Transcript Document

Nitrogen cycles
Nitrate/nitrite
Reductase
Ammonia monooxygenase
Hydroxylamine oxidoreductase
N2 + 3H2 ---> 2NH3
-3
0
∆G’º = -33.5 kJ/mol
However, N-N triple bond is a significant kinetic barrier
Bond energy = 930 kJ/mol
Nitrogen is fixed by anaerobic bacteria
N2 + 10 H+ + 8 e- + 16 ATP ---> 2 NH4+ + 16 ADP + 16 Pi + H2
FeMo Cofactor
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Proposed mechanism for dinitrogen reduction
H+
N
N
NH2
NH
N
NH3+
+ H+
N
Mo
H+
Mo
FeS
FeS
N+
Mo
N
N
N
Mo
Mo
Mo
FeS
FeS
N+
N+
Mo
Mo
N+
Mo
FeS
NH3
H+
NH3
NH2
H+
H+
NH
N
Mo
Mo
Mo
Mo
FeS
FeS
FeS
FeS
N+
Mo
Nitrate:ferredoxin reductase
O
O
N+
S
N
O-
OS
Mo4+ O
O-
OO2-
S
Mo6+ O
Mo6+ O
H+
HS
H2O
Mo4+ O
S
OHMo6+ O
Dissimilatory/Respiratory Nitrite Reductase
Siroheme and ferredoxin
dependent looks and behaves a lot
like cytochrome c oxidase
NO2- ----> NO ----> NH2OH ----> NH4+
+3
+1
-1
-3
Allows anaerobic organisms to respire
electrons onto nitrite instead of oxygen
Dissimilatory Nitrite Reductase
NO2- ----> NO Nitrite reductase, multicopper enzyme
+2
+3
NO ---> N2O
+2
+1
Nitric oxide reductase, heme dependent
N2O ---> N2 Nitrous oxide reductase, multicopper enzyme
+1
0
Produces NO and N2O - potent greenhouse
gasses
Biological Nitrification
NH3 ---> NO2- ---> NO3-3
+3
+5
NH3 + O2 + 2 e- + 2 H+ ---> NH2OH + H2O
Ammonia monooxygenase (related to methane monooxygenase)
NO2- ---> NO3- is non-enzymatic
Hydroxylamine oxidoreductase (heme dependent)
NH2OH + H2O ---> NO2- + 5 H+ + 4 e-
2 of these electrons are transferred to cytochrome bc1
Ultimately they land on O2 and generate proton gradient
2 electrons are returned to AMO to complete the cycle
Assimilation of ammonia
Use glutamate and glutamine as reservoirs
Glutamine synthase
glutamate + NH4+ + ATP ---> glutamine + ADP + Pi + H+
Glutamate synthase
glutamine + KG + NADPH + H+ ---> 2glutamate + NADP+
Net reaction
KG + NADPH + NH4+ + ATP ---> glutamate + NADP+ + H2O + ADP + Pi
Need catalytic amounts of glutamate!
There has to be another way!
Glutamine synthase
COO-
COONH3+
H
CH2
CH2
O
NH3+
H
CH2
COO-
CH2
ATP
O-
O
OPO32-
B
NH3+
H
H
CH2
NH2
CH2
O
NH2
Glutamate synthase
COOCOO-
COO+H
3N
C
H
+H N
3
C
CH2
CH2
E
+H N
3
H
C
H2O
H
CH2
CH2
S
C
O
H
B
COOO
O
C
H
CH2
C
C
A
NH2
E
A
+NH3
CH2
COO-
H
+H
2N
C
S
O
O
COO-
H+H N
3
C
H
CH2
CH2
CH2
CH2
CH2
CH2
C
C
O-
O
O-
C
O-
O
O-
What about the reverse reaction of glutamate
dehydrogenase?
glutamate + NADP+ + H2O ---> KG + NH4+ + NADPH
Normally this reaction favors the right
Glutamine synthetase
is a control point for
growth. Many end
products allosterically
regulate its activity
Amino transferases
Carbamoylphosphate synthase: step 1
NH2
NH2
N
N
N
N
N
O
N
N
O
-O
P
O
O
O
-O
H
H
OH
OH
H
P
O
-O
P
O
O
O
H
-O
H
H
OH
OH
H
P
N
O
H
O
O
O-O
C
HO
P
O
O
O-
O
OC
HO
Bicarbonate
O
P
O-
O-
Carbonic phosphoric anhydride
Carbamoylphosphate synthase: step 2
NH2
Carbonic phosphoric anhydride
N
N
N
O
-O
O
O
O
P
O-
-O
N
H
O
H
H
OH
OH
H
P
O
H
O
OH
O
O
C
HO
P
N
O
H
C
H2N
-O
O
P
O
NH2
O-
H
N
N
B
Carbamate
N
O
-O
P
O
O
O
H
H
OH
OH
O
O
-O
H
P
C
Carbamoylphosphate
H2N
O
P
O-
O-
O-
O
H
N
Ornithine transcarbamoylase
O
H2N
C
O
O
P
+ Pi
O-
O-
H
N+
CH2
CH2
CH2
B
C
H
NH3+
O
NH3+
H
COO-
H 2N
C
H
N
CH2
CH2
CH2
C
H
H
Ornithine
These are -amino acids
Citrulline
COO-
Argininosuccinate synthase: step 1
NH2
NH2
B
N
N
N
N
H
NH
C
N
O
O
-O
NH
P
O
-O
H
H
OH
OH
H
P
O
N
O
O
C
O
O
CH2
NH2+
N
P
O
O
OH
NH
H
H
OH
OH
H
H
CH2
N
H
O-
CH2
-O
CH2
H
C
CH2
O
NH3+
COO-
-O
P
O-
P
H
O
NH3+
C
COO-
-O
P
O-
Citrulline
O
CH2
O
Citrullyl-AMP
O
Argininosuccinate synthase: step 2
NH2
N
NH2
N
N
NH2
+
C
H
O
O
N
O
C
H
CH2
N
O
H
-O
H
H
N
N
O
OH
CH2
H
P
COONH
H
N
P
O
OH
OH
OH
H
H
OH
OH
H
CH2
B
O
H
H
COOCH2
B
H
C
NH3+
NH2+
COOC
COOH
N
C
NH
CH2
CH2
COO-
CH2
CH2
H
Citrullyl-AMP
C
NH3+
COO-
Argininosuccinate
H
N
Argininosuccinate lyase
A
H
NH2+
COO-
NH2+
C
H
N
C
H
NH
H
C
H
C
NH2
-OOC
H
C
NH
C
COO-
CH2
H
CH2
B
CH2
CH2
CH2
CH2
C
NH3+
COO-
Argininosuccinate
H
C
COO-
Arginine
H
NH3+
COO-
Fumarate
Arginase
B
NH2+
B
C
H
O
H
NH2
NH2
NH2
H
O
NH
NH2
H
CH2
A
CH2
CH2
COO-
O
NH2
CH2
CH2
C
NH2
NH
CH2
H
C
C
CH2
CH2
NH3+
H
C
COO-
CH2
NH3+
H
C
COO-
NH3+
The aspartate/malate shuttle
In respiration asp/mal shuttle
Moves NADH into the mitochondrion
with net movement of one H+ in
but also moves NH3 out!
For every asp out a mal goes in
and NH3 is moved out to cytoplasm
Sulfur Assimilation
S2- ---> S ---> SO32- ---> SO42-2
0
+4
+6
Sulfide oxidation by photosynthetic microbes
e- from sulfide
e- from sulfide
Sulfide dehydrogenase
S2- + cyt c553(ox) ---> S + cyt c553(red)
Sulfide:quinone oxidoreductase
S2- + UQ(ox) ---> S + UQH2(red)
S2- ---> S ---> SO42In photosynthetic green sulfur bacteria this
oxidation is the driving force for the rTCA cycle
Elemental sulfur globules are temporarily deposited
outside the cell.
Sulfur oxidation
Sulfur oxygenase reductase (SOR)
Mononuclear non-heme iron enzyme
SOR
S + O2 ----> HSO3- + H+
Non-enzymatic disproportionation
3S + 3H2O ----> HSO3- + 2HS- + 3H+
Sum
4S + O2 + 4H2O ----> 2HSO3- + 2HS- + 4H+
Non-enzymatic reaction with more S
S + HSO3- ----> S2O32- + H+
Sulfite oxidase converts sulfite to sulfate
O
B
O
O-
S
O-
O2-
O
S+
O-
S
S
H
H
O-
4+ O
S Mo
6+ O
S Mo
O-
O
H2O
S
-O
Mo4+
O-
S
O
O
S
Fe3+
-OH
5+ O
S Mo
S
B
H
-OH
OS
Mo6+
S
O
5+ O
S Mo
S
Fe3+
cytochrome c
Thiosulfate:quinone oxidoreductase
2S2O32- S4O62Tetrathionate
Tetrathionate hydrolase
S4O62- + H2O ---> S3O32- + SO42- + 2H+
Spontaneous disproportionation
S3O32- ---> S2O32- + S
How to assimilate sulfate?
typo
PAPS Reductase
B
O
O
H
E
O
E
S
-O
S
S
O
H
O
P
S
S
S
O
O-
A
O-
H
B
H+
H
E
S
E
S
O
S
S
O-
S
O-
H
H-
NADPH reduces the disulfide
Sulfate/ite respiring organisms use dissimilatory
sulfate/ite reductase
This is a respiratory enzyme that couples
sulfate/ite reduction with NADPH
oxidation via ferredoxin
related to cytochrome c oxidase
Catalyzes the reduction of sulfite to sulfide
Sulfide is assimilated by incorporation into cysteine
Acetylserine lyase
H
COO-
H
C
C
NH3+
Serine
H
B
CH3
O
H
H
O
O
Schiff Base
Formation
C
H
3PO
C
C
NH+
O
H
CH3
O
O-
H
COO-
H
H+
C
C
H
NH+
A
2-O PO
3
N
H+
N
H+
H
H
COO-
H
C
C
NH+
SH
H
C
3PO
O2-O PO
3
N
H+
H
HS-
C
O2-O
H
C
O2-O
COO-
N
H+
+ acetate
O
O
O-
C
C
H
+H N
3
COO-
COO-
Aspartate-semialdehyde
Aspartate
C
O
H2C
H
C
C
succinyl-CoA
CH2
H
H
+H N
3
O
HO
CH2
CH2
C
+H N
3
COO-
C
COO-
Homoserine
H
+H N
3
COO-
O-succinyl
homoserine
cysteine
Cystathionine
-synthase
S-adenosylhomocysteine
H
Pyruvate + NH3
succinate
CH2
S
C
+H N
3
COO-
CH2
H
Cystathionine
-lyase
S-adenosylmethionine
C
+H N
3
Cystathionine
-lyase
COO-
Cystathionine
CH3
HS
H
H
C
C
+H N
3
serine
CH2
S
COO-
Methionine
+H N
3
COO-
Homocysteine
cysteine
+
-ketobutyrate
Cystathionine
-synthase
Overview of the transsulfuration reactions
Steegborn, C. et al. J. Biol. Chem. 1999;274:12675-12684
Cystathionine--synthase
COOH2
C
H
H2
C
H2
C
O
COO-
B
O
H2
C
H2
C
H
COO-
H2
C
O
C
H2
C
H2
C
COO-
NH+
H
NH3+
O
N+
H
COOH2
C
H
H
C
N+
H
NH+
COO-
O
H2
C
O
C
H2
C
H2
C
O
H2
C
COO-
H+
NH+
H
N
H
H2
C
O
C
H2
C
H2
C
COO-
Cystathionine--synthase
O
B
COO- H
C
H
H
H
C
COO-
O
H2
C
O
C
H2
C
H2
C
NH+
COO-
-O
C
H
H
H
C
H2
C
C
CH2
COO-
NH+
H
H2
C
S
N+
H
COO-
COOH2
C
H
H
C
N+
H
H
NH3+
B
N+
H
NH+
H2
C
S
H2
C
H+
COOH
NH3
C
H
+
H
H
C
N+
H
NH
H2
C
H2
C
COOS
H2
C
H
NH3+
COO-
Cystathionine--synthase
H+
B
COOCOO-
COOH2
C
H2
C
S
H2
C
H
H2
C
H
H
C
N+
H
H2
C
S
H2
C
H
H
NH+
NH+
COO-
NH3+
HC
N+
H
NH3+
Cystathionine--lyase
COOH2
C
H
H2
C
S
COOH2
C
H
H2
C
NH3+
COO-
B
COO-
S
H2
C
H2
C
H
H
NH+
H
NH3+
NH3+
H+
NH2+
CH2
HN
COO-
H3C
COO-
N+
H
H
B
COOCH2 HS
NH+
H
H+
COOH2
C
COO-
H2
C
H
NH3
H2
C
S
H2
C
H2
C
H
+
NH+
H
Homocysteine
N+
H
COO-
N
H
NH3+
Methionine synthase
H
N
CH3
N
N
Co3+
N
N
N
A
H
CH3
N
HN
N
Co+
N
N
B
H
CH3
S
S
H
C
+H N
3
CH3
N
H
N
Co3+
COON
N
C
+H N
3
COO-
N
N
Co+
N
N
In mammals cysteine is made from methionine
O
O
O-
C
C
H
+H N
3
COO-
COO-
Aspartate-semialdehyde
Aspartate
C
O
H2C
H
C
C
succinyl-CoA
CH2
H
H
+H N
3
O
HO
CH2
CH2
C
+H N
3
COO-
C
COO-
Homoserine
H
+H N
3
COO-
O-succinyl
homoserine
cysteine
Cystathionine
-synthase
S-adenosylhomocysteine
H
Pyruvate + NH3
succinate
CH2
S
C
+H N
3
COO-
CH2
H
Cystathionine
-lyase
S-adenosylmethionine
C
+H N
3
Cystathionine
-lyase
COO-
Cystathionine
CH3
HS
H
H
C
C
+H N
3
serine
CH2
S
COO-
Methionine
+H N
3
COO-
Homocysteine
cysteine
+
-ketobutyrate
Cystathionine
-synthase
SAM-methyltransferase/S-adenosylhomocysteine hydrolase
NuNu-CH3
H
COOH2
C
H
COO-
CH3
H2
C
S+
A
O
H2
C
H
H2
C
H
B
A
S
O
O
NH3+
NH3+
H+
OH
OH
OH
OH
COOH2
C
H
H2
C
SH
A
HO
O
NH3+
OH
OH
Cystathionine--synthase
COOCOOHO
H2
C
HO
B
H2
C
H
H
H
NH+
NH3+
COO-
COOH2
C
H
H2
C
S
H2
C
H
H
NH3+
N+
H
NH+
COON+
H
H+
COO-
H+
H2C
HO
NH+
H2
C
H
NH+
COOH2
C
H
H2
C
S
H
NH3+
B
N+
H
N
H
Cystathionine--lyase
COOCOO-
COOH2
C
H
H2
C
S
H2
C
H
H2
C
COOS
H2
C
B
H2
C
H
H
H
NH3+
NH3+
NH+
NH3+
N+
H
COO-
COOH2
C
H
S
H2
C
H2
C
NH3+
COOH2
C
H
NH+
COOS
H2
C
H2
C
H
NH3+
NH+
H+
N+
H
N
H
Cystathionine--lyase
B
H+
COOCOO-
H
H2
C
H
S
H2
C
COO-
H2
C
H
C
H
NH3+
COOS
H2
C
C
H
NH3+
NH
NH+
N+
H
N+
H
COO-
COO-
H+
NH2+
H3C
H+
COO-
COO-
O
N
COO-
H2
C
H
C
H
COOSH H2C
NH3+
C
H
N
B
H
NH2+
H
N+
H
B
N+
H
Cysteine desulfurylase: looks like serine dehydratase
A
COOH
C
CH2
NH3
+
B
C
SH
SH
H
H
O
C
Schiff Base
Formation
C
CH2
NH+
H
O2-O PO
3
O2-O
COO-
H
3PO
N
H+
N
H+
COO-
COOC
CH2
C
H
Schiff Base
Hydrolysis
O
C
NH2
CH2
NH+
H
C
OO-
2-O PO
3
2-O PO
3
N
H+
N
H+
H2S