H hp k kh s dng Fe3+, S0, v H2 trong ha nng v c t dng
(chemolithoautotrophic ) vi khun Go to:Cc vi khun tc nhn xi
haAcidithiobacillus ferrooxidans c bit n nh mt aerobe rng respires
trn st v lu hunh. y chng ta thy rng cc loi vi khun c th pht trin
chemolithoautotrophically khng ch trn H2/ O2trong iu kin hiu kh m
cn v H2/ Fe3+,H2/ S0,hoc S0/ Fe3+trong iu kin ym kh.H hp k kh s dng
Fe3+hoc S0l cht nhn electron v H2S0hoc nh l mt nh ti tr in t phc v
nh l mt ngun nng lng s cp ca cc loi vi khun.H hp k kh da trn gim
Fe3+do cc loi vi khun tng hp mt lng ng k ca mt loi cytochromec- c
tnh ha nh l mt 28-kDa monomer acid-n nh v ha tan.Cc cytochrome tinh
khit dng oxy ha gim trong s hin din ca cc cht chit th, v cc
cytochrome gim c reoxidized bi Fe3+.H hp da trn gim Fe3+cng vi qu
trnh oxy ha ca mt loi cytochromec-cth c tham gia vo cc c ch chnh ca
sn xut nng lng vi khun h hp trn st k kh.Acidithiobacillus
ferrooxidans.vi khun ha nng v c t dng Acidithiobacillus
ferrooxidans c bit nh 1vi h hp hiu kh trn st v lu hunh. y chng ti
thy vi khun c th ha nng v c t dng khng nhng c th pht trin di H2/O2
iu kin hiu kh m cn c th pht trin di H2/Fe3+, H2/So, hoc S0/Fe0 iu
kin k kh. H hp k kh s dng Fe3+ hoc S0 nh cht nhn in t v H2 hoc S0
nh cht cho in t cung cp ngun nng lng chnh cho vi khun. h hp k kh da
trn gim Fe3+ gip cho vi khun tng hp c s lng loi cytochrome c quan
trng, loi tinh chit ny nh 1acid n nh v ha tan 28-kDa monomer.
Cytochrome tinh khit ny trong dng oxi ha ny gim trong s hin din ca
kim loi nguyn cht c chit xut, v gim cytochrome ny dob oxi ha bi
Fe3+. H hp da trn gim ni Fe3+ n oxi ha cytochrome c c l l c ch sn
xut nng lng chnh ca vi khun trong h hp st k kh.eAcidithiobacillus
ferrooxidans thng c chp nhn l mt chemolithoautotroph hiu kh bt ngun
nng lng cho s pht trin t s h hp oxy ha lin quan n qu trnh oxy ha st
II hoc cc hp cht lu hunh khc nhau.Brock v Gustafson bo co rng cc vi
khun lm gim Fe3+trong s hin din ca S0(10).Tuy nhin, mc gim khng c
cng nhn nh l phn ng ng h hp k t gim st khng cho php s pht trin ca
vi khun (39).Do , n c chp nhn rng gim khp ni ca Fe3+oxi ha ca S0l
mt trong cc bc trong qu trnh chuyn ha lu hunh bi vi khun (39).Pronk
et al.(32,33) v Das et al.(12) cho thy cc vi khun pht trin da trn
cc qu trnh oxy ha ca S0bi Fe3+trong iu kin oxy hn ch.Mc d nhng pht
hin ny a ra kh nng rngA.ferrooxidansc th c th pht trin trong iu kin
ym kh, cc hot ng ca enzyme ni trn khng km theo tng trng (39), v n
vn cha r liu Fe3+phc v nh l cht nhn electron h hp k kh.Mt khc, cc
vi khun s pht trin trn hydro trong iu kin hiu kh (14).Trong trng hp
, H2tng l cht cho in t cho php mt chui h hp oxy ha ly nng lng cho s
pht trin nhn xi ha. Mt khc, trong nhiu heterotrophs tu trong c
haiArchaeavBacteria,k kh h hp lin quan n gim Fe3+hoc S0thng i i vi
qu trnh oxy ha ca H2(25,26,30,38,42).Mt trong nhng vai tr nguyn tc
h hp nh vy s c c h tr nng lng cho chemolithoautotrophy (34), mt loi
autotrophy m thng phc v nh l ch tng trng ca heterotrophs tu nh vy
trongArchaeavBacterianh archaebacteria hyperthermophilic, vi khun
lu hunh gim, v vi khun nguyn s ln men (18,24,36-38,42,44).Tuy nhin,
rt t bit v vai tr ca h hp k kh lin quan n Fe3+hoc S0gim trong tng
trng ca cc vi khun in hnh, trong bao gm mt lot cc lu hunh, st,
ammonia, nitrite oxi ha.Chng ti tm thy rng h hp k kh s dng Fe3+hoc
S0l cht nhn electron v H2nh l mt cht cho in t nh l mt ngun nng lng
chnh cho tng trngcahemolithoautotrophic caA.ferrooxidans.Hn na, nh
h hp st k kh gy ra A.ferrooxidans tng hp mt lng ng k ca mt loi
cytochromec-,n chu trch nhim cho vic gim Fe3+.H hp k kh da trn gim
Fe3+cng vi qu trnh oxy ha ca mt loi cytochromec-cth ng mt vai tr
quan trng trong c ch chnh ca sn xut nng lng vi khun h hp st k kh.VT
LIU V PHNG PHPVi sinh vt v mi trng.Cc chng A.ferrooxidansc cung cp
t cc chng chn lc: ATCC 23270 c ly t cc chng chn lc M;JCM 3865, JCM
3863, v JCM 7811 l t cc chng chn lc ca vi sinh vt Nht Bn;v IFO
14.246 v 14.262 IFO t Vin ln men, Osaka, Nht Bn.Mi chng c tinh ch
bng cch s dng cc phng php phn lp n dng trn mt tm gel silica ca mi
trng Fe2+.Sau khi tinh ch, cc chng ny c duy tr thng xuyn trong 9K
mui c bn mi trng c cha 160 mM Fe2+trong bnh lc v trong iu kin hiu
kh 30 C.Th nghim tng trng k kh.Cc mi trng c s dng cho cc th nghim
tng trng k kh cha cc thnh phn sau (mi lt nc ct): (NH4) 2SO4,133
mg;K2HPO4,41 mg;MgSO47H2O, 490 mg;CaCl22H2O, 9 mg;KCl, 52
mg;ZnSO47H2O, 1 mg;CuSO45H2O, 2 mg;MnSO4H2O, 1 mg;NaMoO42H2O, 0,5
mg;CoCl26H2O, 0,5 mg;Na2SEO410H2O, 1 mg;v NiCl26H2O;1 mg. trung ha
mui c bn ny, thm vo 2,5 g Fe2(SO4) 3 v iu chnh cho pH 2,0 vi 6
NH2SO4.Cc mi trng sau c kh kh bng cch s dng mt my bm ht, sau n c sc
kh nit cho 1 h gim mc oxy ha tan.Cc mi trng c kh oxy ngay lp tc c
lu tr trong mt hp k kh di nit qua m.Trong hp k kh, 30 ml ca mi trng
sau c thm vo 150 ml mi trng k kh 150 ml vi b lc kh trng, v cc chai
c ng gi vi mt stopper butyl tit trng.Cc pha kh ca khong trng trong
chai ng gi sau c thay th bng mt hn hp H2v CO2nh sau.Kh nit trong cc
khong trng u tin c loi b bng cch s dng mt kim tim lp vo stopper
butyl, sau hn hp H2-CO2(80:20) sau c s dng thay th cc pha kh trong
cc khong trng.Cc chu k ht v to p lc c lp i lp li t nht ba ln cho n
khi hn hp kh trong cc khong trng cui cng t c p sut 250 kPa.Khi cc
th nghim nui cy c thc hin vi mt H2O2pha kh, 15 ml khng kh c thm vo
chai bng cch s dng ng tim.Trong tt c cc th nghim, th tch tim l 1 ml
cho 30 ml ca mi trng.nui cy c thc hin 30 C trong mt lng vi lc.St ha
tan.Hm lng st tng ha tan v ca Fe2+c xc nh bng cch s dng cc phng php
phenanthroline.Mu (1 ml) ca mi trng nui cy c thc hin ti tng ln nui
cy thng qua mt mng lc (kch thc l chn lng, 0,2 mm).Dung dch (100 ml)
t mi mu c lc sau c thm vo 2,5 ml ca b m cha 68,0 g natri axetat v
28,8 ml dung dch acid acetic cho mi lt nc ct (pH 4.6).Mt mu 2,5 ml
dung dch phenanthroline 0,2% sau c thm vo hn hp mu xc nh nng ccFe
2+.Trong trng hp st ton phn c xc nh, 1 ml NH2OH 10%.HCl c b sung vo
dung dch m trc khi trn mu gim Fe3+ thnh Fe2+ . Nng ca Fe3+ v ion st
ton phn c xc nh t 1 biu ng cong chun nng st trong mu ny nh mt chc
nng ca s hp th 510 nm.S lng t bo.S lng t bo c xc nh bi mt bung m di
knh hin vi tng phn pha phng i 400.Mt t bo ny sau c tnh l trung bnh
ca hn 25 xc nh ca mi mu nui cy.Thanh lc cytochrome.Cc t bo trng
thnh hiu kh hoc k kh trn mt s phng thc tng trng nh H2/ O2,Fe2+/
O2,H2/ Fe3+,H2/ S0v S0/ Fe3+ c thu hoch mt cch ring bit v c l lng
trong dung dch axit sulfuric pH 2,0.Mt lot cc t bo sau c ph v bi
sonication, v cc thnh phn khng ha tan c loi b hai ln bng ly tm
100.000 gtrong 1 gi.Cc nc ni c s dng cho sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) v hin hnh
(hnh dung ) bng nhum Coomassie brilliant blue (CBB).Cc t bo trng
thnh k kh trn H2hoc Fe3+ni tn mt sau c dng cho cc bc thanh lc ca
cytochrome.Cc bc thanh lc c thc hin nh m t trong mt bo co trc (11)
vi nhng sa i nhn ra.Mt sulfate ammonium c thm vo (dd ni) 20% bo ha
v lng kt ta c loi b bng cch ly tm 10.000 gtrong 30 pht.Nng amoni
sulfat c tng ln n 60% bo ha, v cc vin (bng trn) mu sau khi ly tm
thu c l phn cytochrome.Cc vin l lng trong 50 mM axit
methyleneethanesulfinic (MES) m (pH 4,5), v cc resupension c thm
tch vi cng mt b m.Sau khi thm tch, th vn c p dng cho mt ct
carboxymethyl cellulose cn bng trong 50 mM MES m (pH 4,5).Cc
protein hp th trong cc ct c tch ra vi mt gradient tuyn tnh ln n 500
mM NaCl.Cc phn th hin mu thu c 320 mM NaCl.Sau -kh mui vi mt mng tp
trung, cc phn c p dng cho mt ct MonoQ equibrated trong 50 mM MES m
(pH 4,5).Cc protein ny sau gii hp vi mt gradient tuyn tnh ln n 500
mM NaCl.Phn mu thu c 380 mM NaCl.Ra gii c tp trung vi mt mng tp
trung n 15 ln, t 0,89 107-1,59 108t bo / ml. xc nhn rng s tng trng
quan st c trung gian bi H2,thay i ph thuc thi gian trong mt t bo ca
IFO 14.262 c trng khi H2c cung cp nh cht cho electron duy nht
(Hnh.(Fig.1A).1A).Di nhng iu kin ny, chng ti thy rng mt t bo tng ln
t 2,56 107-2,29 108t bo / ml sau 98 gi , trong khi khng c tng trng
xy ra khi c thc hin trong s vng mt ca H2.Da trn nhng kt qu ny, c th
kt lun rngA.ferrooxidanschng IFO 14.262 l c th s dng H2nh l mt cht
cho electron v O2l cht nhn electron cung cp nng lng cho s pht trin
nhn xi ha.
FIG.1.Tng trng nhn xi ha caA.ferrooxidansv h hp hiu kh v k kh vi
H2l cht cho electron.(A) thay i Thi gian ph thuc vo mt t bo ca chng
IFO 14.262 hiu kh respired trong s hin din () hay khng ()...
BNG 1.Tng trng Aerobic caA.ferrooxidanskhi qu trnh oxy ha ca
H2O2bngMt lot cc th nghim nui cy k kh sau c thc hin cng vi 6
chngA.ferrooxidansvi mi trng c cha Fe3+l cht nhn electron v H2nh l
cht cho electron.Cc s lng t bo v nng st trc v sau 17 ngy c tm tt
trong BngTable2.2.Tng trng ca vi khun c quan st r rng vi cc chng
JCM 7811, IFO 14.246, v IFO 14262. Mt t bo ca mi dng tng ti
thiu> 15 ln, t 2,18 108-4,95 108t bo / ml.chng JCM 3865 cng tng
trng theo cc iu kin, nhng mt t bo cui cng l thp hn so vi cc dng
khc.Vic gim Fe3+thnh Fe2+lin quan mt thit n s pht trin ca vi
khun.Nh vy, Fe2+tch ly trong mi trng ca cc chng JCM 3865, JCM 7811,
IFO 14.246, v IFO 14.262 nhng khng phi trong mi trng ca hai chng m
khng pht trin hoc trong mi trng kim sot ha cht v c khng c cy vi tt
c t bo (d liu khng hin th).Chng ti chn chng JCM 7811 khng nh rng
tng trng quan st c theo hng k kh gim Fe3+bi H2(Hnh.(Fig.1A).1A).Mt
t bo t 4,78 108t bo / ml sau 314 gi , v mt ln na tng trng tng quan
vi s tch t ca Fe2+(Fig.(Fig.1B).1B).thi gian tng trng t n giai on
ngng pht trin ( pha tnh), lng Fe2+tch ly chim 99% st ha tan trong
mi trng.Ngc li, khng c s gia tng mt t bo trong s vng mt Fe3+hoc
H2,cng khng c tch ly ca Fe2+trong mu tim truyn ( cy
).A.ferrooxidanschng JCM 7811 c th pht trin trong iu kin ym kh
autotrophically vi H2l cht cho electron v Fe3+l cht nhn
electron.
BNG 2.Tng trng k kh caA.ferrooxidanskhi gim Fe3+bi H2mtCc hot ng
st gim trn hydro c nghin cu vi cc t bo nguyn vn ca chng JCM 7811
pht trin trn h hp k kh st.Cc mc gim st c o bng cc phn ng hn hp c
cha cc t bo nng c nh v Fe3+ cc nng khc nhau trong iu kin ym kh vi
H2.Mc o c v vi nng Fe3+ban u tng ng (Hnh.(Fig.2A).2A).Gim Fe3+ khng
xy ra trong s vng mt ca cc t bo hoc H2(d liu khng hin th).Ngoi ra,
khng c qu trnh oxy ha ca Fe2+bi cc t bo c quan st thy trong s hin
din ca O2hoc H2(d liu khng hin th).Di nhng iu kin ny,KmvVmaxc tnh
bng 1,51 mM Fe3+v 29,8 10-19mol ca Fe2+sn xut / min / t bo, tng
ng.Tip theo, cc tc ng ca mt vi cht c ch v gim st trong t bo nguyn
vn c nghin cu (Hnh.(Fig.2B).2B).2-heptyl-4-hydroxyquinoline-N-oxide
(HOQNO), hydrazine sulfate, 2,3-dimercapto-1-propanol, vpaxit
-chloromercuriphenylsulfonic (PCMS) c la chn l thuc cho c ch chui h
hp.Hn 90% cc hot ng gim Fe3+vn trong s hin din ca 100 mM HOQNO hoc
100 mM hydrazine sulfate khi gim m khng c s c ch l 100%, trong khi
34% cc hot ng b c ch bi 100 mM 3 dimercapto-1- propanol v hot ng
hon ton b chn bi 100 mM PCMS.Nhng kt qu ny gi rng vic gim st xy ra
nh l kt qu ca s h hp k kh st trn H2.Tip theo chng ta kim tra s tng
trng caA.ferrooxidansJCM 7811 vi s c mt ca H2v S0 diiu kin ym kh
(Hnh.(Hnh 3).3).Chng ti thy rng thc s chng ny c th pht trin
chemolithoautotrophically vi H2l cht cho electron v S0cht nhn
electron, nng sut mt t bo cui cng ca 6,1 107t bo / ml sau 120 gi .
Tng trng khng xy ra trong s vng mt ca mt trong hai S0hoc H2.Cui
cng, kh nng tang trng k kh caA.ferrooxidansJCM 7811 s dng S0 nh cht
cho electron v Fe3+l cht nhn c kim tra.Cc s lng t bo v nng st trc v
sau khi nui cy c tm tt trong BngTable3.3.Di nhng iu kin ny, mt t bo
tng> 12 ln, t 1,05 108t bo / ml sau 14 ngy .Trong thi gian ,
Fe3+thm vo mi trng gim xung cn Fe2+,v li khng c tch ly trong trng
hp khng tim truyn .Nh vy, chng JCM 7811 c th tng trng k kh trn qu
trnh oxy ha ca S0bi Fe3+.
FIG.2.hot ng gim st caA.ferrooxidanschng JCM 7811 nui cy trn h
hp k kh st.(A) Lineweaver-Burk v biu hot ng gim st ca cc t bo trn
hydrogen.(B) nh hng ca cc cht c ch h hp trn hot ng gim st ca cc t
bo.Cc...
FIG.3.Tng trng nhn xi ha caA.ferrooxidanschng JCM 7811 trn k kh
S0h hp vi H2l cht cho electron.Th hin s thi gian thay i ph thuc vo
mt t bo trong s hin din ca H2() hoc N2() vi S0v trong s hin dinH2 m
khng c S0
BNG 3.Tng trng k kh chng JCM 7811 sau khi gim ca Fe3+bi S0a xc
nh cc thnh phn tham gia vo vic gim Fe3+,protein ha tan c trong chit
xut kim loi nguyn cht ca cc t bo trng thnh hiu kh trn Fe2+/ O2hoc k
kh trn H2/ Fe3+c so snh bng SDS-PAGE.Thng tin ca hai nhm t bo khc
bit ng k trong cc vi khun ym kh c hm lng protein 28 kDa
(Fig.(Fig.4A,4A, ng 1 v 2). Sau khi sulfate natri v kt ta acid,
protein ny c tinh chit ng nht in di bng ion v sc k gel
(Fig.(Fig.4A,4A, ng 3), cho ra mt sn phm acid n nh v ha tan cao
trong dung dch ti pH 2,0. Khi lng phn t ca protein tinh khit c xc
nh l 27,4 kDa lc gel. protein ny biu hin( th hin) mt nh hp th 411
nm rng (ng chm chm trong hnh.Fig.4B),4B), v gim lng protein bng cch
s dng mt thuc th gim sn xut nh hp th mi ti 552, 523, v 418 nm (ng
lin nt). Hnh nh ca hnh.Fig.4B4Bcho thy quang ph khc nhau cho cc dng
oxy ha v gim, n thy in hnh ca mt loi cytochromec-.
FIG.4.Biu hin ca mt ha tan, axit cytochrome-n nh trong chng JCM
7811 k kh nui cy vi H2l cht cho electron v Fe3+l cht nhn
electron.(A) CBB-nhum gel SDS-polyacrylamide.Bng 1 v 2 c np vi cc
nc ni tng ng... nghin cu phn ng oxi ha kh ca cytochrome, quang ph c
ghi nhn vi cc chit xut kim loi nguyn cht ca chng JCM 7811 nui cy
trn H2/ Fe3+.Mt nh c quan st 418 nm, cho thy s tn ti ca cc
cytochrome nh l hnh thc gim trong dch chit (dng rn trong
hnh.Fig.5A).5A).Cc dng oxy ha ca cytochrome tinh chit th hin mt nh
hp th 411 nm (ng t trong hnh.Fig.5A)5A) sau c pha trn vi cc chit
xut.Ph ca hn hp cho thy mt nh hp th 418 nm (dng rn trong
hnh.Fig.5B).5B).S dch chuyn nh 411-418 nm m bo rng cc hnh thc oxy
ha ca cytochrome c chuyn i thnh cc hnh thc gim trc s hin din ca cc
chit xut.Tuy nhin, quang ph ca cc hn hp vi vic b sung 4 mM ca
Fe3+cho thy mt nh cao 411 nm (ng t trong hnh.Fig.5B).5B).Khi tip xc
vi Fe3+ dng gim ca cytochrome ngay lp tc reoxidized bi Fe3+.Ngoi
ra, vic sn xut ca Fe2+bi qu trnh oxy ha ca cytochrome gim c xc nhn
bi s gia tng hp th 510 nm, tng ng vi chelating phn ng ca Fe2+vi
phenanthroline (d liu khng hin th).T nhng kt qu ny, loi c-
cytochromeny do c kh nng cung cp nh l cht nhn electron cho qu trnh
oxy ha ca cc chit xut v cht cho electron gim Fe3+.
FIG.5.Phn ng oxi ha kh ca cytochrome trong chit xut t bo v
Fe3+.(A) quang ph hp th ca cc chit xut kim loi ca t bo nui cy trn
H2/ Fe3+(ng lin nt) v dng oxy ha ca cytochrome tinh chit (ng nt
t).Quang ph (B) hp th ca... lm r mi quan h gia cc cytochrome tinh
khit v h hp k kh st, protein ha tan c trong chit xut kim loi nguyn
cht ca cc t bo nui cy trn H2/ O2,H2/ S0hoc S0/ Fe3+c so snh bng
SDS-PAGE (Fig.(Hnh 6).6).Mt s lng ln cc protein 28 kDa c pht hin
trong cc cht chit xut t cc t bo nui trng trn S0/ Fe3+nhng khng c
trong chit xut ca cc t bo nui cy trn H2/ O2hoc H2/ S0. l protein 28
kDa c biu l trong cc t bo h hp k kh trn H2/ Fe3+hoc S0/
Fe3+(Hnh.(Hnh 4)4) cho thy rng biu hin ca protein c gy ra khi cc t
bo h hp vi Fe3+l cht nhn electron, khng phn bit cht cho.
FIG.6.nhum CBB gel SDS-polyacrylamide cho thy mc ca cytochrome
trong chit xut kim loi nguyn cht ca cc t bo pht trin vi H2/ O2(ng
1), H2/ S0(ng 2), hoc S0/ Fe3+(ng 3 ).Mi tn ch ra protein 28 kDa ca
cytochrome.THO LUNN cng c bit rngA.ferrooxidansc th h hp hiu kh bng
Fe2+hoc S0l cht cho electron.Nghin cu ny cung cp bng chng cho
thyA.ferrooxidansc th s dng bn hnh thc khc nhau ca s h hp, bao gm
H2/ Fe3+,H2/ S0v S0/ Fe3+cho s tng trng k kh, H2/ O2cho s tng trng
hiu kh (Hnh.(Hnh 11vand33v BngTables11nTO3).3).Mc d mt vi dng xc nh
bit ca archaebacteria hyperthermophilic (36), ng ch l cc loi vi
khun cng trng by ( biu hin) cc dng khc nhau cho vic h hp hiu kh hoc
k kh bng st, lu hunh, v hydrogen.Trn tt c cc dng h hp, cc loi vi
khun c th pht trin autotrophically.Chemolithoautotrophy ny ca vi
khun c xc nhn bi cc th nghim pht trin thm m khng c CO2trong pha
kh.Khng c s tng trng ng k xy ra m khng c CO2 kiu h hp k kh H2/
Fe3+,H2/ S0v S0/ Fe3+v kiu hiu kh H2/ O2(d liu khng hin th).Khng c
s tng trng ng k cng c quan st thy trong s vng mt ca mt cht cho in t
hoc cht nhn electron (Fig.(Hnh 11vand33v BngTable3).3).Nhng kt qu
ny m bo cc autotrophy ca vi khun trn mt s dng ca h hp.Mt khc, 4
trong s 6 chng c th h hp k kh trn H2/ Fe3+(Fig.(Hnh 11v
BngTable2).2).Ngoi ra, mt dng vi khun cng c th h hp k kh trn S0/
Fe3+v H2/ S0(Hnh.(Hnh 33v BngTable3),3), trong khi ch c mt trong 6
chng c th h hp hiu kh trn H2(Hnh.(Hnh 11v BngTable1).1).Nhiu chng
khng th s dng O2l cht nhn electron cho H2mc d cc vi khun r rng l c
th s dng O2trn st.S bin mt ca cc tim nng s dng O2cng c quan st thy
trong cc t bo nui cy trn H2/ Fe3+.Cc hot ng oxy ha st ca cc t bo
nguyn vn hon ton bin mt (d liu khng hin th).Cc biu hin hoc cc hot
ng ca cc enzym gim O2dng nh c iu ha m trong cc vi khun khi s c mt
ca H2nh l mt cht cho electron.Tuy nhin, hin nay n khng r nh th no
vi khun c kh nng la chn cc cht cho s dng c v cht nhn cho vic h hp
trong cng iu kin. hiu c c ch iu ha ca kiu hnh v ng h hp, nhiu thng
tin v cc gen tng ng vi cc enzym h hp l cn thit.Mc d cc gen cho cc
enzyme chuyn electron nh rusticyanin v cytochrome oxidase cho h hp
hiu kh c bo co (4,5,7), thng tin thm cha c.Mt s cc cytochrome
loictrong mng-rng buc v dng ha tan c tinh ch t nhng t bo A.
ferrooxidans hiu kh pht trin(4,11,16,20,35,40,41,43).Nghin cu hin
nay ln u tin lm sch mt cytochrome t t boA.ferrooxidansk kh pht
trin.Cc protein ha tan tinh khit, trong c mt khi lng phn t l 28
kDa, khc vi kch thc t cytochrome m t trc y
(4,11,16,20,35,40,41,43).Hn na, trnh t ca N-terminal 30 axit amin
ca protein tinh khit c xc nh s b (d liu khng hin th) v tm thy l
khng tng t vi cc trnh t c bit n ca mt trong cc cytochrome tan hoc
mng b rng buc (11,16,20,43) .y khng phi l ng ngc nhin, tuy nhin,
cho rng cytochrome ny c lin quan n gim Fe3+,trong khi nhng ci khc
tham gia vo qu trnh oxy ha Fe2+.Mt trong nhng c tnh vt tri ca
cytochrome ha tan ny l kh nng oxi ha kh.Cc kh nng oxi ha kh trung
im ca mt s loi c- cytochrometA.ferrooxidansthng dao ng trong khong
330 v 360 mV pH 7,0 v gia 610 v 660 mV pH 3,5
(4,11,16,20,35,40,41,43).kh nng ca cytochrome mi l khong 560 mV pH
2,0.kh nng ca cytochrome mi l khc nhau t cc kh nng ca cc cytochrome
khc ca loi vi khun ny.Tuy nhin, kh nng ny m bo rng cc cytochrome c
th gim Fe3+ pH thp.tnh cht ng ch khc ca cytochrome mi l s n nh pH
thp m ti cc cytochrome loicin hnh c th autooxidized.N c bo co rng
cc loi gim cytochromec-cth c reoxidized bi oxy ha tan trong dung
dch pH 2,7 (11).Tuy nhin,> 90% ca cytochrome gim trong dung dch
pH 2.0 c gi li sau vi gi trong iu kin hiu kh, v cc cytochrome gi li
cng c th lm gim Fe3+sau khi (d liu khng hin th).Do , protein c trng
c tm thy trong nghin cu ny l mt cytochrome mi th hin trong vi
khun.Trong cc t bo, cc mi quan h gia gim st v hot ng h hp quan trng
da trn cc thng s tc gim st v gim hot ng ca cc cht c ch h hp
(Hnh.(Hnh 2).2). K kh gim Fe3+l mt qu trnh h hp ca vi khun.Mi quan
h gia cytochrome mi ny v h hp k kh st cng l ng k, v qu trnh chuyn i
t hiu kh h hp k kh r rt upregulated biu hin ca n
(Hnh.(Fig.4A).4A).Tng t nh vy, mc biu hin cao c quan st trong cc t
bo nui cy trn S0/ Fe3+nhng khng phi trn Fe2+/ O2,H2/ O2,hoc H2/
S0(Hnh.(hnh 6) .6).Hn na, cc loi mic-cytochrome gim trong s hin din
ca cc cht chit kim loi nguyn cht ca cc loi vi khun pht trin trn h
hp k kh st v protein gim c reoxidized bi Fe3+(Fig.(Fig.5).5).Cc
cytochrome c th cung cp nh l cht nhn electron chit xut kim loi
nguyn cht v cc cht cho electron gim Fe3+.iu ny cho thy rng cc
loic-ny cytochrome l chc nng h hp k kh lin quan n Fe3+l cht nhn
electron.C bng chng ngy cng tng rng cc cytochrome c tham gia vo vic
gim kim loi k kh, cung cp nh l cc thnh phn trong chui h hp ca rt
nhiu vi khun (1,6,13,23,27-29,31).Tht vy, cc loi cytochromec-m t y
s ng vai tr quan trng trong h hp k kh l mt trong nhng protein chuyn
electron trongA.ferrooxidans.A. ferrooxidans c th pht trin vi H2l
mt cht cho electron v O2,S0,hoc Fe3+l cht nhn electron (Fig.(Hnh
11vand3,3, BnTables11vand2).2).Cc qu trnh oxy ha ca H2s dng mi mt
oxy ha do phi i i vi vic gim NAD (P) cn thit cho s c nh CO2.Cc con
ng vn chuyn in t ton b cho cc h hp vn cn cha r rng, mc d
hydrogenase gn y c tnh ha t vi khun (19), cho thy rng cc electron t
H2u tin s c chuyn giao cho hydrogenase.Tuy nhin, cc hydrogenase
tinh khit c th khng trc tip lm gim NAD+(19).Ngoi ra, NAD+c th khng
c gim bng hnh thc gim ca cytochrome tinh khit (d liu khng hin
th).Nhng kt qu ny c th c ngha l s ti sinh ca NADH l cng vi vic
chuyn electron lin quan n dng chy t hydrogenase thnh Fe3+qua
cytochrome gim thng qua cc thnh phn trong mng bn trn H2/ Fe3+h hp.S
tn ti ca mt electron chuyn ngc li ti to NADH trong ty th ng vt c bo
co.Ngoi ra, cc trng hp chuyn electron ngc vi khun chemoautotrophic
cng c bo co (2,3,22).Gn y, cc electron chuyn ln dc t Fe2+ NAD+v h
hp hiu kh caA.ferrooxidans c xut (17).Vic chuyn giao kh khn c th
lin quan n mt cytochrome gi nhbc1phc tp, theo c ch ho thm, c th
thng qua c ch Q-chu k hot ng ngc li (17,21).Khng c bng chng c mt ti
cc c ch tng t ca mt dng chy ngc cho NADH ti sinh c th c chia s vi h
hp hiu kh v k kh.Tuy nhin, mt lot cc kiu tng trng ca vi khun t dng
s dng H2,O2,S0,hoc Fe3+cho s tn ti ca con ng ph qut cho NADH ti
sinh bi vic chuyn giao kh khn kt ni vi mi chui h hp.Rusticyanin c
bit n nh l mt trong nhng thnh phn chuyn in t c nh gi cao th hin khi
cc t bo c nui cy trn Fe2+trong iu kin hiu kh (8,9).ng ngc nhin,
nhum min dch vi mt khng th anti-rusticyanin tit l s hin din ca
rusticyanin trong cc t bo nui cy trn H2/ Fe3+(d liu khng hin th).Mc
d t bo k kh pht trin khng sm hot ng oxy ha biu hin trong s hin din
ca Fe2+ ha tan,hot ng ny c phc hi sau khi hiu kh vi Fe2+trong vi
tun.S bin mt ca cc hot ng trong s hin din ca rusticyanin l khng ngc
nhin, v mt phn ln ca cc enzyme tn ti nh mt hnh thc apo, m khng c
nguyn t ng cn thit cho Fe2+-oxidizing hot ng (d liu khng hin
th).Dng nh l khng c mi quan h gia cc hot ng v rusticyanin st gim v
cc protein h hp c constitutively hin trong vi khun ny, ty thuc vo
dng h hp trong iu kin ym kh.Tuy nhin, s hin din ca Fe2+c th quan
trng i vi cm ng ca hnh thc hot ng ca rusticyanin, v O2c th ng mt
vai tr tng t.
