iIow me¢+:+++rc,wm,,+ Electron transfer processes between FNR and Fd, or Fld, were studied by stopped-flow methods ushlg an Applied Photophysics SXI7.MV spectmphotometer interfaced with an Acorn 5000 computer using the SX. 17MV software of Applied Photophysics. Samples were made anaerobic by successive evacuation and flushing with O,-free Ar in special tonometers which litted the stoppedflow apparatus. All reactions were carried out in 50 mM Tris-HCI, pH 8.0, at 13 ~'C, and at the wavelengths appropriate to follow the reactions. ]'lie observed rate constants {k,,i,,) were calculated by littin,, the data to mono- or hi-exponential processes, Reduced samples of Fd, Fid and FNR for stopped-flow were prepared by photoreduction with 5-deazariboltavin as described prev iou.,dy ! 241.
3. Results and discussion
3. I. Arra,geme,t +?fa<'Mic residm'.~ +m the sttrf~tce of Amtbaemt .l'erre+hni, Examinatiorl of a side view of the ferredoxin structure using computer graphics indicates that the. iron-sulphur centre fornls a protrusion in the compact protein molecule (figm'e ! ). The cofactor is buried under a nunlbcr of ;.inlint~ acid residues, mainly comprised of the two cysteines which bind one of the two iron atoms ;|11¢1 by polar uncharged anlino acids, such as Thr and Ser 151. No cha,'ged residues are located in the inlnletli+ile vicinity of the ,.-.~t'~F- .... "~ I centre. However. two distinct negative patches are located at both sides of the equatorial position as shown in.ligtoe/. One of the patches is comprised of residues Asp28, Glu31. Glu32 and Asp30. The second patch of negative charge is fimued by residues Asp07. Asp68 and Asp69. Both regions are well conserved among the different cyanobacterial ferredoxins[25, 37. 381. There is also a third region of negative charge, which is conserved in vegetative cyanobacterial ferredoxins but not in the t~rredoxin isozyme t'ound in heterocyst cells 1391, which consists o1" several amino acids located near the carboxyl temfinus, Glu94, Giu95 and Glu96. This third cluster o1" negative charge is also located somewhat ch>ser to the iron-sulphur centre (ligure !). Thus, the ferredoxin molecule is organised as at quasi ~phc,ical nlt~lecule with the reaction site near the surface at the equator of the molecule, and negative charges asymmetrically located at the periphery of the sphere. This distribution of negative charge suggests that these p.'ltches might be involved in the orientation of the ferredoxin molecule as it approaches the reductase. Once the two molecules form a cornplex.
Figure I. Schematic view of Amtl~aemt ferredoxin surface in which the negative charge distribt, ion around the [2Fe-2S[ C¢111i+¢in indicated.
the 12Fe-2SI centre would be po:,itioned close it) the FAD centre of the reductase, ready to perlorm its electron transfer function, in the case of spinach ferredoxin, the presence of a nnolectllar dipole, created by the presence of the two domains of negative potential in tile m~iecule. with its negative end lying just abo~,e the iron-sulphur Celltr¢. has also been proposed 1401+ This charge distribu+ lion has prompled tts tO I'octms t)n the complen/cntar} tlistril~tltion of positive charges ill FNR and has led t~ the ntlg.12eslit+ll tel' il illllllht'r tel nticll sites +In pt+snibl¢ camli dates for involvement in the interaction with !ezre+ doxin 1181.
3.2. Arrangeme,tt o t'a~'idi~' residtu'.s' mz tit+, .~mJ+,'e o/' Amthaemt .[htvoduxin Charge distribution on the surfiice o1" Anubuemt tl',i. vodoxin [6] Ibllows a pattern similar to thai described above I'or ferredoxin (figure 2J. The FMN molecule is at one side of the protein, exposing only the dimethylbcn° zene ring to the solvent. Negative charges are distill utcd on both sides ol' the FMN in two patches which CUmlWlSe Asp65 and Giu67 on use side. and Asp144, Glu 145 and Asp146, located at lhc opposite side of the molecule. ()lher negative an-fine acid re.~dduet,, which ctmld also bc
involved in the interaction of llavodoxin with the rcduc-
tase (Glu16 and Asp129) are distributed on the stlrl'ace around the FMN molecule (fig,re 2). Chemical modilication studies have also indicated that the region containing Asp144. G!u !45. Asp146 and Asp 129 must he involved in the interaction with FNR 141 I.
8~
G6me~-Moreno el al. that laser llash photolysis of solutions containing flaw~doxin and FNR resulted in the reduction of the FAD cofactor of FNR followed by a rapid elecmm transfer from FNR semiquinone to the flavodoxin to generate its FMN semiquinone form I421, as shown in equation (2):
+
xd
[Fldo,,-FNRml -
+o,.. Fld+,m +
FNRo,, (2)
In some cases saturation is not obtainable in the accessible FNR concentration range. In these situations second-order rate constants m'c measured, which include the complex association constant as well as kc.. Mutations of a number of different acidic amino acids present on the surface of the fcnedoxin and flavodoxin molecules have been perfimned and the effect of tile mutation ev:fluated through the determination of the kinetic pal'ametcrs lk)r the electron translk'r reaction.
3.4. Arrcmgemem of'basic ivsi&ws o, the smfuce o l'Amtbaem+ +"VR F|gu~ 2. Schematic view of A,a&+em+ flav~oxin surt~ce in ~hich the negative charge distributionaround the FMN cofacmr i~ indicated.
3,3, The role <)fnegative charges it)ji'rwdoxin ~and,ih+v+&t~'inl in &termh+iog the rate ,+i'ele(+lmt+ o'(m,q~t" with FNR Electron transfer iva~dons t~tween Amtbm+m+ ferre+ doxin (or flavt~loxin) and FNR have been proposed to occur via a mechanism that invol~es tile fornlation of a transient complex I~tween the two pmtein~ prior It) the actual ete~:trou tr~mst'erpr~:ess, Th~ laser t!ash photolysis technique has allowed the study of s.uch, fast pro+ t e s t s [21 I. Tt~ laser flash pr~uces the rapid t~duction o f fe~doxin p~sent in a solution with FNR, The Sl~ctral changes ~vccunmg . . . . . ' , after the laser ttash enable one to tbllow the electron transfer ~actions and to determine the rate constant {/g~0 which, in some cases, can ~ measured at m~turatingFNR concentrations, where the rate ~ c o m e s FNR concentration indel~ndent, Under these latter conditions, the ~action rate constant that is determined is due to intracomplex electron transfer (k,,,), which includes factors such as structural rearrangement and changes in hydration of ~ t h protein and ~dox cofactor during transition slate t'ormation (rem~anisation energy), Overall, the reactio++ is descfil~d by the (minimat~ tv, o+step mechanism shm~n in equation I i3fq: Fdra + ~ R o ~
[Fd~-FNRoJ " ~
Fdo,, ÷ FNR~(1)
A similar sc~me would apply when Ilavt~oxin acts as the electron donor to FNR, It has ~ e n previously shown
The FNR three-dimensional structure shows a cavity at the bottom of which is located the isoaUoxazine ring of the FAD molecule (iigmv3t. Such a cavity could easily accommodate small protein molecules such as ferredoxin or flavt~oxin, since the concave cavity |bund in the FNR complements the convex surface of either of the two electron transport proteins that intentct with h. Taking into account the data available li'om cross-linking and chemical modification studies, using eill!er spinach or At+abaem+ FNR l I3~ 161, and t!~e three+dimensiolud structures of tile molectdes, a nunlber of' basic amino acid residues are clem'ty distinguisiaed o n the pcrhnetcr of the aftu+enlen+ tioned cax'ity which can potentially be involved in the protein°protein interaction. Again. there arc t~.o groups of charged residues which are at opposite sides of the FAD
cotactat. One patch is tbrmed by I#s72, Lys75 and Aru,7, while the other is brined by Lys290, Lys293 and Lys294. Moreover, a num~r of other positively charged residues, Argl6, Lysi38, A ~ 2 ~ , are distributed around the FAD cofactor forming a crown of positive charge (tigure 3J.
3,5. Charge reversal mtetatiotts hi the 67~69 neg+aive p
Recognition site in FNR for its protein pal'u~ers
841 ration of |hese results points to a mode~ in which t~'rredoxin and FNR form a complex which i., stabilized by electrostatic interactions. Howe~er. the fact that the bindin,,~, in tight does not necessarik', mean that the two protein col'actors are in the correct orientation for the eflicient transfer o| the electron. In fact. the ionic stren,,th~_ dependencies show that if the complex is too tight the mobility of fen'edoxin is hindered in such a way that it cannot re-orient properly after collisional contact to produce a productive complex with FNR [31 ]. It can also be concluded that the role of the negative patch in the 67-69 region of ferredoxin is to produce a dipole that orients the t~rredoxin molecule as it approaches FNR to start the catalytic cycle.
3.6. ('h,rge reversal re,ration.s in the ('arboxvl termi,,~ regio, o.f Amd~aemt .li'rredo.~i,.
Figure 3. Schematic ~,i¢v¢of Amd~,emt FNR surf:ice in v,hich the positive charge distributitm around the FAD colactor is indicated.
the differential equation describing the process (equalion I). The results obtained (talkie !1 show tilat the charge reversal at these positions produced a slight decrease in the k,.t values obtained at low ionic strength, except when Asp68 in replaced by a lysine. It in expected that the introduction of a positive charge otl ferredoxin at a position that in involved in the interaction with its partner would prt)duce all impairment of complex l'orrnation and, therel't~re, of the electron transfer ability. The behaviour of the single inula|lts, Asp671.ys and Asp691..ys. and the doul~le illtltant Asl~671.ys/Asp6tJi.ys arc in agreement with this idea. However, the Asp68Lys Fd mutanl is anomalous c,~ rates of electron transl~r in that it s h o w e d ' ItlCled, "~' s,d (table l). Consistent with this, the double mutant Asp68Lys/Asp69Lys, had wild°type like activity, probably because the impairment due to the Asp69Lys mutation is ol'l~et by the anomalous increased activity of the Asp68Lys mutation. To explain the:;e results, it has been suggested that there is a specilic interaction between Asp68 and a complementary (presumably positively charged) re.,;idue on FNR which forces the proteins to assume a less favourable orientation at low ionic strengths (where electrostatic Ibrces are stronger) during electron transfer [23[. In this context, it is interesting to note that tim Aspf7Lys and Asp6t}Lys mutants are also impaired ill higher ionic strengths, whereas the Asp68Lys inutant still slaows wild-type activity 1231. No correlalions between complex thermodynamic stabilities {K,I)and reaction rate constants were observed (table !), indicating thai the stability of the complex is not directly related to the efficiency of the electron transfer reaction. The interpre-
Glulanlic acid residues in positions 94 and 95 in ..An,b,c,a ferredoxin ha~e also been replaced by residues bearing a positive c'harge [! 9, 20]. The results obtained lot these two Fd mutalllS were completely different. Elimination or reversal of the negative charge at position 94 results in almost complete abolition of ils ability to transfer electrons to FNR. whereas the corresponding replacements at position 95 do not have any effect on the electron transfer reaction. Thus. the ket l'~r the reaction between Glu94GIn Fd and FNR was 0.34 s -~. as compared to 3600 s I for the wild type Fd ~tabl¢ !,. and the value for Glu941.ys Fd was to~ small to be estimated. Comparison of the second order ,'ate t?OllStalitS for Glu94GIn ~md Glu041+ys (l,bh' !) clearly show++~that the removal of a negative charge at that position produces a inuch less reactive protein, itowever, Ih¢ Ft!,,,:FNR,,, disst~ciali~m COllSlalllS for Iho cotnplexes with these IIIlllalll Fds ;ll'C only threeofold and sixolOId lower, respectively, thnn thglt for wild-type Fd. This indicates that decreases ill the stability of the complex cannot account t'o~" the drastic decrease of the electron transl~r activity. Therefore, a very important role in the electron transfer 1ruction is indic~_~ted for the acidic residue in position 94 of Fd. That role cotdd be related to the hydrogen bond which exists between Glu94 and Ser47. Thus, it has been found that the Ser47Ala mutant is also highly impaired in its electron transfer activity with FNR (whereas the Ser47Thr and Thr48Aia mutants are not). and that both replacements. Ghi94Lys and Ser47Ala. cause a signilicant positive shift in the reduction potential of the iron-sulphur centre of these proteins, altht)ugh this shift in not the cause ol the activity changes [431. These results point to the requirement I o r a highly specilic protein-protein geometrical interface, i.e.. the effect of making identical replacements of two contiguous residues {Glu94Lys and Giug51,ys. as well as Ser47Ala and Thr48Ala) produces completely dit'l~rent effects on the reactivity of ferredoxin.
842
G6mez-Moreno et al.
T~t~ I. Second o~er rate constants and COtT~ted'~ kinetic parameters tbr the oxidation of reduced fcn'edoxms by oxidiscd native FNRh. b~l lbrm Ka [Fd,,,:FNR,,] Ct~M~ k x IO s tM ~s ~y k,., ts ~ Ka ll"d,a:FNR,.,] ff~M~ WiM ty~ As~7~s
4.5 +_0.6~" 1.8 + O.1I"
1.2 _+O.1a 0.8 + O.! '
3600 + 4(D~" 2300 + 3(X}~"
2.2 + 3~" 0.5 +_O.!*"
As~Lys
Asp~Lys Asp67t68Lys Asp68/69Lys As~7168/figLys Glu95Lys GIu94Lys Glu94GIn
0,7 +_0.2 ~
!.9 +_O.~Jt
9 0 ~ ) + 2(XX) ~
i .8 _+0.3*' 4.0 + 0,7 ~ 1.9 :t: 0.3*' n.d. ~ n.d. 26 ± 3a 13 ± 4'
0.9 + O.i* 0.4 + 0, ! ~ i. 1 +_0. |d 0.5 _+0.2" !.2 :t: 0. !'* O.(~)(R)5a
2902) + 200* 2000 +_ 100.'
0.5 _+0. ! ~ 0.9 + O.I ~
0.0001Y
3.8 + 0.2*' 1.2 + O.I*' n.d. n.d. n,d.
4400 + 400*'
n.d.*' n.d. n.d. 0.34 _+0.03 ''~
n,d.
"The data were corrected for nonoproductive complex formation as descril~d in 131 I. *'Ex~riment~ were carried out at an ionic strength of 12 mM except where otherwise indicatcd~ ~Second order rate constants, "Taken from 1191. ' ~ k e n t~ma 1311, *Taken thm~ 1221. ~Complex fiwmation could not be detected. *'No( de!ennined, 'Taken tiom 1211. iThis ex~riment was carried ou~ at 1(~) mM ionic strength. *~Takenfrom 1231,
3, 7, Mutation of negatively chm~ed amim~ acid residues on the surface ~[ Amd~¢m~ flav,~doxi~ Several negatively cha~ed residues at the surface of AP~at~wna flawrdoxin laave previously been replaced by neutral residues: Glu61GIn, Glu67Ala, Asp!(~)G!n, Asp126Ala and Glu!45A!a1441, Only relalive!y small differences in the ra~es of electron tnmst~r between FNR,,~ and these mutated flavt~loxins were observed when c~m~pared |o the w i l d q y ~ ilavodoxin, These changes probably ~flect alterations in the mama! orientations of the two proteins within a prefom~ed complex, The largest deceases in ~activity were t~bserved for the Asp126Ata and Glu67Ala flavodoxin mutanls, With the limited arnount of inibm~alion thus far available lbr this p~teln it is not possible to conclude if the~ charged amino acid residues on ~ surf~e of Analmena flav~oxin play a significant fide in tke electron transfer reaction, The small effects o/k~rv~ may ~ a con~quence of the tact that no cha~e ~ve~als have ~ e n made in these mutants, but only cha~e t~eutralisations, 3,& Ch~o~e rerersai of ~ s i c amino acid s~,sidt~es
o~ the slo:tiwe ~Amd~em~ FNR $ou~ of t ~ residues loaning the positive patches around the FAD of Amd~aena FNR have ~ e n replaced, using site~dir~,cted mutagenesis, by both negatively charnel ~ ~utral amino acids 1181, The ability of the correspo~ting mutat~,xl proteins to transfer electrons with
both Fd and FId has been studied using steady-state and rapid kinetic techniques, and the stability of their complexes with FNR has ~ e n determined, For the Arg77Glu and Arg77GIn rep!acemen|s, no active mutants that proper!y bind FAD ha c ~ e n obtained I! 81 and thus the study of !!!e involvement of Arg77 in the interaction ol' FNR with fen-edoxin or flavodoxin has not been possible. These resul!s indicate |he hllportant ftulction of the guankliniun) group of Arg77 in the electrostatic stabilisation o f Ihe pyrophosphate group of tile FAI) col'at for 141. The steadystate kmettc cha ~ctensatlon of the Arg!6G!u, Lys72Glu, Lys75Glu, Lys!38Glu, Arg2fvtGlu, Lys290Glu and Lys294Glu FNR mutants revealed three different types of ~haviour in those reactions in wlfich complex tbrmation and e,I-ccutn -c~ transt~r to/from ferredoxin were involved, whe~as no major changes were detected for any of the mutants with ~ ~ c t to the wild type FNR in their reactivity with NADP ÷ INADPH 118]. These three types of FNR mutants, distinguished by their bellaviour towm'd Fd, were also found when the laser tlash photolysis technique was applied to the study of the electron transfer reaction fr~m reduced ferredoxin to the different FNR,,,, mutants ~tahk, ll~, While replacement of amino acid icstdu~s 138, 264, 290 and "94 by negatively charged amino acids did not markedly affect the rate ConShtld.,, h~r electron transfer from Fd,.a, the introduction of a negative cl~urge at positions 16 and 72 px'oduced substantial decreases in the electRm tt'ansfer rate which could be ,~s,t.er,~ed~"by. the laser photolysis technique (see the second order rate constants in tabh, !!). The third type of ~' V
"~
.
.
.
.
.
Recognition site in FNR for its protein partners
843
TaNle l|. Fast kine|ic parameters of electron transtk, r from reduced Fd to oxidised v, ild bpe and mulant FNRs studied b'~ laser flash photolysis".
l.WRform Wild type~" Arg 16Glu Lys72G|u Lys75Glu Lys138Glu Arg264Giu Lys29t)Glu Lys294Giu
k'°~ [Fd,,,:I.'NR+,] ~.++31~
~, x I0 '+ tM ~,~ ~r;
k, I,~"tj
k',~ [Fd,,/f,XR,,,] qt/tlj
0.3 -t O. t u 120 +_21) 5tt + 15 n.d5 4. ! +_0.8 3.3 _+ !. I 4.0 + i.6 7.2 + 1.3
7~ I
3h110 _+41111
2.2 +_(I.3
n.d. b
n.d. b
2
n.d. t'
n.d?'
0.05 v
> 165 3700 + 400 3800 + 500 3500 + 600 4800 _+300
n.d." 1.8 + 0.2 1.3 + 0.2 i.9 _+0.3 2.9 + (:).2
!0 !3 9 9
"in addition to protein, solutions also con|ained O. I mM dRf and I mM EDTA in 4 mM potassium phospimte, pH 7.0 (~l = 12 raM). ~'Nnt able to be accurately determined. 'Complex Ikmnation was not detected, '~Estimated from Ihe initial slopes of the k,,, vs. [FNR] ¢ur~c.,, alter correction for pre-formed complex (see [311 for details). "Estimated as in 13 ! I, Waken fl'Otll1311. ~This value was not correc|cd for pre-exisling complex since K u could not be measured.
behaviour is that shown by Lys75Glu FNR, which is ahnost completely impaired in its electron transfer reaction with Fd. The dissociation constants measured for the complexes Ibrmed between the Fd mutants and the reductase ~table I!) reflect the involvement of lhese amino acids in the stab|lisa|ion of the complexes. This is especially true for the Lys75Glu mutant Ibr which the binding constant Ior the complex was too weak to be measured. These results provide strong evidence that all of the mutated residues in A n a b a e m t FNR are involved in Fd binding, with Lys75 being n l o s t important, Ar~16 and Ly:;72 being mmewlmt less important, and the other Ik)ul" residues far less important Ior the billding process. Further protein engineering on I.ys75 has also been carried out in
order to investigate its role in tile electron transfer interaction, and the arginine, glutamine and serine mutants have been prepared. Laser flash photolysis experiments in which reduction of the different FNR mutants at position Lys75 by Fd,t at / = i00 mM indicates that the Lys75Arg mutant (tabh" !111 behaves similarly to the wild-type FNR, whereas saturation couh! ~x)t be detected for tile Lys75GIn and Lys75Ser FNR forms to allow k,:t and Kd for the Fd,d:FNR,, ~ complex to be calculated. The observed second order rate constants for tile electron transfer processes with tile dilTerent FNR forms show that Lys75Ghl and Lys75Ser are kinetically quite similar to each other, and that l.ys75Glu in severely impaired in its electron tnmsfcr interaction with Fd.
Tahle | | i . Kinetic paranleters fi)r the reduction of the diffelvnt Lys75 FNR mutants studied by lasm" flash plmtolysb¢'
FNR tim. Wild type u
Lys75Arg Lys75GIn Lys75Ser Lys75Glu
K,t 1 i'd,,,, IWR,,, 1 qdt¢)
k x !0* fM i,~. t ),
k,,, (s t)
!'~a [I'd,a:Y NR,,, ] qd~l)
3.3 :k 0.6 4.8 -± 0.5 380 4: 20* 200 .+_60* n.d.~
2.7 ± n 4
5500 °J: 400 d''
1.7 k O. I'*'"
1.5 5:0.2 1t.38 -±0. I 1 0.56 + 0.02 0.0082 + 0.0003
4900 +, 600'* . . --
3.0 J: 0.4" .
. = =
Deaerated solutions also contained !00 ttM 5-deazaribollavin and I mM ED'FA in 4 mM Imta.,,.dum l~hosrflmte buffer, pH 7.0. The ionic strength was adjusted to I00 mM using 5 M NaCI. "Taken from [241. ~Second order rate constants were estimated for wild type and Lys75Arg from initial slopes of tile hYl~erbolic dependence of k,,t,, w. [FNR] curves (|lOt corrected for the concentration of pre-fornled complex). Secoltt] order rate constatltS for the othel nlutallts were nleasured directly fronl the linear ploln. dCorrected ibr the concentration of preformed complex. "Taken from 13 II. 'Binding was It,o weak to observe saturation in the experimentally accessible protein concentration range. Therefore. K, wits detennined from difference spectra, assuming that the dilTerence extintion coefficient for the complex was the same as ihal A+: calculated Ibr the complexes of Lys75Arg and wiki type FNR with wild type Fd (At: value is 2000 5_ 300 M t cmT), t~The spectral perturbation due to binding was l e o weak to be measured for thin mutant.
844
G6nle~-Morello et al. IV. Fast kinetic parameters tbr electron transfer i~actioris of wild type and charged-rever.,,al mulanl FNR form.,; sludied by ........................................................................................................................................................................................................
k,,t,, tstJ.for the mLt'ing o.f FNR,,, with FNR jorm WiM t y ~ ~ Arg204Giu Ly>~75Glu
k,,t,, ts ~l for the relying <~1"FNR,a wi6t
"Fd~
dFIdra
"Fd,,,
"Fid,,,
n.d." 250 ± 30 n.d. ~ 161 ± i0 2.7 ± 0.4 0.50 ± 0.05
n.d."
n.d."
14.6 ± 0.4 3.1 +0.2 0.52 +_0.06 0.08 _+0.02
>200
2.5 +_ i.0 1.0 + i.0 I ± 0.2 0.75 +0.5 O. I ! + 0.02 0.018 _+.0.001
~iiT ~mples were mixed in the stopped-flov¢ cell at the indicated redox states. T Tfis-HCI, pH 8.0.
< O.OO! !
13 <"C. All samples were prepared in 50 mM
~Data frol~ !241. cReacliotl followed at 507 rim, '~Re~tion titllowed at 61111nm, ~Reacihm occurred within the dead time of the instrument.
We have previously shown that kil,ctic data for the electron transfer ~actions from FNR~ to Fd,,~ or FId<,.. and from either Fd,~aor F!d,~ato FNR.x. can ~ tb!lowed using anaerobic stopped.flow techniques t241, Although this technique is not appropriate lbr the determination of the kinetic parameters of sonic of the reactions involving the wild type proteins, we have shown that it can provide important information for those mutants with alteced properties either with regard to complex formation or electron transfer processes ~tween FNR and its protein t'larl~rs. The study of such ~actions for the Lys75Glu and Arg2~Glu FNR charge-reversal mutants tt.blelVj shows diffel~nt ty~s of ~haviour Ibr ~ t h mira!sis, either with Fd or Fld, that um in agl~elnent with those fimud i~i the above mentioned slead),osla!e and laser Ihixh phololyo sis studies, As shown hy laser tlash pholo!ysis (labh' Ilj, ,~aiu~ti:on ot' FNR by Fd~,l is greatly iJtl)¢c!ed by tile m t r t ~ t t o n of a negative cha~e tit l~isiiion 75, whereas ¢ha~e mve~al at !~sition 2 ~ results in wild type:like reactivity, Similar results wee obtained for the two ¢o~slx~nding ~actions when F!d is the mdox panner (table IVL These ~sults indicate that mutation at these ~)sitkms has similar el/'~ts on ~ t h electron transfer proteins, fenxxloxin and flaw+idoxin, and thus p ,idc e v i ~ t ~ e thai fcr~doxin and flavt~oxil/both must lille'"r'actwith FNR at Ihe ~ n ~ region of the ~duclas¢, Further mo~. it is quite evi~nt thin different amino acid residues in FNR are involved to different extents in the interaction with both ferr~loxin and flav~loxin. All of lhe~ data den~strate that the charge-mve~al FNR mutations which al~ l ~ a l ~ at the FAD..binding dom~fin (Argl~lu. Lys72Glu and L)+s75Glu) (ti,l~uw3J cause dramatically a l t e ~ e l ~ i m n tmnst'er properties with I~rh protein redox piitl~rs, w~reas those mutations situated in the NADP+. binding domain iLys 138Glu, A~2t~Glu, Lys290Glu and L y s 2 ~ l u l cause considerably less impairment in the electlx~ll transfec pix~ces~s to Fd and Fld.
The inter.action between redox partners participating in electron transfer reactions has been the subject of study in several biological systems. In the photosynthetic electron transfer chain it was shown that interactions between acidic patches in plastocyanin with positive charges in cytochrome f conifibute to the stabilisation of the complex 1451. Very recently, the dynamic structure of the transient complex l'vetween these two proteins has been elucidated and electrostatic interactions are proposed to "guide' the partners into the op!ima! position lot electron transfer1461. ~evera! other reports also indicate that electrostatic interaclions produce the siabilisation of dectixm lninsl~r complexes in biological sys!elns. They include the interaction between milochtmdri'd l~'n~doxin and ferred~xin rcducia>,e 1471, cyiochrt~mc t' and cylochmil!e c ~roxidase1481, cylochrome P,I$1I and lhi: ~_~uxh!1491, or !4FeqSI ferredoxhi and niirt_!gcaase I501, According to this, it is tempting t~J propose thai the ~t17¢1s oil the i'~_iieof electron translTr thai we have observed in the Fd-FNR system upon rephicement of charged amino acids oil the protein sarl'aces could also be the case ibr otker protein-protehl electron transfer reactions, 4. Conelu..uons The ~sults presented in this study have assessed tile iml~rlance of electrostatic interactions in tile formation of protein-protein complexes and subsequent electron transfer in a physiologically relevanl rcdox system, it has been dead)' demonstrated that an exceptionally high degree of specificity exists at the interlace of the intermediate complex ~tween reduced Fd and oxidised FNR. and that the mutual orientation ~tween the redox cof.,-:tors is a crucial I~arameter in determining electron transfer rates. Furthermore. some of the mutated residues are shown to
Recognition site in FNR for its protein partners be essential for achieving rd~e correct docking orienlation, while olhers are only moderately involved. Moreover, evid~,~.e thai fe~redoxin and flavodoxin must both imeract with FNR at the same ~gion of the ~ductasc has also been shown. The combination of site-directed mutagenesis of At~abaena FNR and stopped-flow techniques have allowed fast kinetic studies of electron transfer between Fld and FNR mutant forms which were too fast to be followed for the wild type enzyme. Although the results obtained ttms fur are not sufficient to unambiguously determine the structure of the intermediate complex, we are hopeful that additional studies will permit this to be accomplished,
845 Ulll I!21
1131 114] 115l
[161
Aeknow|cdgmenls This work was made possible in part by grants from the National Institutes of Health to G.T. (DKI5057) and grant BIO94 from Comisi6n lnterministetial de Ciencia y Tecnoiogfa to C. G-M. M. M-J. was a recipient of a fellowship fi'om the DGA.
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8~
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13OI
1431 Hurley J,K,, Weber-Mare A.M., Stankovich M,T., Benning M.M., Thoden J.B.. Vanhooke LL., Holden H.M,, Chae Y.K., Xia B,, Cheng H., Markley J.L., Marffnez-Jfilvez M., G6mez-Moreno C., Schmeits J,L, Tollin G., Structure-function relationships in Anabaena ferredoxin: correlations between X-ray crysta| structures, reduction potentials, and rote constants of electron Iranslbr to ferredoxin:NADP' reductase for sitc-spcciiic ferrcdoxin mulants, Biochemistry 36 (1997) i!i00--11117,
lson D.E., L a i r transfer in milk ~279. 1311 Hurley J.K,. Filial M.E. G6mez-Moreno C.. Tollin G.. Electro~ath: and hydrophobic interactions during complex formation and electron tnmsfer in the ferredoxin:NADP" reductase system from A ~ n a , J, Am, Chem. Soc. 118 1 ! ~ 5526-5531. 1~2! Prz~ieeki C,T,, Bhattachaff~a A,K.. Tollin G,, Cusanovich M.A,. Kinetics of r e d , lion of Cla~trMium I~terianum rubredoxin by I~q' pholo~dueed spinach h;rredoxin:NADP" reductase and I've flavins, Electron transfer within a protein-protein complex. J. Biol. Chem, 260 11985) 1452-1458. 1331 To|lin G,. U ~ of la~r flash pholotysi~ time-re~lved spectmphotomelr~ to invemigate imerpmtein and inlraprotein electron transfe~ mechanisms, J. Biacnerg Biomembr, 27 119951 303-309. 1341 Totlin G,, H~tard J.l;, Intra~ and intermolecular electron transfer processes in redox proteins. Arch. Biochem, Biophys. 287 t 1991 ) 1~7. Tollin G,. tturley J.K., ttazzard I.T.. Meyer TE.. Use of laser flash pl~olysis tim¢ore~lved sp¢c!ropho~ometry Io investigate inletprotein and intraprotein elecm~n transfer mechanisms, Biophys, Chem. 48 (19931 259~279, 1361 Simondmn R,P., lbllin G., Transient kinetics of redox reactions of flavod~xin: effects of chemical modification of the flavin mononu¢leolide pin, the.de group on tl~ dynamics of intermediate ~omplex formation and electron transfer, Biochemist D, 22 ( 19831 3~8-3016, 1~71 V ~ Den Plus J,, De G r ~ R.E, W¢isbeek E J.. VanArkel G,A.. Coding ~quenee of a f¢rrcdoxin gene from Amd~wtm variahi!is ATCC 29413, Nucleic A¢id,s Res, !4 119861 7803, Rcilh M,~., L~qden~¢h D,E,, Slrau,~ N,A,. l~olation and nudeolide ~qu~,¢e anat)'~t~ of the t~rredoxin I gene from t!~e cyanoh~¢lcfium Amwystls , i d , l~m.~ R2., .1. Bacterml, Its8 l!gSf;~ t319--1324, i;10h|I~ H,, H ~ l k o m R,, M~leeu!ar c!tming and ilucleotid¢ ~equen~e analysis o[ tl~e gtme ¢~Ming t~t h¢lclawy~l lencd~xin f~om tt~ qyano!~lerlum ¢t,,l~¢nd rip, slt~ain I~'C 7120. M,L Gen. Goner, 214 11988t 27~-2~5. i401 J¢lcs~v I., B~h~ard H,R,. Th~m~.~lyna_ml~ of !~n~doxin hind1.8 to f~. ~)~tm NADP* ~ d u c t ~ ~t.d th~ rol~ of v~a~cr a~ the ¢o~pl¢~t int¢rfa¢¢, Biochemistry 33 ( 1 ~ ) !3321~13328, l~ttl M~Mina M,, l~le~o M,L,, M~nd¢t E,, G6,~oMor~no C,, Identio ltealtlo~ of s ~ i f i c ead~)xyl groups on h,at~;etta I~"C 7119 flavt~doxin which are involved in tt~ interaction with fertcdoxin: NADP' reducing, Eur, J, Bi~hem, 203 (1992] 373-379,
[44] Navarro J.A,, Hervfis M., Gen~or C.G., Cheddar G., Filial M.F., De La Rosa M.A., Gdmez-Morcno C., Cheng H., Xia B., Chae Y.K., Yah H., ~kmg B., Straus N.A., Markley .I.L.. Hurley LK,, 'Ibllin G., Site-specific mutagene~is dcm~mstrales thin the slluctural requirements ton efficient electron transl;er in Amd~wm~ I/'nvdoxm and flav~xtoxh~ arc highly dependem on tile reaction partner: kinetic studies ~'ith photosyslem I, ferrcdoxin:NADP' rcduclase, and cytochrome c. Arch. Biochem. Biophys. 321 (1995~ 229-238. 1451 Qin L, Kostic N.M., Enlorced interaction of one molecule of plasmc).anin with two molecules of cytt~.'hrome ¢' and an electrontransfer reaction involving lhe hydrophobic palch on the plaslocyanm surface, Bio,-hemistry 35 { 19961 3379-3386. 1461 Ubbink M.o Ejdeback M., Karsson B.G., Bendall D.S., The struclure of the complex of plaslocyanin and cytochrome j~ delemfincd b~. pammagne|ic NMR and restrained rigid-body molecular dynamics, S~ructurc 6 (1998} 323-335. [47] Coghlan V.M., VickeD' L E , Electrostatic interactions stabilising l~rredoxm electron transfer complexes. J. Biol. Chem. 267 11992~ 8932-8935, 1481 Ctnin A~F., MeLendtm G,. Zang Q.. Hake R.A., Falvo J., lal K.S.. ('iccarelli R,B,, Hoizschu It,. Effects of surlhce amino acid t ~ p ! a c e ~lle fit n i ~! C }"i ~ ' 11t t ~! ~ ¢ t L pen~xidase on complex Ihrmathm t~i|h cy|oclinm~e ~,, Biochemislry 311 ( lq91 ) I 1585 11595. 14~Jj Jenkins C.M,~ (;entt~l C,(L, Ioilht| M.I(, Wi||e|llliin M,R,, (;tilllCtM~,+em~ C,, N~gitti~cly citalgcd Anahm+ma Itavodoxm rt'sidues ~A~p144 altd (~hlll.|5~ ~||c in|p~.t;UH It~r rc¢;HlMitulio|l tff Cy[O chrome P~,30 17whydmxylaseactivity. J. Biol0 Chem. 272 1!9971
~501 Naud I,, Meyer C., David L., Breton J.. Galliard J.. Jouanneau Y.. Idenlilication of residue of Rho&dmcter caps,huus fcrredoxin ! imporlam for its interaction with nitrogenase, Eur. J. Biochem. 237 19961 3~D405.
Protein-protein imeraction in e|ectron transfer reactions: The ferredoxhfffiavodoxirdferredoxin:NADP + reductase system from Anabaena Carlos G6mez-Moreno"*, Marta Martfnez-Jdlvez'L Milagros Medina". John K. Hurley b, Gordon Tollin b "l)¢7ro't,mento de Bioquh,ica y Biologht Molecuhtr v Cehdat: l'~r'ultad de Ciem'ius. U, iversichM de Z~IFtI~OSgl,50009 ZtIFO~OSO. Spat, t'Delmrtmenl r!t'Biochemislry; I/,iversitv ~!/'Ari-mm. Tin'so,. AZ 85721. USA
(Received 3 April 1998: accep|ed I 7 September ! 998) Abslracl - - Electron Iransl'cr reactions involving protcin-I'wolein inlera¢|ions require Ihe I'ornlalion of a Irat]sient complex ~,~.hich brings together Ihe Ivvo redox celllres exchanging elecll'olls. This is Ihe ¢;.lse for Ih¢ l|axoprotein ferredoxin:NADP + Iedu¢iase t I::NR! fl'onl the Cyallohactel'itllll Amd~.em~. an enzyme which inlcracts with l'erredoxill in lhe photosynthetic pathway to receive the electrons requh'¢d for NADP' redu¢lion. The reduclase show.,, a ¢oncaxe cavi|y in ils slructure inlo vduch small proleins such as lcrrcdoxin can lit. Flavodoxm, an FMN-containmg protein Ihal is synthesined ill c),at~obacl¢i'ia ultder iron-delicient conditions, pla3s the Silllle role as ferredoxin in its interaction with FNR in spite of its different strttclure, size and redox cofactor. There are a number of negafi',ely charged mnino acid residues on the surface of IL'rredoxin m~d Ilavodoxin that play a role in the electron lransfer reaction with the reductase. Thus far, in only one case has charge replacemem of one of the acidic residues produced an increase in the rate of electron transl~'r, whereas in several other cases a decrease in the rate is observed. In the most dramatic example, replacement of Glu at position 94 ol'Amlbaem~ fer,'edoxirt results in virlually the cornplele loss of ability to Iranst~r electrons. Charge-reversal of positively charged amino acid residues in the rcductase also produces strong effects on the rate of electron Iiallsft21". Several degrees oI' impairment have been obsel'~;ed, tile nlost signilicant involving a positively charged Lys at position 75 which appears to be essential for tile stabili|y of the cotnplex between the reductase and fcrredoxin. The results presented in this paper provide it clear dernonstratitm of the inlportance of electrostatic interacliotls o n the stability of the transient complex l'ormed during electron transfer b~ the protein,, presently under study. 0) Stlcit~lO l'rant;aise tie hiochimie el biologic mol,~ctdaire 1 Elsevier, Paris
FNR / ferredoxin / Ilavodoxin / protein-protein interae|ion I. intnJductio,i
Eleclron~,lransfer reaclions involving proleinoprolein interactions require the I'orlualion el t~ ti;ansient ¢o|nplex which brings together the two redox cemres exchanging electrons. During oxygenic plr~losytlthesis four electrons are removed l'rom two water molecules, yieldi~Ig molecuo lar oxygen and protons. The two high potential electrons removed fi'om water are used to reduce one NADP + molecule. In the course of the oxidation-reduction teat-
; C'ii:,:eSpi"id i' :e iii'd ,: p':i'iiS AIH~reviations: FNR. ferredoxiil:NADP' reduclase; FNR,,,. FNR in the oxidised Male; FNR,d. FNR in lhc reducc, l s1~lw: FNR ,,. FNR in the semiquinone slale; l'd, l¢rrcdoxin; I d,,~. l'd in the oxidised slale; Fd,d, Fd in the reduced slale~, Fld. flavodoxin; Fld,,~. Fld in lhe oxidised slale; Fld,,~. Fld in lhe reduced slale; Fld~,r Fld in the semiquinone slal¢; llrrG, isopropyl-l:bl)thiogalacloside: dRf. 5-deazariboflavin: dRill, semiquinone form of dRf: el. electron transfer.
lions involved in electron transfer from water Io N A D P ' . enough energy is also lihcrated Io synthesisc one ATP luoiccul¢. Fcrrcduxhl;N ADP ' rcdtl¢ l;,|s¢ { FN R} catal), sc~, the lransl~r of two eleelrons fronl reduced l~rredoxin (Fd) to N A D P + I l l . This enzyme ¢onlaitls a nonocov;d~ntly bound FAD with a midpoint redox potential of ~344 mV (pH 7.0)121. Plant and cyanobacterial ferredo×in.~ atv small globuhn" proteins (!1 kDa). which contain one [2Fe-2SI centre which participates in reactions in which electrons are u'ansferred at low potentials (E+o = =4,20 mV) 13 I. Under iron-delicient conditions+ certain strains of cyanobacteria arid eukaryotic algae synthesise an FMNo containing Ilavoprotein, llavodoxin (Fld), which replaces I'crredoxin in those reactions in whict~ the ironoprotein ix itwolved 13 ]. Its parlicipation in pholosynlhesis is. then. to substitute for Fd in the transfer of one electron from PSI |o FNR. for which it u~c~ the half reaction in which lhc protein oscillates between the semiquinone and hydro° quinone Ik~rms. al a midpoint redox potential o f - 4 3 6 mV (at pH 7)121. The reaction in which these three proteins are involved requires the I'ommtion of a Irarisietll complex between Fd and F:NR. or Fkl and FNR. so that electron
838 transfer between the two proteins can occur. It is now well established that the rate of interprotein electron transfer is modulated by parameters that include: a) a thermodynamic term that takes into consideration the difference in dox ~ e n t i a l s between the prosthetic groups which are i~ting in the reaction: b) the distance between the two centres that exchange electrons; and c) what is called " the it reo~am,' S atton energy,• a term that refe~ to the change in geometry that protein and solvent molecules undergo during the formation of the transition state of the reaction. This implies that the rate of electron transfer would depend on the structural characteristics of the prt~ein-protein interface during the electron transfer step, ;o; well as distance, relative orientation and redox potential differences between the two centres. three dimensional structu~s of,4m~/r+emr FNR 141, Fd 151 and FId 161 have been detemained at high resolution, providing invaluable intbmlation for the study of this inte~sting problem. The three-dimensional structure of Ana~ena FNR re~mbles that reported lbr the spinach enzyme 171,consisting of two domains. One of these binds IAD, and is made up of a scaffold of six antiparallel strands arranged in two perl~ndicu!ar ~-sheets, tile bottom of which is caplx,~l by a short or-helix and a long ltx~p [41. ~ e NADP+ binding domain consists of a core of live parallel ~--strands surrounded by seven ~Jt-helices. This arrangement corresponds to a variant of the Rossman fold typical of dinucleotide binding proteins 181. Mo~over, tile th~e~dimert~ional struc!u~ of this enzyme has ~ e n pro~sed to be the p~rotyl~ of a large thmi!y of tlavind¢~ndent oxido~ductases that fimetion as transducer-, I~tween nicotinamide dhlu¢leotides (two-electron t~l! ' .... ' 1 !ers) and oneoelectron carriers i7, 9, II)[, The FNR molo ecule has a very noticeable concave cavity, comprising FAD and N A D P binding domains, where the Fd {and FId) is ptt~pos~l lo bind !4, 1!, !21, Fhe I ~ D mol~ule i:s k~ated in the centre of this cavity with its dimethyl~n~e~ ring, the part of the c"~~lactor ',.... molecule through w h~ch " the electrons must ~ exchanged, pointing I o w a , s the solvent, Chemical cross-linking o f F N R to
G6mez-Moreno et al. 2. Materials and methods
2. !. Oligonucleoti~h'-(fire,'ted mtttagenesis Mt~tants of recombinant Amd~aem~ PCC 7 ! 20 Fd were made using the TranslbrmerT M site-directed mutagenesis kit from Clontech and a construct of the petF gene, pAn662 I251, cloned into the plB125 vector (International Biotechnologies Inc., New Haven, CF, USA) as template. E, colt strain JM !09 was translbrmed with this plasmid lbr protein expression. Mutants of recombinant A,abaemt PCC 7119 FNR were produced using a construct of the petH gene which had been previously cloned into the expression vector pTrc99a 1261. Amino acid substitutions were also canted out using 1he Translbrmer si|e-dil'ected mutagenesis kit fi'om Clontech (Palo Al|o, CA, USA ~. Tile constructs containing the mutated FNR gene were used to wansfonn the E. (:oil PC 0225 strain 1271. Mutations were verified by DNA sequence analysis.
2.2. Put'~lication ~.i /'~teins Anahaena PCC 7119 wild type and mutant FNR forlns were purified from IPTG induced cultures as previously descried 124, 271. Ataalnlena PCC 7120 wild type and mutant ferredoxins were purified as previously descri~d[19]. Recombinant flavodoxin from Anahaem~ ~ C 7119 was prepared as described 128]. UV-visible ab.~orption spectra and SDS-PAGE electrophorcsis wet~ used as purity criteria. ....~. !limlit~g ~'ott.~la!m, [ o r the I.~1,,: FNRo,, compl¢.~e.~ "
Dissocia|iolt ¢~tll|stallls, binding energies and extinclion c~flicienls of tile complexes helween oxidised FNR species and oxidised !crrcdoxin or oxidised l!avodoxi!) were obtained as previously d~acri~d 1291. Experimental data were fit to a theoretical equation for I:1 stoichiometry by means of non-linear regression.
2.4. lxtser,lhtsh photolysis measurements
F'dll31, c~mical m~ilicationl!2, 14-161 and site, di~x:t~ mutagenesis II7, 181 exl~riments have identilied, in bolh the spinach and the Am~l~ena enzymes, a numi~r of iml~rtant a~inine and iysine t~sidues in the area where the Fd interaction is proposed to occur 14, ! !1, in ~ e n t yea~, a large amount of structural, kinetic and e l e c t r ~ m i c a l data that demonstrate the crucial role that certain amino acid residues in i~rredoxin play in the re~li~n with FNR have ~ e n t~'el~rtCd ~!~.~231, Simihu~ studies a~ now in progress to study the role of residues at t ~ FNR surface 118, 241, The data presented herein summarises our wo~ concerning the role of acidic amino acid residues in Ana~etm Fd, or FId, and basic residues in Anatatetm FNR at the protein-protein docking interfaces.
The puled laser photolysis apparatus used to obtain transient electron transfer kinetics has been described previously 130-321, as has the photochemical reaction which initiates electron transfer133-351. Laser flashinduced kinetic measurements were performed at room temperature, in addition to protein, samples also contained I mM EDTA and 95-1(~) ltM dRf in 4 mM potassium phosphate buffer, pH 7.0. When necessary, the k~nic s|~ngth oi the solution was adjusted to IO0 mM using 5 M NaCi, Samples were made anaerobic by bubbling for I h with H20-saturated Ar gas prior to addition of protein. Binding constants for the transient Fd.,~:FNR,.,, complexes were determined by fitting the laser flash photolysis data to the exact solution of the differential equation describing
Recognition nile in FNR for its protein partners
839
a mhfimal ttwo-siep} mechanism mvol~hlg complex formation followed by electron ti'~,insli{'l 13{>t.
2.5. St~q~ped-iIow me¢+:+++rc,wm,,+ Electron transfer processes between FNR and Fd, or Fld, were studied by stopped-flow methods ushlg an Applied Photophysics SXI7.MV spectmphotometer interfaced with an Acorn 5000 computer using the SX. 17MV software of Applied Photophysics. Samples were made anaerobic by successive evacuation and flushing with O,-free Ar in special tonometers which litted the stoppedflow apparatus. All reactions were carried out in 50 mM Tris-HCI, pH 8.0, at 13 ~'C, and at the wavelengths appropriate to follow the reactions. ]'lie observed rate constants {k,,i,,) were calculated by littin,, the data to mono- or hi-exponential processes, Reduced samples of Fd, Fid and FNR for stopped-flow were prepared by photoreduction with 5-deazariboltavin as described prev iou.,dy ! 241.
3. Results and discussion
3. I. Arra,geme,t +?fa<'Mic residm'.~ +m the sttrf~tce of Amtbaemt .l'erre+hni, Examinatiorl of a side view of the ferredoxin structure using computer graphics indicates that the. iron-sulphur centre fornls a protrusion in the compact protein molecule (figm'e ! ). The cofactor is buried under a nunlbcr of ;.inlint~ acid residues, mainly comprised of the two cysteines which bind one of the two iron atoms ;|11¢1 by polar uncharged anlino acids, such as Thr and Ser 151. No cha,'ged residues are located in the inlnletli+ile vicinity of the ,.-.~t'~F- .... "~ I centre. However. two distinct negative patches are located at both sides of the equatorial position as shown in.ligtoe/. One of the patches is comprised of residues Asp28, Glu31. Glu32 and Asp30. The second patch of negative charge is fimued by residues Asp07. Asp68 and Asp69. Both regions are well conserved among the different cyanobacterial ferredoxins[25, 37. 381. There is also a third region of negative charge, which is conserved in vegetative cyanobacterial ferredoxins but not in the t~rredoxin isozyme t'ound in heterocyst cells 1391, which consists o1" several amino acids located near the carboxyl temfinus, Glu94, Giu95 and Glu96. This third cluster o1" negative charge is also located somewhat ch>ser to the iron-sulphur centre (ligure !). Thus, the ferredoxin molecule is organised as at quasi ~phc,ical nlt~lecule with the reaction site near the surface at the equator of the molecule, and negative charges asymmetrically located at the periphery of the sphere. This distribution of negative charge suggests that these p.'ltches might be involved in the orientation of the ferredoxin molecule as it approaches the reductase. Once the two molecules form a cornplex.
Figure I. Schematic view of Amtl~aemt ferredoxin surface in which the negative charge distribt, ion around the [2Fe-2S[ C¢111i+¢in indicated.
the 12Fe-2SI centre would be po:,itioned close it) the FAD centre of the reductase, ready to perlorm its electron transfer function, in the case of spinach ferredoxin, the presence of a nnolectllar dipole, created by the presence of the two domains of negative potential in tile m~iecule. with its negative end lying just abo~,e the iron-sulphur Celltr¢. has also been proposed 1401+ This charge distribu+ lion has prompled tts tO I'octms t)n the complen/cntar} tlistril~tltion of positive charges ill FNR and has led t~ the ntlg.12eslit+ll tel' il illllllht'r tel nticll sites +In pt+snibl¢ camli dates for involvement in the interaction with !ezre+ doxin 1181.
3.2. Arrangeme,tt o t'a~'idi~' residtu'.s' mz tit+, .~mJ+,'e o/' Amthaemt .[htvoduxin Charge distribution on the surfiice o1" Anubuemt tl',i. vodoxin [6] Ibllows a pattern similar to thai described above I'or ferredoxin (figure 2J. The FMN molecule is at one side of the protein, exposing only the dimethylbcn° zene ring to the solvent. Negative charges are distill utcd on both sides ol' the FMN in two patches which CUmlWlSe Asp65 and Giu67 on use side. and Asp144, Glu 145 and Asp146, located at lhc opposite side of the molecule. ()lher negative an-fine acid re.~dduet,, which ctmld also bc
involved in the interaction of llavodoxin with the rcduc-
tase (Glu16 and Asp129) are distributed on the stlrl'ace around the FMN molecule (fig,re 2). Chemical modilication studies have also indicated that the region containing Asp144. G!u !45. Asp146 and Asp 129 must he involved in the interaction with FNR 141 I.
8~
G6me~-Moreno el al. that laser llash photolysis of solutions containing flaw~doxin and FNR resulted in the reduction of the FAD cofactor of FNR followed by a rapid elecmm transfer from FNR semiquinone to the flavodoxin to generate its FMN semiquinone form I421, as shown in equation (2):
+
xd
[Fldo,,-FNRml -
+o,.. Fld+,m +
FNRo,, (2)
In some cases saturation is not obtainable in the accessible FNR concentration range. In these situations second-order rate constants m'c measured, which include the complex association constant as well as kc.. Mutations of a number of different acidic amino acids present on the surface of the fcnedoxin and flavodoxin molecules have been perfimned and the effect of tile mutation ev:fluated through the determination of the kinetic pal'ametcrs lk)r the electron translk'r reaction.
3.4. Arrcmgemem of'basic ivsi&ws o, the smfuce o l'Amtbaem+ +"VR F|gu~ 2. Schematic view of A,a&+em+ flav~oxin surt~ce in ~hich the negative charge distributionaround the FMN cofacmr i~ indicated.
3,3, The role <)fnegative charges it)ji'rwdoxin ~and,ih+v+&t~'inl in &termh+iog the rate ,+i'ele(+lmt+ o'(m,q~t" with FNR Electron transfer iva~dons t~tween Amtbm+m+ ferre+ doxin (or flavt~loxin) and FNR have been proposed to occur via a mechanism that invol~es tile fornlation of a transient complex I~tween the two pmtein~ prior It) the actual ete~:trou tr~mst'erpr~:ess, Th~ laser t!ash photolysis technique has allowed the study of s.uch, fast pro+ t e s t s [21 I. Tt~ laser flash pr~uces the rapid t~duction o f fe~doxin p~sent in a solution with FNR, The Sl~ctral changes ~vccunmg . . . . . ' , after the laser ttash enable one to tbllow the electron transfer ~actions and to determine the rate constant {/g~0 which, in some cases, can ~ measured at m~turatingFNR concentrations, where the rate ~ c o m e s FNR concentration indel~ndent, Under these latter conditions, the ~action rate constant that is determined is due to intracomplex electron transfer (k,,,), which includes factors such as structural rearrangement and changes in hydration of ~ t h protein and ~dox cofactor during transition slate t'ormation (rem~anisation energy), Overall, the reactio++ is descfil~d by the (minimat~ tv, o+step mechanism shm~n in equation I i3fq: Fdra + ~ R o ~
[Fd~-FNRoJ " ~
Fdo,, ÷ FNR~(1)
A similar sc~me would apply when Ilavt~oxin acts as the electron donor to FNR, It has ~ e n previously shown
The FNR three-dimensional structure shows a cavity at the bottom of which is located the isoaUoxazine ring of the FAD molecule (iigmv3t. Such a cavity could easily accommodate small protein molecules such as ferredoxin or flavt~oxin, since the concave cavity |bund in the FNR complements the convex surface of either of the two electron transport proteins that intentct with h. Taking into account the data available li'om cross-linking and chemical modification studies, using eill!er spinach or At+abaem+ FNR l I3~ 161, and t!~e three+dimensiolud structures of tile molectdes, a nunlber of' basic amino acid residues are clem'ty distinguisiaed o n the pcrhnetcr of the aftu+enlen+ tioned cax'ity which can potentially be involved in the protein°protein interaction. Again. there arc t~.o groups of charged residues which are at opposite sides of the FAD
cotactat. One patch is tbrmed by I#s72, Lys75 and Aru,7, while the other is brined by Lys290, Lys293 and Lys294. Moreover, a num~r of other positively charged residues, Argl6, Lysi38, A ~ 2 ~ , are distributed around the FAD cofactor forming a crown of positive charge (tigure 3J.
3,5. Charge reversal mtetatiotts hi the 67~69 neg+aive p
Recognition site in FNR for its protein pal'u~ers
841 ration of |hese results points to a mode~ in which t~'rredoxin and FNR form a complex which i., stabilized by electrostatic interactions. Howe~er. the fact that the bindin,,~, in tight does not necessarik', mean that the two protein col'actors are in the correct orientation for the eflicient transfer o| the electron. In fact. the ionic stren,,th~_ dependencies show that if the complex is too tight the mobility of fen'edoxin is hindered in such a way that it cannot re-orient properly after collisional contact to produce a productive complex with FNR [31 ]. It can also be concluded that the role of the negative patch in the 67-69 region of ferredoxin is to produce a dipole that orients the t~rredoxin molecule as it approaches FNR to start the catalytic cycle.
3.6. ('h,rge reversal re,ration.s in the ('arboxvl termi,,~ regio, o.f Amd~aemt .li'rredo.~i,.
Figure 3. Schematic ~,i¢v¢of Amd~,emt FNR surf:ice in v,hich the positive charge distributitm around the FAD colactor is indicated.
the differential equation describing the process (equalion I). The results obtained (talkie !1 show tilat the charge reversal at these positions produced a slight decrease in the k,.t values obtained at low ionic strength, except when Asp68 in replaced by a lysine. It in expected that the introduction of a positive charge otl ferredoxin at a position that in involved in the interaction with its partner would prt)duce all impairment of complex l'orrnation and, therel't~re, of the electron transfer ability. The behaviour of the single inula|lts, Asp671.ys and Asp691..ys. and the doul~le illtltant Asl~671.ys/Asp6tJi.ys arc in agreement with this idea. However, the Asp68Lys Fd mutanl is anomalous c,~ rates of electron transl~r in that it s h o w e d ' ItlCled, "~' s,d (table l). Consistent with this, the double mutant Asp68Lys/Asp69Lys, had wild°type like activity, probably because the impairment due to the Asp69Lys mutation is ol'l~et by the anomalous increased activity of the Asp68Lys mutation. To explain the:;e results, it has been suggested that there is a specilic interaction between Asp68 and a complementary (presumably positively charged) re.,;idue on FNR which forces the proteins to assume a less favourable orientation at low ionic strengths (where electrostatic Ibrces are stronger) during electron transfer [23[. In this context, it is interesting to note that tim Aspf7Lys and Asp6t}Lys mutants are also impaired ill higher ionic strengths, whereas the Asp68Lys inutant still slaows wild-type activity 1231. No correlalions between complex thermodynamic stabilities {K,I)and reaction rate constants were observed (table !), indicating thai the stability of the complex is not directly related to the efficiency of the electron transfer reaction. The interpre-
Glulanlic acid residues in positions 94 and 95 in ..An,b,c,a ferredoxin ha~e also been replaced by residues bearing a positive c'harge [! 9, 20]. The results obtained lot these two Fd mutalllS were completely different. Elimination or reversal of the negative charge at position 94 results in almost complete abolition of ils ability to transfer electrons to FNR. whereas the corresponding replacements at position 95 do not have any effect on the electron transfer reaction. Thus. the ket l'~r the reaction between Glu94GIn Fd and FNR was 0.34 s -~. as compared to 3600 s I for the wild type Fd ~tabl¢ !,. and the value for Glu941.ys Fd was to~ small to be estimated. Comparison of the second order ,'ate t?OllStalitS for Glu94GIn ~md Glu041+ys (l,bh' !) clearly show++~that the removal of a negative charge at that position produces a inuch less reactive protein, itowever, Ih¢ Ft!,,,:FNR,,, disst~ciali~m COllSlalllS for Iho cotnplexes with these IIIlllalll Fds ;ll'C only threeofold and sixolOId lower, respectively, thnn thglt for wild-type Fd. This indicates that decreases ill the stability of the complex cannot account t'o~" the drastic decrease of the electron transl~r activity. Therefore, a very important role in the electron transfer 1ruction is indic~_~ted for the acidic residue in position 94 of Fd. That role cotdd be related to the hydrogen bond which exists between Glu94 and Ser47. Thus, it has been found that the Ser47Ala mutant is also highly impaired in its electron transfer activity with FNR (whereas the Ser47Thr and Thr48Aia mutants are not). and that both replacements. Ghi94Lys and Ser47Ala. cause a signilicant positive shift in the reduction potential of the iron-sulphur centre of these proteins, altht)ugh this shift in not the cause ol the activity changes [431. These results point to the requirement I o r a highly specilic protein-protein geometrical interface, i.e.. the effect of making identical replacements of two contiguous residues {Glu94Lys and Giug51,ys. as well as Ser47Ala and Thr48Ala) produces completely dit'l~rent effects on the reactivity of ferredoxin.
842
G6mez-Moreno et al.
T~t~ I. Second o~er rate constants and COtT~ted'~ kinetic parameters tbr the oxidation of reduced fcn'edoxms by oxidiscd native FNRh. b~l lbrm Ka [Fd,,,:FNR,,] Ct~M~ k x IO s tM ~s ~y k,., ts ~ Ka ll"d,a:FNR,.,] ff~M~ WiM ty~ As~7~s
4.5 +_0.6~" 1.8 + O.1I"
1.2 _+O.1a 0.8 + O.! '
3600 + 4(D~" 2300 + 3(X}~"
2.2 + 3~" 0.5 +_O.!*"
As~Lys
Asp~Lys Asp67t68Lys Asp68/69Lys As~7168/figLys Glu95Lys GIu94Lys Glu94GIn
0,7 +_0.2 ~
!.9 +_O.~Jt
9 0 ~ ) + 2(XX) ~
i .8 _+0.3*' 4.0 + 0,7 ~ 1.9 :t: 0.3*' n.d. ~ n.d. 26 ± 3a 13 ± 4'
0.9 + O.i* 0.4 + 0, ! ~ i. 1 +_0. |d 0.5 _+0.2" !.2 :t: 0. !'* O.(~)(R)5a
2902) + 200* 2000 +_ 100.'
0.5 _+0. ! ~ 0.9 + O.I ~
0.0001Y
3.8 + 0.2*' 1.2 + O.I*' n.d. n.d. n,d.
4400 + 400*'
n.d.*' n.d. n.d. 0.34 _+0.03 ''~
n,d.
"The data were corrected for nonoproductive complex formation as descril~d in 131 I. *'Ex~riment~ were carried out at an ionic strength of 12 mM except where otherwise indicatcd~ ~Second order rate constants, "Taken from 1191. ' ~ k e n t~ma 1311, *Taken thm~ 1221. ~Complex fiwmation could not be detected. *'No( de!ennined, 'Taken tiom 1211. iThis ex~riment was carried ou~ at 1(~) mM ionic strength. *~Takenfrom 1231,
3, 7, Mutation of negatively chm~ed amim~ acid residues on the surface ~[ Amd~¢m~ flav,~doxi~ Several negatively cha~ed residues at the surface of AP~at~wna flawrdoxin laave previously been replaced by neutral residues: Glu61GIn, Glu67Ala, Asp!(~)G!n, Asp126Ala and Glu!45A!a1441, Only relalive!y small differences in the ra~es of electron tnmst~r between FNR,,~ and these mutated flavt~loxins were observed when c~m~pared |o the w i l d q y ~ ilavodoxin, These changes probably ~flect alterations in the mama! orientations of the two proteins within a prefom~ed complex, The largest deceases in ~activity were t~bserved for the Asp126Ata and Glu67Ala flavodoxin mutanls, With the limited arnount of inibm~alion thus far available lbr this p~teln it is not possible to conclude if the~ charged amino acid residues on ~ surf~e of Analmena flav~oxin play a significant fide in tke electron transfer reaction, The small effects o/k~rv~ may ~ a con~quence of the tact that no cha~e ~ve~als have ~ e n made in these mutants, but only cha~e t~eutralisations, 3,& Ch~o~e rerersai of ~ s i c amino acid s~,sidt~es
o~ the slo:tiwe ~Amd~em~ FNR $ou~ of t ~ residues loaning the positive patches around the FAD of Amd~aena FNR have ~ e n replaced, using site~dir~,cted mutagenesis, by both negatively charnel ~ ~utral amino acids 1181, The ability of the correspo~ting mutat~,xl proteins to transfer electrons with
both Fd and FId has been studied using steady-state and rapid kinetic techniques, and the stability of their complexes with FNR has ~ e n determined, For the Arg77Glu and Arg77GIn rep!acemen|s, no active mutants that proper!y bind FAD ha c ~ e n obtained I! 81 and thus the study of !!!e involvement of Arg77 in the interaction ol' FNR with fen-edoxin or flavodoxin has not been possible. These resul!s indicate |he hllportant ftulction of the guankliniun) group of Arg77 in the electrostatic stabilisation o f Ihe pyrophosphate group of tile FAI) col'at for 141. The steadystate kmettc cha ~ctensatlon of the Arg!6G!u, Lys72Glu, Lys75Glu, Lys!38Glu, Arg2fvtGlu, Lys290Glu and Lys294Glu FNR mutants revealed three different types of ~haviour in those reactions in wlfich complex tbrmation and e,I-ccutn -c~ transt~r to/from ferredoxin were involved, whe~as no major changes were detected for any of the mutants with ~ ~ c t to the wild type FNR in their reactivity with NADP ÷ INADPH 118]. These three types of FNR mutants, distinguished by their bellaviour towm'd Fd, were also found when the laser tlash photolysis technique was applied to the study of the electron transfer reaction fr~m reduced ferredoxin to the different FNR,,,, mutants ~tahk, ll~, While replacement of amino acid icstdu~s 138, 264, 290 and "94 by negatively charged amino acids did not markedly affect the rate ConShtld.,, h~r electron transfer from Fd,.a, the introduction of a negative cl~urge at positions 16 and 72 px'oduced substantial decreases in the electRm tt'ansfer rate which could be ,~s,t.er,~ed~"by. the laser photolysis technique (see the second order rate constants in tabh, !!). The third type of ~' V
"~
.
.
.
.
.
Recognition site in FNR for its protein partners
843
TaNle l|. Fast kine|ic parameters of electron transtk, r from reduced Fd to oxidised v, ild bpe and mulant FNRs studied b'~ laser flash photolysis".
l.WRform Wild type~" Arg 16Glu Lys72G|u Lys75Glu Lys138Glu Arg264Giu Lys29t)Glu Lys294Giu
k'°~ [Fd,,,:I.'NR+,] ~.++31~
~, x I0 '+ tM ~,~ ~r;
k, I,~"tj
k',~ [Fd,,/f,XR,,,] qt/tlj
0.3 -t O. t u 120 +_21) 5tt + 15 n.d5 4. ! +_0.8 3.3 _+ !. I 4.0 + i.6 7.2 + 1.3
7~ I
3h110 _+41111
2.2 +_(I.3
n.d. b
n.d. b
2
n.d. t'
n.d?'
0.05 v
> 165 3700 + 400 3800 + 500 3500 + 600 4800 _+300
n.d." 1.8 + 0.2 1.3 + 0.2 i.9 _+0.3 2.9 + (:).2
!0 !3 9 9
"in addition to protein, solutions also con|ained O. I mM dRf and I mM EDTA in 4 mM potassium phospimte, pH 7.0 (~l = 12 raM). ~'Nnt able to be accurately determined. 'Complex Ikmnation was not detected, '~Estimated from Ihe initial slopes of the k,,, vs. [FNR] ¢ur~c.,, alter correction for pre-formed complex (see [311 for details). "Estimated as in 13 ! I, Waken fl'Otll1311. ~This value was not correc|cd for pre-exisling complex since K u could not be measured.
behaviour is that shown by Lys75Glu FNR, which is ahnost completely impaired in its electron transfer reaction with Fd. The dissociation constants measured for the complexes Ibrmed between the Fd mutants and the reductase ~table I!) reflect the involvement of lhese amino acids in the stab|lisa|ion of the complexes. This is especially true for the Lys75Glu mutant Ibr which the binding constant Ior the complex was too weak to be measured. These results provide strong evidence that all of the mutated residues in A n a b a e m t FNR are involved in Fd binding, with Lys75 being n l o s t important, Ar~16 and Ly:;72 being mmewlmt less important, and the other Ik)ul" residues far less important Ior the billding process. Further protein engineering on I.ys75 has also been carried out in
order to investigate its role in tile electron transfer interaction, and the arginine, glutamine and serine mutants have been prepared. Laser flash photolysis experiments in which reduction of the different FNR mutants at position Lys75 by Fd,t at / = i00 mM indicates that the Lys75Arg mutant (tabh" !111 behaves similarly to the wild-type FNR, whereas saturation couh! ~x)t be detected for tile Lys75GIn and Lys75Ser FNR forms to allow k,:t and Kd for the Fd,d:FNR,, ~ complex to be calculated. The observed second order rate constants for tile electron transfer processes with tile dilTerent FNR forms show that Lys75Ghl and Lys75Ser are kinetically quite similar to each other, and that l.ys75Glu in severely impaired in its electron tnmsfcr interaction with Fd.
Tahle | | i . Kinetic paranleters fi)r the reduction of the diffelvnt Lys75 FNR mutants studied by lasm" flash plmtolysb¢'
FNR tim. Wild type u
Lys75Arg Lys75GIn Lys75Ser Lys75Glu
K,t 1 i'd,,,, IWR,,, 1 qdt¢)
k x !0* fM i,~. t ),
k,,, (s t)
!'~a [I'd,a:Y NR,,, ] qd~l)
3.3 :k 0.6 4.8 -± 0.5 380 4: 20* 200 .+_60* n.d.~
2.7 ± n 4
5500 °J: 400 d''
1.7 k O. I'*'"
1.5 5:0.2 1t.38 -±0. I 1 0.56 + 0.02 0.0082 + 0.0003
4900 +, 600'* . . --
3.0 J: 0.4" .
. = =
Deaerated solutions also contained !00 ttM 5-deazaribollavin and I mM ED'FA in 4 mM Imta.,,.dum l~hosrflmte buffer, pH 7.0. The ionic strength was adjusted to I00 mM using 5 M NaCI. "Taken from [241. ~Second order rate constants were estimated for wild type and Lys75Arg from initial slopes of tile hYl~erbolic dependence of k,,t,, w. [FNR] curves (|lOt corrected for the concentration of pre-fornled complex). Secoltt] order rate constatltS for the othel nlutallts were nleasured directly fronl the linear ploln. dCorrected ibr the concentration of preformed complex. "Taken from 13 II. 'Binding was It,o weak to observe saturation in the experimentally accessible protein concentration range. Therefore. K, wits detennined from difference spectra, assuming that the dilTerence extintion coefficient for the complex was the same as ihal A+: calculated Ibr the complexes of Lys75Arg and wiki type FNR with wild type Fd (At: value is 2000 5_ 300 M t cmT), t~The spectral perturbation due to binding was l e o weak to be measured for thin mutant.
844
G6nle~-Morello et al. IV. Fast kinetic parameters tbr electron transfer i~actioris of wild type and charged-rever.,,al mulanl FNR form.,; sludied by ........................................................................................................................................................................................................
k,,t,, tstJ.for the mLt'ing o.f FNR,,, with FNR jorm WiM t y ~ ~ Arg204Giu Ly>~75Glu
k,,t,, ts ~l for the relying <~1"FNR,a wi6t
"Fd~
dFIdra
"Fd,,,
"Fid,,,
n.d." 250 ± 30 n.d. ~ 161 ± i0 2.7 ± 0.4 0.50 ± 0.05
n.d."
n.d."
14.6 ± 0.4 3.1 +0.2 0.52 +_0.06 0.08 _+0.02
>200
2.5 +_ i.0 1.0 + i.0 I ± 0.2 0.75 +0.5 O. I ! + 0.02 0.018 _+.0.001
~iiT ~mples were mixed in the stopped-flov¢ cell at the indicated redox states. T Tfis-HCI, pH 8.0.
< O.OO! !
13 <"C. All samples were prepared in 50 mM
~Data frol~ !241. cReacliotl followed at 507 rim, '~Re~tion titllowed at 61111nm, ~Reacihm occurred within the dead time of the instrument.
We have previously shown that kil,ctic data for the electron transfer ~actions from FNR~ to Fd,,~ or FId<,.. and from either Fd,~aor F!d,~ato FNR.x. can ~ tb!lowed using anaerobic stopped.flow techniques t241, Although this technique is not appropriate lbr the determination of the kinetic parameters of sonic of the reactions involving the wild type proteins, we have shown that it can provide important information for those mutants with alteced properties either with regard to complex formation or electron transfer processes ~tween FNR and its protein t'larl~rs. The study of such ~actions for the Lys75Glu and Arg2~Glu FNR charge-reversal mutants tt.blelVj shows diffel~nt ty~s of ~haviour Ibr ~ t h mira!sis, either with Fd or Fld, that um in agl~elnent with those fimud i~i the above mentioned slead),osla!e and laser Ihixh phololyo sis studies, As shown hy laser tlash pholo!ysis (labh' Ilj, ,~aiu~ti:on ot' FNR by Fd~,l is greatly iJtl)¢c!ed by tile m t r t ~ t t o n of a negative cha~e tit l~isiiion 75, whereas ¢ha~e mve~al at !~sition 2 ~ results in wild type:like reactivity, Similar results wee obtained for the two ¢o~slx~nding ~actions when F!d is the mdox panner (table IVL These ~sults indicate that mutation at these ~)sitkms has similar el/'~ts on ~ t h electron transfer proteins, fenxxloxin and flaw+idoxin, and thus p ,idc e v i ~ t ~ e thai fcr~doxin and flavt~oxil/both must lille'"r'actwith FNR at Ihe ~ n ~ region of the ~duclas¢, Further mo~. it is quite evi~nt thin different amino acid residues in FNR are involved to different extents in the interaction with both ferr~loxin and flav~loxin. All of lhe~ data den~strate that the charge-mve~al FNR mutations which al~ l ~ a l ~ at the FAD..binding dom~fin (Argl~lu. Lys72Glu and L)+s75Glu) (ti,l~uw3J cause dramatically a l t e ~ e l ~ i m n tmnst'er properties with I~rh protein redox piitl~rs, w~reas those mutations situated in the NADP+. binding domain iLys 138Glu, A~2t~Glu, Lys290Glu and L y s 2 ~ l u l cause considerably less impairment in the electlx~ll transfec pix~ces~s to Fd and Fld.
The inter.action between redox partners participating in electron transfer reactions has been the subject of study in several biological systems. In the photosynthetic electron transfer chain it was shown that interactions between acidic patches in plastocyanin with positive charges in cytochrome f conifibute to the stabilisation of the complex 1451. Very recently, the dynamic structure of the transient complex l'vetween these two proteins has been elucidated and electrostatic interactions are proposed to "guide' the partners into the op!ima! position lot electron transfer1461. ~evera! other reports also indicate that electrostatic interaclions produce the siabilisation of dectixm lninsl~r complexes in biological sys!elns. They include the interaction between milochtmdri'd l~'n~doxin and ferred~xin rcducia>,e 1471, cyiochrt~mc t' and cylochmil!e c ~roxidase1481, cylochrome P,I$1I and lhi: ~_~uxh!1491, or !4FeqSI ferredoxhi and niirt_!gcaase I501, According to this, it is tempting t~J propose thai the ~t17¢1s oil the i'~_iieof electron translTr thai we have observed in the Fd-FNR system upon rephicement of charged amino acids oil the protein sarl'aces could also be the case ibr otker protein-protehl electron transfer reactions, 4. Conelu..uons The ~sults presented in this study have assessed tile iml~rlance of electrostatic interactions in tile formation of protein-protein complexes and subsequent electron transfer in a physiologically relevanl rcdox system, it has been dead)' demonstrated that an exceptionally high degree of specificity exists at the interlace of the intermediate complex ~tween reduced Fd and oxidised FNR. and that the mutual orientation ~tween the redox cof.,-:tors is a crucial I~arameter in determining electron transfer rates. Furthermore. some of the mutated residues are shown to
Recognition site in FNR for its protein partners be essential for achieving rd~e correct docking orienlation, while olhers are only moderately involved. Moreover, evid~,~.e thai fe~redoxin and flavodoxin must both imeract with FNR at the same ~gion of the ~ductasc has also been shown. The combination of site-directed mutagenesis of At~abaena FNR and stopped-flow techniques have allowed fast kinetic studies of electron transfer between Fld and FNR mutant forms which were too fast to be followed for the wild type enzyme. Although the results obtained ttms fur are not sufficient to unambiguously determine the structure of the intermediate complex, we are hopeful that additional studies will permit this to be accomplished,
845 Ulll I!21
1131 114] 115l
[161
Aeknow|cdgmenls This work was made possible in part by grants from the National Institutes of Health to G.T. (DKI5057) and grant BIO94 from Comisi6n lnterministetial de Ciencia y Tecnoiogfa to C. G-M. M. M-J. was a recipient of a fellowship fi'om the DGA.
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1171 1181
1191
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1241
1251
1261
1271
Karplus EA., thins C.M., S~ructure-function relalions for ferredoxin reductase, J, Bi~energ, Biomembr. 26 (t994)89-99. Jelesarov W., DePascalis A.R., Koppenol W.H., Hirasawa M.. Knafl" D.B., Bosshard H.R., Ferredoxin binding site on ferredoxin: NADP' reductase. Differential chemical modification of free and ferredoxm:bound enzyme, Eur. J. Biochem, 216 (1993) 57--66. Zaneui G.. Morelli D., Ronchi S. Negri A., Aliverti A., Curti B.. Structural studies on the interaction between ferredoxin and lerredoxin:NADP+ reductase, Biochemistry 27 (1988) 3753-3759. Medina M., M6ndez E., G6mez-Moreno C., Lysine residues on ferredoxin:NADP ÷ reductase from Anabaena sp. PCC 7119 involved in substrate binding, FEBS Lea. 298 (1992) 25-28. Medina M., M6ndez E.. G6mez-Moreno C., Identification of arginyi residues involved in the binding of ferredoxin:NADP + reductase from Anabaena sp. PCC 7119 to its substrates, Arch. Biochem. Biophys. 299 (1992) 281-286. Zanetti G., Aliverti A., Curti B., A cross-linked complex between fcrredoxin and fcrredoxin:NADP' rcductase, J, Biol. Chem. 259 (1984) 6153.--6157, Alive~aiA., Con'ado M.E., Zanetti G., Involvement of !' sine-88 of spinach fcrredoxin:NADP ~ reductase in the interac,mn with ferredoxin, FEBS Leu. 343 (1994) 247-250. Schmitz S., Martincz-Jtilvez M., Gomez-Moreno C., Bohme H. Interaction of positively changed amino acid residues of recombinant cyanobacteria ferredoxin:NADP' reductase with ferredoxm probed by site directed mutagcnesi:;, Biochim. Biophys. Acta 1363 (1998) 85-93. tturley J.K., Salarnon Z., Meyer T.E., Fitch J.C., Cusanovich M.A., Markley J.L., Cheng H., Xia B., Chac Y.K., Medina M.. G6mez-Moreno C., Tollin G.,, Amino acid residues in Anabaena fcrredoxin crucial to interaction with ferredoxin:NADW reductase: site-directed mutagenesis and laser flash photolysis, Biochemistry 32 (1993) 9346-9354. Hurley J.K., Cheng H., Xia B., Markley J.L., Medina M., G6mez-Morcno C., Toilin G., An aromatic amino acid is required at position 65 in Anabaena fen'edoxin for rapid electron transler to l'en'cdoxin:NADP' reductase, J. Ant. Chem. Soc. !15 (1993} I 1698- I 1701. Hurley J.K.. Medina M.. G6mcz-Moreno C., Tollin G.. Further characterisation by site~directed mutagencsis of the protein-protein interface m the ferredoxin/fcrredoxin:NADP' reductase .,;ystcm fi'onl AmJbaena: requirement of a negative charge at positioll 94 in ferrctloxin for rapid electron transfer, Arch. Itiochcm. lliophys, 312 ( 1994 ) 480.,~'186. Hurley J.K.. Fiilat M.E, G6mezoMoreno C., Tollin G., Structurefunction relationships in the fen'edoxin/fcrredoxin: NADP + reduco tase system from Anabaena, Biochimie 77 (1995) 539~5480 Hurley J.K., Schmeits J.L., Genzor C,. G6mezoMoreno C., Tollin G., Charge reversal mutations in a conserved acidic patch in Anabaena fert,edoxin can attenuate or enhance electron transler m ferredoxin:NADP + reduetase by altering protein/protein orieutao tion within the intermediate complex, Arch. Biochem. Btophys. 333 (1996) 243-250. Medina M., Marffnez-Jfilvez M., Hurley J.K.. Tollin G.. G6meZo Moreno C., Involvement of glutamic 301 in the catalytic mechanism of I~rredoxin:NAl3P' reductase from Anabaena PCC 7119. Biochemistry 37 (1998) 2715=2728. Alam J., Whitaker R.A., Krogman D.W,. Curtis S.E., Isolation and sequence of the gene for ferredoxin I from the cyanobacterium Anabaena sp. strain PCC 7120, J. Bacteriol. 168 (1986) 1265=127 I. Fillat MF.. Bakker Ii.A.C., Wcisbeck P,J., Sequence of the ferredoxin:NADP' reductase gent from Anabaena PCC 7119, Nucleic Acids Res. 18 (1990) 7161. G6rnez-Moreno C., Marffnez-Jfilvez M., Fillat M.F., Hurley J,K.. "lbllin G,, Molecular recognition in electron transfer proteins, in: Mathis P. (Ed.), Photosynthesis: from Light to Biosphere, Kluwcr Academic Publishers, the Netherlands. !!, 1995, pp. 62%632,
8~
G6mez-Morcno ctal.
1281 Filial M.E. Bortia~ W.E.. Weisbozk PJ.. Isolation and overexpression in £:~cherichia coti of the flavtxloxin genc tYom Amdmt, na PCC 7119. Bioehem. J. 280 (19911 187-191. 1291 Sancho J., G6mevMoreno C., Interaction of ferreduxtn:NADP' reduata~ from Am~baena with its substrates. Arch, Biochem,
1421 Walker M.C., Pueyo J.J., Navarro J.A,, Gdmez-Moreno C., lbllin G.. Laser flash photolysis studies of the kinetics of reduction of ferrcdoxins and fcrredoxin:NADP' reductases from Am~haem~ PCC 7119 and spinach, Arch. Bit~:henl, Biophys. 287 11991~ 351-358,
13OI
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