„novel mechanisms of anaerobic methane...
TRANSCRIPT
„Novel mechanisms of anaerobic methane oxidation"
Rudolf K. Thauer
Max Planck Institute for Terrestrial Microbiology
06. 01. 11
0
50
100
150
CH4 mmol dm3
Depthmm
2 4
0 10 20 30SO4
2 mmol dm3
0
SO42
Archaea and Bacteria
CH4
Zone ofAnaerobic oxidation of methane(AOM)
Oxic
Pioneering work (1970s):C. Barnes, E. Goldberg;C. Martens, W. Reeburgh
CH4 + SO42- + 2H+ CO2 + H2S +
2H2O
ΔGo´= - 21 kJ/molInhibited by BES
-10 20100Temperature °C
30
15
10
5
0Methane gas+ water ice
Methane gas+ water
Methanehydrate
Methanehydrate +water ice
Ove
rpre
ssur
e (
bar)
22 ml CH4per l H2O
22 l CH4per l H2O
220 ml CH4per l H2O
-10 20100Temperature °C
30
15
10
5
0Methane gas+ water ice
Methane gas+ water
Methanehydrate
Methanehydrate +water ice
Ove
rpre
ssur
e (
bar)
22 ml CH4per l H2O
22 l CH4per l H2O
220 ml CH4per l H2O
Methane seep area in the western Black Sea
methane seeping area
The largest anoxic water body on earth
No oxygen below 130 m
Anaerobic microorganisms are not restricted to the sediment
High High sulfatesulfate (25 (25 mMmM))
TemperatureTemperature nearnear thethe bottombottom10 10 ooCC
Working Plattform and sampling equipment
The Russian research VesselProf. Logachev
The German submersible JagoImages: GHOSTDABS
CH4 + SO42- + Ca2+ = CaCO3 + H2S +H2O
Microbial mats in the Black Sea (composed mainly of methanotrophicarchaea and sulfate reducing bacteria).
Archaea (ANME-1)Bacteria
In the laboratory microbial mats from the Black Seacatalyze (no pure culture available):
CH4 + SO42- + 2H+ ⇌ CO2 + H2S + 2H2O
ΔGo´= - 21 kJ/mol
apparent Km for CH4 10 bar
specific rate of AOM at 1 bar CH4 1 nmol/min/mg protein
The methanotrophic archaea in the Black Sea mats contain high concentration of methyl-coenzyme M reductase (three lines of evidence)
Methane oxidation with sulfate is inhibited by bromoethane sulfonate, a specific inhibitor of methyl-coenzyme M reductase
Conclusion:First step in AOM with sulfate is catalyzed by methyl-coenzyme M reductase
HS NH
O H CO2-
CH3
H O
CH4
+
Methyl-coenzyme M Coenzyme B
Heterodisulfide
+
SS N
H
O H CO2-
CH3
H O
-O3S
-O3SS
CH3
PO32-
PO32-
Methyl-CoM Reductase from methanogenic archaeaΔGo´= -30±10 kJ/mol
αα22ββ22γγ22
F430Ni(II)
F430Ni(I)
EEoo´= ´= -- 650 mV650 mV1 e1 e--
1 e1 e--
F430Ni(III)
EEo o > + 1> + 1VV
F430Ni(II)
F430Ni(I)
EEoo´= ´= -- 650 mV650 mV1 e1 e--
1 e1 e--
F430Ni(III)
EEo o > + 1> + 1VV
F430Ni(II)
F430Ni(I)
EEoo´= ´= -- 650 mV650 mV1 e1 e--
1 e1 e--
F430Ni(III)
EEo o > + 1> + 1VV
F430Ni(II)
F430Ni(I)
EEoo´= ´= -- 650 mV650 mV1 e1 e--
1 e1 e--
F430Ni(III)
EEo o > + 1> + 1VV
F430Ni(II)
F430Ni(I)
EEoo´= ´= -- 650 mV650 mV1 e1 e--
1 e1 e--
F430Ni(III)
EEo o > + 1> + 1VV
F430Ni(II)
F430Ni(I)
EEoo´= ´= -- 650 mV650 mV1 e1 e--
1 e1 e--
F430Ni(III)
EEo o > + 1> + 1VV
N
N N
N
H
H
HOOC
O
HN
O
Ni
905 Da905 Da
1512
13
19
172
1
5
1020
18
H2NOC
COOH
COOH
COOH
COOH
H3CCH3
3
N
N N
N
H
H
HOOC
O
HN
O
N
905 Da905 Da
1512
13
19
172
1
5
1020
18
H2NOC
COOH
COOH
COOH
COOH
H3
CH3
3
173
++
HS NH
O H CO2-
CH3
H O
CH4
+
Methyl-coenzyme M Coenzyme B
Heterodisulfide
+
SS N
H
O H CO2-
CH3
H O
-O3S
-O3SS
CH3
PO32-
PO32-
Methyl-CoM Reductase from methanogenic archaea
ΔΔGGoo´́= = -- 30 30 ±±10 kJ/mol10 kJ/mol
•The presence of coenzyme M andcoenzyme B in methanotrophicarchaea has not been shown.
•MCR has not been shown to catalyze the oxidation of methane
?
©Michaelis/GHOSTDABS, Hamburg
CH4 + SO42- + Ca2+ = CaCO3 + H2S +H2O
Isolation of MCR from
the microbial mats
Microbialmat
Cellextraction
Pellet Supernatant
Chromatographyon anion exchangeresins
MCR
MCR crystalls
Methyl-coenzym M reductase from ANME-1
Coenzyme B
Coenzyme M
6.3 Å
2.4 Å
Shima et al. 2010
Coenzyme F430
Coenzyme B
Coenzyme M
Coenzyme F430
positiveelectrondensity
COOH
N
N N
N
HH3C
H2NOC
H
HOOC
O
COOH
COOH
HN
O
H3
COOH
Ni
C
SSCHCH33
++
951.28 Da951.28 Da
H COOH
N
N N
N
HH3C
H2NOC
H
HOOC
O
COOH
COOH
HN
O
H3
COOH
Ni
C
SSCHCH33
++
951.28 Da951.28 Da
H
F430 in methyl-coenzyme M reductase from ANME-1J. Am. Chem. Soc. 130, 10758-10767 (2008)
HS NH
O H CO2-
CH3
H O
CH4
+
Methyl-coenzyme M Coenzyme B
Heterodisulfide
+
SS N
H
O H CO2-
CH3
H O
-O3S
-O3SS
CH3
PO32-
PO32-
ΔGo´= -30 kJ/molCan MCR catalyze the back reaction and ifyes at sufficient rates to account for the in vivomethane oxidation rates?
?
13CH4 + CoM-S-S-CoB ⇌13CH3-S-CoM + HS-CoB ∆Go = + 30 kJ/mol
12CH3-S-CoM + HS-CoB ⇌ 12CH4 + CoM-S-S-CoB ∆Go = – 30 kJ/mol
13CH4 + 12CH3-S-CoM ⇌ 12CH4 + 13CH3-S-CoM ∆Go = 0 kJ/mol
Nature 2010
Nature 2010
Nature 2010
CH4 +S
S NH
O H CO2-
CH3
H O
-O3SPO3
2-
HS NH
O H CO2-
CH3
H OMethyl-coenzyme M Coenzyme B
+-O3SS
CH3
PO32-
Methyl-CoM Reductase from M. marburgensis
Specific rate 12 nmol/min/mgat 1 bar CH4
Apparent Km 10 bar)
In the laboratory microbial mats from the Black Seacatalyze (no pure culture available):
CH4 + SO42- + 2H+ ⇌ CO2 + H2S + 2H2O
ΔGo´= - 21 kJ/mol
apparent Km for CH4 10 bar
specific rate of AOM at 1 bar CH4 1 nmol/min/mg protein
CH4
H2CO2
CH3COOH
Biomass(3 Gt/a) methano-
genicarchaea
bacteriaprotozoafungi
Methane deposits(> 10,000 Gt)
anox
ic e
nviro
nmen
ts
o
xic
tr
opos
pher
e
Lignin(0.3 Gt/a) thermogenic
formation- both very slow -
microbial or
BiomassBiomass(140 (140 GtGt/a)/a)
CO2 (380 ppm)
net primary production via oxygenic photo-
synthesis + .OH
+ O2
+ NO2-
+ FeIII
+ MnIV
+ SO42-
photochemical oxidation(0.5- 0.6 Gt CH4/a)
aerobic bacteria ( 0.6 Gt CH4/a)
N2-forming bacteria (?Gt CH4/a)
bacteria ? (? Gt CH4/a)
methanotrophic archaea with sulfate-reducing bacteria (up to 0.3 Gt CH4/a)
oxid
atio
n (1
GtC
H4/a
)
(1 Gt CH4/a)
diffusion(0.5 Gt CH4/a)
CH4(1.8 ppm)
geochemicalformation
aerobic oxidation
anaerobic oxidation
CO32- + 8 [H] from
serpentinization
sedi
mta
tion
bein
gbu
ried
+ O2
+ NO3-, FeIII, MnIV, or SO4
2-
2 NO2- 2 NO
N2
O2
CH4
CH3OH CO22e-
2e-
NO dis-mutase?
pMMO
Methane formation/oxidationMeike BrefortReinhard BöcherAnne KasterSeigo Shima
ETH ZürichBernhard JaunStefan MayrSilvan Scheller
University of OxfordJeffry Harmer
MPI für Biophysik FrankfurtUlrich ErmlerKristian Parey
MPI BremenMPI BremenFritz WiddelMartin KruegerMartin Krueger