Anabaena sp. PCC 7119 Fla odoxin as Elec on Ca ie om
Pho osys em I o Fe edoxin-NADP
ⴙ
Reduc ase
ROLE OF TRP
57
AND TYR
94
*
Recei ed o publica ion, Decembe 21, 2001, and in e ised o m, Ap il 10, 2002
Published, JBC Pape s in P ess, Ap il 11, 2002, DOI 10.1074/jbc.M112258200
Jose´ L. Casaus‡, Jose´ A. Na a o§, Manuel He a´s§, Anabel Los ao‡, Miguel A. De la Rosa§,
Ca los Go´mez-Mo eno‡, Ja ie Sancho‡, and Milag os Medina‡¶
F om he ‡Depa amen o de Bioquı´mica y Biologı´a Molecula y Celula , Facul ad de Ciencias, Uni e sidad de Za agoza,
Za agoza 50009 and §Ins i u o de Bioquı´mica Vege al y Fo osı´n esis, Uni e sidad de Se illa-CSIC,
CIC Isla de la Ca uja, Ame´ ico Vespucio s/n, Se illa 41092, Spain
The in luence o he amino acid esidues sandwiching
he la in ing in la odoxin (Fld) om he cyanobac e-
ium Anabaena sp. PCC 7119 in complex o ma ion and
elec on ans e (ET) wi h i s na u al pa ne s, pho o-
sys em I (PSI) and e edoxin-NADP
ⴙ
educ ase (FNR),
was examined in mu an s o he key esidues T p
57
and
Ty
94
. The mu an s’ abili y o o m complexes wi h ei-
he FNR o PSI is simila o ha o wild- ype Fld. How-
e e , some o he mu an s exhibi al e ed kine ic p op-
e ies in hei ET p ocesses ha can be explained in
e ms o al e ed la in accessibili y and/o he mody-
namic pa ame e s. The mos no iceable al e a ion is
p oduced upon eplacemen o Ty
94
by alanine. In his
mu an , he p ocesses ha in ol e he ans e o one
elec on om ei he PSI o FNR a e clea ly accele a ed,
which migh be a ibu able o a la ge accessibili y o
he la in o he educ an . Howe e , when he opposi e
ET low is analyzed wi h FNR, he educed Y94A mu an
ans e s elec ons o FNR sligh ly mo e slowly han
wild ype. This can be explained he modynamically
om a dec ease in d i ing o ce due o he signi ican
shi o 137 mV in he educ ion po en ial alue o he
semiquinone/hyd oquinone couple (E
1
) o Y94A, ela i e
o wild ype (Los ao, A., Go´ mez-Mo eno, C., Mayhew,
S. G., and Sancho, J. (1997) Biochemis y 36, 14334–14344).
The beha io o he es o he mu an s can be explained
in he same way. O e all, ou da a indica e ha T p
57
and
Ty
94
do no play any ac i e ole in la odoxin edox e-
ac ions p o iding a pa h o he elec ons bu a e a he
in ol ed in se ing an app op ia e s uc u al and elec-
onic en i onmen ha modula es in i o ET om PSI o
FNR while p o iding a igh FMN binding.
Fla odoxins a e small
␣
/
␤
la op o eins in ol ed in ET
1
e-
ac ions in mic oo ganisms and ce ain algae. They can be syn-
hesized cons i u i ely o induced by a lack o i on in he
cul u e medium (1, 2). The p incipal ea u e o la odoxins is
ha hey con ain a nonco alen ly bound low po en ial la in
co ac o (FMN) ha con e s edox p ope ies o he p o ein.
The binding o FMN o la odoxin conside ably inc eases he
s abili y o he co ac o semiquinone s a e by al e ing i s e-
duc ion po en ials. Typically, he midpoin po en ial o he
quinone/semiquinone couple, E
2
, is inc eased, whe eas ha o
he semiquinone/hyd oquinone couple, E
1
, is dec eased. This
allows la odoxin o beha e as a one-elec on ans e cen e
ha cycles in i o be ween he semiquinone and he ully
educed o ms (1, 3).
The h ee-dimensional s uc u es o se e al la odoxins a e
known (4–10). In mos la odoxins, he isoalloxazine ing ( he
edox-ac i e moie y o FMN) is s acked be ween wo a oma ic
esidues. One o hem is a widely conse ed y osine ha
makes ex ensi e con ac s wi h he isoalloxazine, whe eas he
o he a oma ic esidue is usually a yp ophan ha in e ac s
mainly wi h he wo me hyl g oups o he isoalloxazine (11).
The p oximi y o he wo a oma ic esidues o he la in ing
makes hem in e es ing candida es o play a ole in modula ing
he educ ion po en ials o la odoxin and, he e o e, modula -
ing he p o ein’s abili y o play speci ic unc ions in he elec on
ans e chains whe e i is in ol ed. The in luence o hese
a oma ic esidues on FMN binding, la odoxin educ ion po-
en ials, and elec on spin densi y dis ibu ion o he semiqui-
none s a e has been s udied by si e-di ec ed mu agenesis using
la odoxins om di e en sou ces (11–14), and s uc u es o
some o hese mu an s a e a ailable (15). Howe e , o he bes
o ou knowledge, he in luence o hese a oma ic esidues on
la odoxin ET eac ions has no been analyzed. In Anabaena
Fld, he unc ionali y o si e-di ec ed mu an s wi h emo al o
su ace nega i e side chains has been epo ed, no only wi h
i s physiological pa ne s (PSI and FNR) bu also wi h o he
ET p o eins (16, 17). The in oduced mu a ions exe ed only
li le e ec s on he eac ions wi h FNR and PSI, whe eas hey
showed la ge e ec s on he nonphysiological eac ions wi h
cy och ome cand cy och ome P450.
In Anabaena sp. PCC 7119 Fld, he la in-sandwiching es-
idues a e T p
57
and Ty
94
( he only side chains in con ac wi h
he la in ing) (9). P e ious cha ac e iza ion o se e al
Anabaena sp. PCC 7119 Fld mu an s a posi ions 57 and 94 has
* This wo k was suppo ed by Comisio´n In e minis e ial de Ciencia y
Tecnologı´a G an BIO2000-1259 ( o C. G.-M.), CONSI⫹D, Dipu acio´n
Gene al de A ago´n, G an P006/2000 ( o M. M.), Comisio´n In e minis-
e ial de Ciencia y Tecnologı´a G an BQU2001-2520 ( o M. M.), Di ec-
cio´n Gene al de Es udios Supe io es G an PB97-1027 and G an BMC
2001-2522 ( o J. S.), Di eccio´n Gene al de In es igacio´n G an
BMC2000-0444 ( o M. A. R.), Eu opean Union Ne wo ks ERB-FM-
RXCT98-0218 and HPRN-CT1999-00095 ( o M. A. R.), and Jun a de
Andalucı´a Plan Andaluz de In es igacio´n G an CVI-0198 ( o M. A. R.).
The cos s o publica ion o his a icle we e de ayed in pa by he
paymen o page cha ges. This a icle mus he e o e be he eby ma ked
“ad e isemen ” in acco dance wi h 18 U.S.C. Sec ion 1734 solely o
indica e his ac .
¶To whom co espondence should be add essed: Dep . de Bioquı´mica
y Biologı´a Molecula y Celula , Facul ad de Ciencias, Uni e sidad de
Za agoza, Za agoza 50009, Spain. Tel.: 34-976-762476; Fax: 34-976-
762123; E-mail: [email p o ec ed].
1
The abb e ia ions used a e: ET, elec on ans e ; dR , 5-deaza i-
bo la in; dR H䡠, semiquinone o m o dR ; Fld, la odoxin; Fld
ox
, Fld in
he oxidized s a e; Fld
d
, Fld in he educed s a e; Fld
sq
, Fld in he
semiquinone s a e; FNR, e edoxin-NADP
⫹
educ ase; FNR
ox
, FNR in
he oxidized s a e; FNR
d
, FNR in he educed s a e; FNR
sq
, FNR in he
semiquinone s a e; PSI, pho osys em I; WT, wild- ype; T icine,
N-[2-hyd oxy-1,1-bis(hyd oxyme hyl)e hyl]glycine.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 25, Issue o June 21, pp. 22338–22344, 2002
© 2002 by The Ame ican Socie y o Biochemis y and Molecula Biology, Inc. P in ed in U.S.A.
This pape is a ailable on line a h p://www.jbc.o g22338
This is an Open Access a icle unde he CC BY license.
shown ha hese a oma ic esidues, in pa icula Ty
94
, in lu-
ence he co ac o educ ion po en ials and i s abso p ion spec-
um. In addi ion, measu emen o he apo la odoxin-FMN
complex binding ene gies showed ha bo h Ty
94
and T p
57
s eng hen he in e ac ion o apo la odoxin wi h FMN in he
di e en edox s a es and a e esponsible o se ing he cha -
ac e is ic educ ion po en ials exhibi ed by his p o ein (11). In
con as , i has been shown ha eplacemen o Ty
94
and
T p
57
only e y sligh ly in luences he elec on spin densi y
dis ibu ion in he la odoxin semiquinone s a e (14). In he
p esen s udy, we epo he unc ional cha ac e iza ion o
Anabaena sp. PCC 7119 Fld mu an s ob ained by eplacemen
o T p
57
and Ty
94
by o he a oma ic esidues as well as by
alanine and leucine (11). Complex o ma ion and ET be ween
PSI and Fld and be ween Fld and FNR ha e been s udied o
he WT and mu a ed o ms o Fld in o de o analyze he
in ol emen o Ty
94
and T p
57
in hese p ocesses. So a , none
o he h ee-dimensional s uc u es o he mu an s he e cha ac-
e ized is a ailable. Howe e , ecen ly, he s uc u es o wo Fld
mu an s om Desul o ib io ulga is a he equi alen posi ion o
Ty
94
om Anabaena Fld, Y98H and Y98W, ha e been epo ed
(15). Since p ecise main-chain and side-chain supe posi ion is
obse ed be ween he s uc u es o WT Flds om bo h species,
specially a ound he la in ing, he unc ional beha io o he
Anabaena mu an s is discussed based on he s uc u al ea u es
obse ed o hese wo D. ulga is mu an s uc u es.
EXPERIMENTAL PROCEDURES
Biological Ma e ial—Fld mu an s om Anabaena sp. PCC 7119 we e
p epa ed by oligonucleo ide-di ec ed mu a ion o he Fld gene, o e p o-
duced in Esche ichia coli, and pu i ied as p e iously desc ibed (10, 11).
Anabaena sp. PCC 7119 WT FNR was pu i ied om E. coli isop opyl-
␤
-D- hiogalac oside-induced cul u es ha had been p e iously ans-
o med wi h he p c99a ec o con aining he FNR gene om
Anabaena sp. PCC 7119, as p e iously desc ibed (18). UV- isible spec-
a and SDS-PAGE elec opho esis we e used as pu i y c i e ia. PSI
pa icles om Anabaena sp. PCC 7119 we e ob ained by
␤
-dodecyl
mal oside solubiliza ion as desc ibed by Ro¨gne e al. (19) and modi ied
by He a´se al. (20). The P700 con en in PSI samples was calcula ed
om he pho oinduced abso bance changes a 820 nm using he abso p-
ion coe icien o 6.5 mM
⫺1
cm
⫺1
de e mined by Ma his and Se´ i (21).
Chlo ophyll concen a ion was de e mined acco ding o A non (22). The
chlo ophyll/P700 a io o he esul ing PSI p epa a ions was 140:1. The
same ba ches o PSI and la op o eins we e used h oughou his s udy.
Spec al Analysis—UV- isible spec a we e eco ded on a KON-
TRON U ikon 942 spec opho ome e . Dissocia ion cons an s, binding
ene gies, and changes in ex inc ion coe icien s o he complexes be-
ween WT FNR
ox
and he di e en Fld
ox
a ian s we e ob ained by
di e en ial spec oscopy as p e iously desc ibed (18). The expe imen s
we e pe o med on solu ions con aining ⬃20
␮
MFNR in 4 mMpo as-
sium phospha e, 1 mMEDTA bu e (pH 7.0) a 25 ⫾1°C, o which
aliquo s o he di e en Fld o ms we e added. Dissocia ion cons an s
and di e en ial ex inc ion coe icien s o he complexes we e ob ained
by i ing he expe imen al da a o he heo e ical equa ion o a 1:1
s oichiome ic complex by means o nonlinea eg essions.
S eady-s a e Enzyma ic Assays—The NADPH-dependen cy och ome
c educ ase ac i i y o FNR was de e mined using he di e en Fld
mu an s as he elec on ca ie om FNR o cy och ome c(18, 23). To
calcula e he FNR K
m
alues o each Fld a ian , he s anda d eac ion
mix u es con ained 50
␮
MNADPH, 2.5 nMFNR, 0.75 mMcy och ome c
(Sigma), and di e en concen a ions o he co esponding Fld mu an .
All measu emen s we e ca ied ou a 25 ⫾1°Cin50m
MT is/HCl,
pH 8.0.
S opped- low Kine ic Measu emen s—ET eac ions be ween FNR
and Fld we e s udied by s opped low unde anae obic condi ions in an
Applied Pho ophysics SX17.MV spec opho ome e in e aced wi h an
Aco n 5000 compu e , using he SX.18MV so wa e o Applied Pho o-
physics as p e iously desc ibed (18). The appa en obse ed a e con-
s an s (k
app
) we e calcula ed by i ing he expe imen al da a o mono-
o biexponen ial kine ics. Samples we e made anae obic (in specially
designed onome e s, which i ed he s opped- low appa a us) by suc-
cessi e e acua ion and O
2
- ee a gon lushing. Reduced samples o FNR
and Fld o s opped low we e p epa ed by pho o educ ion wi h 5-dR as
desc ibed (18). All eac ions we e ca ied ou in 4 mMpo assium phos-
pha e, 1 mMEDTA, pH 7.0, a 13 ⫾1°C and ollowed a 600 nm, he
wa eleng h a which semiquinone o ma ion o bo h la op o eins can
be ollowed. FNR and Fld we e mixed a 1:1 a ios wi h inal p o ein
concen a ions anging om 7 o 9
␮
M. Each kine ic ace was he
a e age o 4–10 independen measu emen s. E o s in he de e mina-
ion o he appa en obse ed a e cons an alues (k
app
) we e o ⫾10%.
Lase Flash Abso p ion Spec oscopy—The lase lash sys em was
based on a PTI PL-2300 ni ogen lase (emission wa eleng h, 337 nm;
lash du a ion, 0.6 ns; ou pu ene gy, 1.4 mJ). The lase beam was
a enua ed by 80% wi h calib a ed neu al densi y il e s. Kine ic
aces o lash-induced abso bance changes a 580 nm we e eco ded
using a Nicole 450 digi al oscilloscope. The analysis ligh sou ce was a
100-wa ungs en-halogen lamp, which passed h ough an O iel mono-
ch oma o (model 77250, bandwid h 10 nm). The de ec ion pho omul-
iplie (Hamama su R928) was p o ec ed om ac inic ligh by a second
monoch oma o (Jobin-Y on; model H-20, bandwid h 1 nm). The wa e-
leng h o 580 nm was chosen o moni o Fld semiquinone o ma ion
wi h he lowes possible con ibu ion o PSI o he abso bance change.
All o he expe imen s we e ca ied ou in a 1-cm pa h leng h cu e e a
22 ⫾1°C. Unless o he wise s a ed, he s anda d eac ion mix u e
con ained, in a inal olume o 1 ml, 20 mMT icine/KOH, pH 7.5, 0.03%
␤
-dodecyl mal oside, an amoun o PSI-en iched pa icles equi alen o
35
␮
g o chlo ophyll ml
⫺1
, 0.1
␮
Mphenazine me hosul a e, 2 mMMgCl
2
,
2m
Msodium asco ba e, and Fld a he indica ed concen a ion. Asco -
ba e and phenazine me hosul a e addi ion ensu es ha PSI is o ally
e educed be ween lashes. Each kine ic ace was he a e age o 40–50
independen measu emen s wi h 30-s spacing be ween lashes. Sam-
ples we e p o ec ed om ac inic ligh be ween lashes by an elec onic
shu e synch onized wi h he lase igge . Fo mos expe imen s, he
es ima ed e o in he obse ed a e cons an s was less han 20%, based
on ep oducibili y and signal- o-noise a ios. Da a collec ion was as
p e iously desc ibed (24). Exponen ial analyses o he oscilloscope
aces we e pe o med using he Ma qua d me hod wi h he so wa e
de ised by P. Se´ i (Saclay, F ance). Kine ic analyses we e ca ied ou
acco ding o he wo-s ep eac ion mechanism p e iously p oposed (24,
25). E o s in he es ima ed alues o cons an s o complex associa ion
(K
a
) and elec on ans e (k
e
) we e o ⫾15 and ⫾5%, espec i ely. The
pu a i e p o ona ion a e cons an o Fld semiquinone (k
p o
) a e e-
duc ion by dR H䡠was de e mined by ollowing he abso bance dec ease
and inc ease a 465 and 580 nm, espec i ely (26).
RESULTS
S eady-s a e Kine ics Analysis o he Di e en Fld Mu an s—
The ca aly ic p ope ies o FNR o he la odoxin-media ed
NADPH-dependen cy och ome c educ ase assay ha e been
de e mined unde s eady-s a e condi ions using WT and mu-
a ed la odoxins. This ac i i y in ol es, in addi ion o FNR
educ ion by he py idine nucleo ide, he in e ac ion and ET
be ween FNR and Fld and he inal educ ion o cy och ome c
by he semiquinone o m o Fld (18). As shown in Table I, mos
Fld mu an s yield k
ca
alues wi hin a ac o o 2 o WT, bu
signi ican di e ences a e obse ed when Y94A o Y94W a e
used. The alue o k
ca
wi h Y94A inc eases 5- old ela i e o
TABLE I
Midpoin educ ion po en ials o he di e en Flds (da a om Re . 11)
and s eady-s a e kine ics pa ame e s o WT FNR in he NADPH-
dependen cy och ome c educ ase ac i i y using WT and mu an Flds
as elec on ca ie
Fld
Reduc ion
Po en ials
a
S eady-s a e kine ic pa ame e s
E
2
E
1
k
ca
K
m
k
ca
/K
m
mV s
⫺1
mMmM
⫺1
s
⫺1
WT ⫺212 ⫺436 43 ⫾142⫾1 1.0 ⫾0.1
Y94A ⫺203 ⫺299 222 ⫾720⫾512⫾3
Y94F ⫺186 ⫺436 24 ⫾448⫾8 0.5 ⫾0.2
Y94W ⫺182 ⫺462 ⬍5
b
ND
b
ND
W57F ⫺152 ⫺432 37 ⫾339⫾4 1.0 ⫾0.1
W57L ⫺212 ⫺407 20 ⫾321⫾5 1.0 ⫾0.5
W57A ⫺173 ⫺417 18 ⫾212⫾4 1.5 ⫾0.9
W57Y ⫺139 ⫺447 36 ⫾427⫾6 1.4 ⫾0.5
a
Da a om Re . 11.
b
Only an es ima ion o k
ca
was possible due o he e y low amoun
o eac ion obse ed, which was independen o Fld concen a ion.
A oma ic Residues in Fla in-media ed Elec on T ans e 22339
WT, whe eas Y94W displays li le ac i i y and an es ima ed
k
ca
alue a leas 7- old lowe han WT. Unlike WT Fld, which
is much less e icien han e edoxin in his assay, he Y94A
Fld mu an (k
ca
⫽222 ⫾7s
⫺1
) is an ET media o as e icien
as he i on-sul u p o ein (k
ca
⫽200 ⫾10 s
⫺1
(18)).
FNR K
m
alues o mos Fld mu an s we e wi hin a ac o o
2 o WT (Table I). Only W57A showed a signi ican dec ease in
K
m
(4- old). These da a sugges ha he a ini y o he FNR䡠Fld
complex is ha dly modi ied by he mu a ions. The K
m
o Y94W
could no be de e mined due o he lack o ac i i y obse ed in
he assay.
When he ca aly ic e iciency (k
ca
/K
m
) o FNR was de e -
mined using di e en Fld a ian s in he cy och ome cassay, i
u ned ou ha mos Flds lead o an FNR ca aly ic e iciency
simila o ha o WT. In con as , Y94A appea ed o be 1 o de
o magni ude mo e e icien han WT, and Y94W was almos
non eac i e. The highe e iciency o Y94A e lec s he much
as e ET p ocess, whe eas he low e iciency o Y94W could be
due ei he o a much slowe ET p ocess o o he o ma ion o a
nonp oduc i e in e ac ion be ween Fld and ei he FNR o cy-
och ome c.
In e ac ion o Fld
ox
Va ian s wi h FNR
ox
—To in es iga e he
e ec o he Fld mu a ions a posi ions 57 and 94 on i s in e -
ac ion wi h FNR, di e ence abso p ion spec oscopy was used
o di ec ly e alua e he dissocia ion cons an s o he oxidized
complexes (Fig. 1). The di e en ial spec um o he WT
Fld
ox
䡠FNR
ox
complex ha has been p oposed o a ise om
al e a ion o he la in en i onmen s upon Fld䡠FNR associa ion
shows abso p ion maxima a ound 390 and 465 nm (18). Simila
spec al pe u ba ions we e de ec ed o all o he Fld mu an s
s udied he e upon mixing wi h FNR
ox
. I is wo h no ing he
simila shape o he di e en ial spec a ob ained wi h he
di e en Fld mu an s, wi h bands a ound 390 and 464–480 nm
wi h he same ela i e in ensi y as o he WT Fld complex
(Fig. 1) despi e he ac ha some Fld mu an s (i.e. Y94W and,
especially, Y94F) p esen unusual la in abso p ion spec a
(Fig. 1D) (11). This sugges s ha he Fld
ox
䡠FNR
ox
di e ence
spec um is mainly due o al e a ion o he FAD abso p ion in
FNR and indica es ha he o ien a ion o he wo p o eins in
he complex is no al e ed by he mu a ions he e s udied.
Acco dingly, all Fld
ox
mu an s showed simila K
d
alues o he
WT complex (Table II). Mo eo e , he ⌬
⑀
alues o ansi ion
band I a e no a om he alue ob ained o he WT Fld
complex, wi h he maximal changes obse ed o Y94A and
Y94W, whose ⌬
⑀
alue a e ⬃2- old la ge . This inc emen ed ⌬
⑀
could be ela ed wi h he al e a ions desc ibed in he abso p-
ion spec a o hese mu an s (11). Taken oge he , hese da a
indica e ha T p
57
and Ty
94
exe li le in luence on he
o ien a ion and s eng h o he FNR
ox
䡠Fld
ox
associa ion.
Fas Kine ic Analysis o he In e ac ion and ET P ocess be-
ween FNR and Fld Mu an s as S udied by S opped Flow—To
u he in es iga e he ole o Fld T p
57
and Ty
94
in associa-
ion and ET o FNR, anae obic s opped- low kine ic expe i-
men s we e ca ied ou . The eac ions be ween he di e en
edox s a es o Fld and FNR we e analyzed by ollowing
semiquinone o ma ion a 600 nm by mixing equimolecula
amoun s o bo h p o eins, each in a di e en edox s a e, ei he
oxidized o educed. When FNR
ox
was mixed wi h Fld
d
,an
inc ease in abso bance a 600 nm, p e iously asc ibed o o -
ma ion o bo h semiquinones (18), was de ec ed o WT Fld (no
shown) and all o he mu an s (Fig. 2). Fo WT, Y94F, and
W57L Fld, mos o he eac ion akes place wi hin he ins u-
men al dead ime (⬃3 ms) (Table III), whe eas slowe p ocesses
a e obse ed o he es o he mu an s. When he eac ion o
Y94A was analyzed, he kine ic aces we e bes i ed by a
wo-exponen ial equa ion, wi h k
app
alues o 356 and 64 s
⫺1
(Fig. 2A). A simila beha io was obse ed o W57A, al hough
in his case he i s p ocess akes place mainly wi hin he
ins umen al dead ime (Fig. 2B, Table III). In bo h cases, he
ini ial p ocess is consis en wi h he o ma ion o he wo
semiquinones (Reac ion 1), ollowed by educ ion o FNR
sq
o
he ully educed s a e by Fld
d
, which is s ill in he mix u e
(Reac ion 2) (18).
FNRox ⫹Fld d 兵-
|
0
Kd
关FNRox···Fld d兴
O
¡
ke
其FNRsq ⫹Fldsq
P 1
kapp1
Fld d ⫹FNRsq 兵-
|
0
Kd⬘
关FNRsq···Fld d兴
O
¡
ke ⬘
其Fldsq ⫹FNR d
P 1
kapp2
REACTIONS 1AND 2
Mo e no iceable a e he kine ics o Y94W, W57F, and W57Y
(Fig. 2B). In hese mu an s, a lag phase wi h no abso bance
changes is obse ed, bo h a 600 nm (Fig. 2B) and a 460 nm
(no shown). This indica es ha he accumula ion o an oblig-
a o y in e media e species akes place p io o ET. Mo eo e ,
when he in e media e accumula es and he eac ion s a s,
p ocesses much slowe han ha wi h WT Fld ake place (Table
III), and hey appea o occu only o a mino ex en , as de-
FIG.1. Spec oscopic cha ac e iza ion o he complexes
o med be ween selec ed Fld
ox
o ms and FNR
ox
.A, abso p ion
spec a o WT Fld
ox
.B, abso p ion spec a o Y94F Fld
ox
.C, di e ence
abso p ion spec a ob ained du ing he i a ion o WT FNR
ox
(17.8
␮
M)
wi h WT Fld
ox
.D, di e ence abso p ion spec a ob ained du ing he
i a ion o WT FNR
ox
(16.4
␮
M) wi h Y94F Fld
ox
.
TABLE II
Dissocia ion cons an s, di e en ial ex inc ion coe icien s, and ee
ene gies o complex o ma ion o oxidized WT FNR wi h oxidized
WT and mu a ed Flds
Fld K
d
⌬␧
465 nm
⌬G
0
mMmM
⫺1
cm
⫺1
Kcal mol
⫺1
WT 3.6 ⫾1.0 1.5 ⫾0.2 7.4 ⫾0.1
Y94A 2.3 ⫾0.5 2.6 ⫾0.4 7.7 ⫾0.3
Y94W 6.0 ⫾1.5 3.2 ⫾0.6 7.2 ⫾0.3
Y94F 1.0 ⫾0.5 1.6 ⫾0.5 8.2 ⫾0.3
W57F 4.0 ⫾0.8 1.0 ⫾0.1 7.4 ⫾0.2
W57L 2.8 ⫾0.5 1.2 ⫾0.1 7.5 ⫾0.1
W57A 4.5 ⫾1.0 1.0 ⫾0.3 7.4 ⫾0.6
W57Y 2.2 ⫾0.8 2.0 ⫾0.6 7.7 ⫾0.3
A oma ic Residues in Fla in-media ed Elec on T ans e 22340
duced om hei low ampli udes (Fig. 2B). The ini ial lag phase
could e lec a eo ganiza ion o he ini ial complex, equi ed o
achie e an o ien a ion compe en o e icien ET.
The e e se eac ion has also been in es iga ed. P e ious
s opped- low s udies on he eac ion o FNR
d
wi h WT Fld
ox
indica ed ha his eac ion was a ela i ely slow p ocess o
which wo phases we e de ec ed (18). These we e assigned o
he ollowing p ocesses.
Fldox ⫹FNR d 兵-
|
0
Kd
关FNR d···Fldox兴
O
¡
ke
其Fldsq ⫹FNRsq
P 1
kapp1
Fldox ⫹FNRsq 兵-
|
0
Kd⬘
关FNRsq···Fldox兴
O
¡
ke ⬘
其Fldsq ⫹FNRox
P 1
kapp2
REACTIONS 3AND 4
When his eac ion was pe o med using di e en Fld mu-
an s, a biphasic beha io , wi h wo phases o simila ampli-
ude, was de ec ed o mos mu an s (Fig. 3A), as in he WT Fld
eac ion. Thus, o Y94F, Y94W, and W57L Fld, he eac ion
akes place wi h k
app
alues wi hin a ac o o 2 o hose wi h
WT Fld (Table III), al hough he k
app
o he second eac ion o
Y94W is signi ican ly slowe . Replacemen o T p
57
ei he by
phenylalanine, alanine o y osine p oduced la ge k
app
alues
(up o a ac o o 7 o W57A in he i s p ocess; Table III). In
all o hese cases, he yp ophan side-chain has been eplaced
by sho e esidues. Finally, we no ice he e ec p oduced by
eplacemen o Ty
94
by alanine. I s educ ion by FNR
d
i s o
a monoexponen ial p ocess wi h a k
app
alue 175- old highe
han ha obse ed o WT Fld (Fig. 3B, Table III), indica ing
ha emo al o he a oma ic side chain a posi ion 94 p oduces
an impo an e ec in he ET mechanism om FNR o Fld. The
monoexponen ial beha io obse ed o his mu an migh be
due ei he o an almos ull consump ion o FNR
d
by he i s
p ocess (Reac ion 3) o o a change in he mechanism o ET
om he educed FAD o he Fld FMN.
Kine ic Analysis o he Lase Flash Fas Reduc ion o Fld
Va ian s by PSI—P e ious s udies ha e shown ha he educ-
ion o Anabaena Fld
ox
o he semiquinone s a e by spinach PSI
pa icles can be ollowed using lase - lash abso p ion spec os-
copy (27). The s udy o he semiquinone o ma ion cons i u es a
use ul eac ion model o analyze he in e ac ion o ces and
elec on ans e pa ame e s in ol ed in he educ ion o Fld by
PSI (16, 27, 28). In he p esen wo k, we ha e analyzed he
eac i i y o Anabaena Fld owa d PSI om he same species.
Reduc ion o Fld mu an s by PSI pa icles ollowed monoexpo-
nen ial kine ics o WT and all o he mu an s (e en a high
la op o ein concen a ion). Fig. 4 ep oduces he oscilloscope
aces ob ained o Y94A, W57Y, and WT Flds and shows ha
he eac ion is much slowe wi h W57Y han wi h Y94A o WT.
The obse ed pseudo- i s o de a e cons an s (k
obs
)o WTand
mu an Fld educ ion by PSI depend nonlinea ly on he con-
cen a ion o la op o ein and show a sa u a ion p o ile (Fig. 5).
This sugges s ha a bimolecula ansien (PSI䡠Fld) complex is
o med p io o ET, acco ding o he ollowing minimal wo-s ep
eac ion mechanism, as desc ibed p e iously in o he PSI/Fld
sys ems (16, 27, 28).
FIG.2. Time cou se o he anae obic eac ions be ween WT
FNR
ox
wi h di e en Fld
d
mu an s as measu ed by s opped
low. The moni o ing wa eleng h was 600 nm. Final concen a ions a e
gi en in pa en heses.A, eac ion o WT FNR
ox
(8.5
␮
M) wi h Y94A Fld
d
(8.5
␮
M) ( esidual o his i ing is shown a he bo om). B, eac ion o
WT FNR
ox
(7.5
␮
M) wi h W57A Fld
d
(7.5
␮
M)( illed squa es; esidual
o his i ing is shown a he bo om); WT FNR
ox
(8.1
␮
M) wi h W57F
Fld
d
(8.6
␮
M)( illed iangles); WT FNR
ox
(8.2
␮
M) wi h W57Y Fld
d
(8.6
␮
M)( illed diamonds) and WT FNR
ox
(8.4
␮
M) wi h Y94W Fld
d
(8.6
␮
M)
( illed ci cles).
TABLE III
Kine ic pa ame e s o he eac ions o WT FNR wi h WT and
mu a ed Flds as s udied by s opped low
Samples we e mixed in he s opped- low ins umen a equimolecula
concen a ions (⬃8
␮
M inal concen a ion) and a he indica ed edox
s a es, T
a
⫽13 ⫾1°C. Reac ions we e ollowed a 600 nm.
Fld FNR
ox
⫹Fld
d
FNR
d
⫹Fld
ox
k
app1
k
app2
k
app1
k
app2
s
⫺1
s
⫺1
WT ⬎600
a
1.8 0.8
Y94A 356 64 400.0
Y94F ⬎600
a
1.8 1.1
Y94W 54
b
0.5 0.06
W57F 35
b
12.0 1.9
W57L ⬎600
a
3.8 2.3
W57A ⬎600
a
146 14.0 0.5
W57Y 34
b
5.1 0.8
a
Mos o he eac ion occu ed wi hin he ins umen al dead ime.
b
A lag phase is obse ed a 600 nm un il 30–50 ms; he k
app
was
es ima ed a e his phase.
FIG.3. Time cou se o he anae obic eac ions be ween WT
FNR
d
wi h di e en Fld
ox
mu an s as measu ed by s opped
low. The moni o ing wa eleng h was 600 nm. Final concen a ions a e
gi en in pa en heses.A, eac ion o WT FNR
d
(8
␮
M) wi h WT Fld
ox
(8.5
␮
M)( illed diamonds); WT FNR
d
(8.7
␮
M) wi h W57A Fld
ox
(8.2
␮
M)
(open diamonds); WT FNR
d
(8.7
␮
M) wi h W57F Fld
ox
(9
␮
M)( illed
ci cles); WT FNR
d
(10
␮
M) wi h W57Y Fld
ox
(9
␮
M)( illed iangles;
esidual o his i ing is shown a he bo om); WT FNR
d
(8.1
␮
M) wi h
W57L Fld
ox
(8
␮
M)(open squa es); WT FNR
d
(7.8
␮
M) wi h Y94F Fld
ox
(8
␮
M)( illed squa es) and WT FNR
d
(8.5
␮
M) wi h Y94W Fld
ox
(8.5
␮
M)
(open ci cles). B, eac ion o WT FNR
d
(7.5
␮
M) wi h Y94A Fld
ox
(7.5
␮
M)
( esidual o his i ing is shown a he bo om).
A oma ic Residues in Fla in-media ed Elec on T ans e 22341
PSI d ⫹Fldox -
|
0
Ka
关PSI d···Fldox兴
O
¡
ke
PSIox ⫹Fldsq
REACTION 5
in which he elec on ans e i s -o de a e cons an (k
e
) can
expe imen ally be in e ed om he limi ing k
obs
a in ini e Fld
concen a ion, whe eas K
a
is he equilib ium cons an o he
complex. Minimal alues o K
a
and k
e
, which can be es ima ed
om da a like ha in Fig. 5 using he o malism de eloped by
Meye e al. (25), a e shown in Table IV o WT and mu an
Flds. We ha e p e iously epo ed (26) a pH-dependen a e-
limi ing s ep o WT Fld educ ion by he dR adical, which
was assigned o Fld semiquinone p o ona ion. The pu a i e
p o ona ion a e cons an o Fld semiquinone (k
p o
) a e e-
duc ion by dR has hus been de e mined wi h all mu an s
he ein desc ibed, bu no signi ican di e ences a e obse ed
be ween mu an s and WT Fld (Table IV). F om hese indings
we can in e ha he ET a e cons an de e mined a sa u a -
ing Fld concen a ion (k
e
) and he p oposed p o ona ion a e
cons an (k
p o
) co espond o wo un ela ed, independen p o-
cesses. Thus, ou da a indica e ha whe eas none o he e-
placemen s pe o med a posi ion 94 o 57 p omo es signi ican
changes in he K
a
alue o he PSI
d
䡠Fld
ox
complex unde ou
s anda d condi ions, some o he mu a ions p omo e d as ic
changes in he k
e
om PSI o Fld. Rema kably, he k
e
o Y94A
Fld is 1 o de o magni ude highe han ha o WT, whe eas
eplacemen o Ty
94
by a yp ophan p oduces a i ually
non eac i e p o ein (Table IV). Replacemen o Ty
94
by phe-
nylalanine has li le e ec on he eac i i y o he la op o ein,
as compa ed wi h he o he mu an s a posi ion 94, as expec ed
o his conse a i e change. Wi h ega d o posi ion 57, e-
placemen o yp ophan wi h any smalle hyd ophobic esidue
imp o es he ET om PSI o Fld. This e ec is pa icula ly
ele an in W57F, whose k
e
is 6 imes highe han WT. The
unexpec ed esul is ha eplacemen o yp ophan by y o-
sine p omo es he e e se e ec , he ET a e cons an dec eas-
ing 5 imes ela i e o WT.
DISCUSSION
T p
57
and Ty
94
, which sandwich he isoalloxazine ing in
Anabaena sp. PCC 7119 Fld, ha e been shown o in luence i s
educ ion po en ials and abso p ion spec a and o s eng hen
he FMN apo la odoxin in e ac ion (11). In he p esen s udy,
we ha e analyzed hei in luence on he Fld in e ac ion and ET
by measu ing he unc ionali y o di e en Fld mu an s a
posi ions 57 and 94 wi h i s wo physiological edox pa ne s,
FNR and PSI.
S eady-s a e and as kine ic s udies ha e been ca ied ou o
analyze he eac ions o Fld wi h FNR. The K
m
alues ob ained
o FNR, using he di e en Fld a ian s as p o ein ca ie s in
FIG.5. Dependence upon Fld concen a ion o he k
obs
o
educ ion o WT and mu a ed Fld o ms by Anabaena PSI. Ex-
pe imen al condi ions we e as desc ibed unde “Expe imen al P oce-
du es.”The solid lines ha e been ob ained by i ing he expe imen al
poin s o he o malism de ised by Meye e al. (25).
TABLE IV
Kine ic pa ame e s o he educ ion o WT and mu a ed Flds by
Anabaena PSI and by deaza ibo la in as s udied by
lase lash pho olysis
Fld Reduc ion by PSI Reduc ion by
dR (k
p o
)
a
K
a
k
e
M
⫺1
s
⫺1
s
⫺1
WT 6.0 ⫻10
5
500 450
Y94A 7.0 ⫻10
5
5500 730
Y94F 8.0 ⫻10
5
135 610
Y94W ND
b
⬍100 410
W57F 3.0 ⫻10
5
2950 625
W57L 1.0 ⫻10
5
950 440
W57A 6.0 ⫻10
5
1480 580
W57Y 4.0 ⫻10
5
105 505
a
The a e cons an s we e es ima ed om he Fld concen a ion-
independen kine ic aces ollowed a bo h 465 and 580 nm (see “Ex-
pe imen al P ocedu es”).
b
No de e mined, due o ex emely slow kine ics.
FIG.4.Kine ic aces showing WT and mu an Flds educ ion
by Anabaena PSI. Fla op o ein concen a ion was 20
␮
M, and he pH
alue was 7.5. All kine ic aces we e well i ed o single exponen ial
cu es. O he condi ions we e as desc ibed unde “Expe imen al
P ocedu es.”
A oma ic Residues in Fla in-media ed Elec on T ans e 22342

he NADPH-dependen cy och ome c educ ase assay (Table I),
as well as he da a de i ed om di e en ial spec oscopic anal-
ysis o he FNR
ox
-Fld
ox
in e ac ion (Fig. 1, Table II) indica e
ha he in oduced mu a ions do no p oduce any majo e ec
in he s abili y o he FNR
ox
䡠Fld
ox
complex o in he unc ion-
ali y o he FNR
d
䡠Fld
ox
complex. Howe e , some o he mu a-
ions clea ly al e he ET be ween he wo p o eins. The mos
no iceable al e a ion is p oduced upon eplacemen o Ty
94
by
alanine. In his mu an , p ocesses in ol ing ET om FNR
d
o
Fld
ox
show a es up o 5- old highe han hose ob ained wi h
WT Fld when moni o ing he FNR cy och ome c educ ase
ac i i y (Table I) and up o 222- old highe when ollowing he
di ec eac ion by s opped low (Fig. 3B, Table III). P e ious
s udies, howe e , had shown ha FNR
d
is appa en ly no so
well sui ed o ans e ing a single elec on o WT Fld
ox
as i
is o Fd
ox
(E
m
⫽⫺384 mV) (29), whe eas FNR
sq
is much mo e
e icien ( a e cons an ⬎7000 s
⫺1
(30)). These indings e eal
he impo ance o he s uc u al ea u es o he p o eins pa -
icipa ing in ET p ocesses o e he modynamic ac o s (18).
Since, in he case o he Y94A mu an , he in oduced side
chain does no p o ide an elec onic en i onmen ha can
imp o e by i sel he ET p ocess, s uc u al and he mody-
namic aspec s ha e o accoun o he enhancemen obse ed.
The epo ed educ ion po en ials o his mu an show an E
1
alue conside ably less nega i e han ha o WT Fld (⫺299
mV e sus ⫺436 mV), indica ing ha in his mu an he en-
ance o he second elec on is he modynamically a o ed,
which migh allow a ull educ ion o Y94A Fld
sq
by FNR
d
o
e en by FNR
sq
. This could explain he high k
ca
alue obse ed
o FNR in he NADPH-dependen cy och ome c educ ase
assay when using Y94A; once enough Y94A Fld
d
is o med, i
migh cycle be ween he hyd oquinone and semiquinone s a es
when ans e ing elec ons om FNR o cy och ome c, hus
wo king unde a he modynamic o ce close o ha ope a ing
in he Fd assay. Howe e , an enhancemen o he ET om
FNR
d
o Fld
ox
should no be disca ded. To cla i y his, we ha e
ollowed he FNR
d
/Fld
ox
eac ion by s opped low. The inc ease
in abso bance obse ed a 600 nm (Fig. 3B) indica es a e y as
accumula ion o semiquinone s a es, which is no consis en
wi h a wo-elec on ans e p ocess and sugges s a single
elec on ans e and accumula ion o he wo semiquinone
species. The e o e, he la ge e iciency obse ed when analyz-
ing he ET om FNR
d
o Y94A Fld
ox
has o be due o a as e
single ET p ocess o gene a e Y94A Fld
sq
. Since complex o -
ma ion is appa en ly no a ec ed and he E
2
alue o his
mu an is simila o ha o WT Fld, s uc u al aspec s ha e o
accoun o he enhancemen o he p ocess. We sugges ha
eplacemen o he la ge y osine esidue by he small alanine
migh help by simply making he la in co ac o mo e accessi-
ble o educ ion. Consis en ly, eplacemen o y osine by yp-
ophan (wi h a la ge side chain) in he Y94W mu an would
make he la in co ac o less accessible, he eby explaining he
lowe ET a e om FNR
d
o he mu an , as shown by bo h
s eady-s a e and as kine ic s udies (Tables I and III). This
hypo hesis is suppo ed by he s uc u es epo ed o wo
mu an s om D. ulga is Fld, Y98H and Y98W, a a posi ion
equi alen o ha o Ty
94
in Anabaena Fld (15). These s uc-
u es show ha , al hough he o e all olding emains he same
as ha o na i e Fld, he en i onmen o he isoalloxazine ing
is modi ied by he in oduced mu a ions. No iceably, in bo h
mu an s he 60–64 loop, (T p
60
co esponds o T p
57
in he
Anabaena Fld) is displaced and esembles mo e he s uc u e o
he Fld semi educed s a e han ha o he oxidized s a e.
Mo eo e , he hyd ogen-bonding ne wo k and he wa e
scheme a ound he FMN a e di e en , and bo h mu an s uc-
u es show a sligh bending in he FMN isoalloxazine ing. In
pa icula , he s uc u e o he Y98W mu an clea ly shows a
la ge shielding o he la in om he sol en , especially a
posi ions N-5, whe e a p o on has o en e in o de o p oduce
he educed and semiquinone s a es o FMN, and C-6 o he
la in ing. This s ongly sugges s ha he obse ed dec eased
eac i i y o Y94W Anabaena Fld in he ET p ocesses wi h FNR
is due o a dec eased accessibili y o he la in co ac o . On he
o he hand, he s uc u e o he Y98H mu an om D. ulga is
Fld shows less shielding o he la in ing om he sol en ,
wi h he benzene ing ully exposed. This lack o shielding has
been ela ed wi h he no iceable E
1
shi o mo e posi i e al-
ues exhibi ed by his mu an (15). A simila E
1
shi has been
ound in ou Y94A mu an (11), sugges ing also a la ge acces-
sibili y o he isoalloxazine ing in his mu an , as expec ed o
a Ty o Ala eplacemen .
Howe e , when analyzing he opposi e p ocess, educ ion o
FNR by Fld a ian s, Y94A ET is hinde ed ela i e o WT Fld.
This is so because he in oduced mu a ion p oduces an impo -
an al e a ion o he la in E
1
alue, which is 137 mV mo e
posi i e han ha o WT, making educ ion o FNR (E
m
⫽⫺323
mV (29)) much less a o able om he he modynamic poin o
iew. The Y94W, W57F, and W57Y Fld mu an s (Fig. 2B)do
no show any majo al e a ions o he educ ion po en ials, ye
hey display a lag phase and slowe and diminished p ocesses.
In hese mu an s, s uc u al aspec s mus accoun o he ob-
se ed lag phase, indica i e o he accumula ion o an in e me-
dia e p io o ET, and sugges ing ha complex eo ganiza ion
canno achie e he op imal o ien a ion in he ansi ion s a e
o e icien ET.
In e ac ion and ET be ween educed PSI and he di e en
Flds we e analyzed by using lase lash pho olysis. Fo WT Fld,
he ob ained kine ic pa ame e s a e o he same o de o mag-
ni ude as hose p e iously desc ibed o he in e ac ion o his
la op o ein wi h PSI om di e en sou ces (16, 28). The sim-
ila associa ion cons an s ob ained wi h ei he WT Fld o he
di e en mu an s o he PSI
d
䡠Fld
ox
complex sugges ha he
binding abili y o Fld o PSI is no a ec ed by he in oduced
mu a ions (Table IV). As obse ed when analyzing educ ion by
FNR
d
, educ ion o Y94A Fld by PSI p esen s a alue o k
e
much highe han WT. This mus , again, be due o a highe
accessibili y o he la in co ac o o educ ion in he Y94A
mu an . Consis en wi h his in e p e a ion, Y94W is i ually
non eac i e wi h PSI (Table IV). I is no iceable ha whe eas
mos eplacemen s o T p
57
wi h smalle hyd ophobic esidues
op imize he elec on ans e om PSI o Fld wi h espec o
WT, an impo an dec ease is obse ed o he W57Y a ian
(Table IV). This inding clea ly indica es ha he hyd oxyl
g oup is impai ing he ET eac ion.
The a o emen ioned s udies indica e ha emo al o he
a oma ic side chain in he Y94A mu an inc eases he accessi-
bili y o he co ac o o he FMN one-elec on educ ion p ocess
(Tables I, III, and IV), whe eas he modynamic ac o s would
a o he en ance o he second elec on (11), which would
p oduce accumula ion o Fld
d
in he cells upon ligh i adia-
ion. Howe e , he he modynamic d i ing o ce would oppose
ET om ully educed Fld o i s physiological subs a e, FNR.
Thus, ou da a clea ly indica e ha Ty
94
is no equi ed o ET
om PSI o FNR ia Fld; howe e , he occu ence in Fld o a
y osine esidue a ha posi ion is necessa y o modula e he
FMN E
1
alue and o a s ong FMN binding (11). Mo eo e , i
p o ides a comp omise be ween e icien PSI/Fld and Fld/FNR
ET. P e ious s udies sugges ed a possible ole o T p
57
in he
kine ics o Fld edox eac ions (11). Howe e , ou da a indica e
ha mu an s a his posi ion can accep elec ons om FNR
d
as e icien ly as WT (Table I and III) and much mo e e icien ly
om PSI (Table IV). Mo eo e , eplacemen o T p
57
by leucine
A oma ic Residues in Fla in-media ed Elec on T ans e 22343
o alanine does no p oduce majo al e a ions in he Fld abili y
o educe FNR, al hough almos no educ ion o W57F o W57Y
Flds is de ec ed. Ou da a, hus, indica e ha T p
57
canno be
di ec ly in ol ed in he ET p ocess i sel bu a he p o ides an
app op ia e s uc u al and elec onic en i onmen while coop-
e a ing wi h Ty
94
in FMN binding (11).
In summa y, in he ET p ocess om PSI o FNR ia Fld, he
a oma ic esidues s acking he FMN co ac o appea no o be
di ec ly in ol ed in he ET pa hway. Thus, in he case o
Anabaena Fld, he la in en i onmen , p o ided by T p
57
and
Ty
94
, migh no be he mos “p oduc i e”one o each indi id-
ual p ocess, bu i ep esen s an e icien comp omise o he
di e en ET s eps and allows a igh FMN binding in o de o
op imize he o e all p ocess o ET om PSI o NADP
⫹
.
REFERENCES
1. Mayhew, S. G., and Tollin, G. (1992) in Chemis y and Biochemis y o Fla-
oenzymes, Vol. III (Mu¨lle , F., ed) pp. 389–426, CRC P ess, Inc., Boca
Ra on, FL
2. Roge s, L. J. (1987) in The Cyanobac e ia (Fay, P., and Van Baalen, C., eds) pp.
35–67, Else ie Science Publishing Co., Inc., New Yo k
3. Ludwig, M. L, and Luschinsky, C. L. (1992) in Chemis y and Biochemis y o
Fla oenzymes, Vol. III (Mu¨lle , F., ed) pp. 427–466, CRC P ess, Inc., Boca
Ra on, FL
4. Wa enpaugh, K. D., Sieke , L. C., and Jensen, L. H. (1973) P oc. Na l. Acad.
Sci. U. S. A. 70, 3857–3860
5. Bu ne , R. M., Da ling, G. D., Kendall, D. S., Lequesne, M. E., Mayhew, S. G.,
Smi h, W. W., and Ludwig, M. L. (1974) J. Biol. Chem. 249, 4383–4392
6. Smi h, W. W., Pa idge, K. A., Ludwig, M. L., Pe sko, G. A., Tse noglou, D.,
Tanaka, M., and Yasunobu, K. T. (1983) J. Mol. Biol. 165, 737–753
7. Fukuyama, K., Wakabayashi, S., Ma suba a, H., and Roge s, L. J. (1990)
J. Biol. Chem. 265, 15804–15812
8. an Mie lo, C. P. M., Lijnzaad, P., Ve oo , J., Mu¨lle , F., Ve endsen, H. J.,
and de Vlieg, J. (1990) Eu . J. Biochem. 194, 185–198
9. Rao, S. T., Sha ie, F., Yu, C., Sa yshu , K. A., S ockman, B. J., Ma kley, J. L.,
and Sunda alingan, M. (1992) P o ein Sci. 1, 1413–1427
10. Genzo , C. G., Pe ales-Alco´n, A., Sancho, J., and Rome o, A. (1996) Na . S uc .
Biol. 3, 329–332
11. Los ao, A., Go´mez-Mo eno, C., Mayhew, S. G., and Sancho, J. (1997) Biochem-
is y 36, 14334–14344
12. Swenson, R. P., and K ey, G. D. (1994) Biochemis y 33, 8505–8514
13. Zhou, Z. M., and Swenson, R. P. (1996) Biochemis y 35, 15980–15988
14. Medina, M., Los ao, A., Sancho, J., Go´mez-Mo eno, C., Cammack, R., Alonso,
P. J., and Ma ı´nez, J. (1999) Biophys. J. 77, 1712–1720
15. Reynolds, R. A., Wa , W., and Wa enpaugh, K. D. (2001) Ac a C ys allog . Sec.
D57, 527–535
16. Na a o, J. A., He a´s, M., Genzo , C. G., Chedda , G., Filla , M. F., De la
Rosa, M. A., Go´mez-Mo eno, C., Cheng, H., Xia, B., Chae, Y. K., Yan, H.,
Wong, B., S aus, N. A., Ma kley, J. L., Hu ley J. K., and Tollin, G. (1995)
A ch. Biochem. Biophys. 321, 229–238
17. Jenkins, C. M., Genzo , C. G., Filla , M. F., Wa e man, M. R., and Go´mez-
Mo eno, M. (1997) J. Biol. Chem. 36, 22509–22513
18. Medina, M., Ma ı´nez-Ju´l ez, M., Hu ley, J. K., Tollin, G., and Go´mez-Mo eno,
C. (1998) Biochemis y 37, 2715–2728
19. Ro¨gne , M., Nixon, P. J., and Dinne , B. A. (1990) J. Biol. Chem. 265,
6189–6196
20. He a´s, M., O ega, J. M., Na a o, J. A., De la Rosa, M. A., and Bo in, H.
(1994) Biochim. Biophys. Ac a 1184, 235–241
21. Ma his, P., and Se´ i , P. (1981) Is . J. Chem. 21, 316–320
22. A non, D. I. (1949) Plan Physiol. 24, 1–15
23. Shin, M. (1971) Me hods Enzymol. 23, 440–442
24. He a´s, M., Na a o, J. A., Dı´az, A., Bo in, H., and De la Rosa, M. A. (1995)
Biochemis y 34, 11321–11326
25. Meye , T. E., Zhao, Z. G., Cusano ich, M. A., and Tollin, G. (1993) Biochemis y
32, 4552–4559
26. Na a o, J. A., He a´s, M., Pueyo, J. J., Medina, M., Go´mez-Mo eno, C.,
De la Rosa, M. A., and Tollin, G. (1994) Pho ochem. Pho obiol. 60, 231–236
27. Medina, M., He a´s, M., Na a o, J. A., De la Rosa, M. A., Go´mez-Mo eno, C.,
and Tollin, G. (1992) FEBS Le . 313, 239–242
28. Mu¨hlenho , U., and Se´ i , P. (1996) Biochemis y 35, 1367–1374
29. Hu ley, J. K., Webe -Main, A. N., S anko ich, M. T., Benning, M. M., Thoden,
J. B., Vanhooke, J. L., Holden, H. M., Chae, J. K., Xia, B., Cheng, H.,
Ma kley, J. L., Ma ı´nez-Ju´l ez, M., Go´mez-Mo eno, C., Schmei s, J. L., and
Tollin, G. (1997) Biochemis y 36, 11100–11117
30. Walke , M. C., Pueyo, J. J., Go´mez-Mo eno, C., and Tollin, G. (1990) A ch.
Biochem. Biophys. 281, 76–83
A oma ic Residues in Fla in-media ed Elec on T ans e 22344