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Nitrogen cycles Nitrate/nitrite Reductase Ammonia monooxygenase Hydroxylamine oxidoreductase.

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Nitrogen cycles Nitrate/nitrite Reductase mmonia monooxygenase ydroxylamine oxidoreductase
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Nitrogen cycles

Nitrate/nitriteReductase

Ammonia monooxygenaseHydroxylamine oxidoreductase

N2 + 3H2 ---> 2NH3 ∆G’º = -33.5 kJ/mol

However, N-N triple bond is a significant kinetic barrier

Bond energy = 930 kJ/mol

N2 + 10 H+ + 8 e- + 16 ATP ---> 2 NH4+ + 16 ADP + 16 Pi + H2

Nitrogen is fixed by anaerobic bacteria

0 -3

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

FeMo Cofactor

Proposed mechanism for dinitrogen reduction

Mo

FeS

N

N

Mo

N

N

FeS

H+

Mo

N

NH

FeS

H+

Mo

N

NH2

FeS

+ H+

Mo

N

NH3+

FeS

NH3

Mo

N

FeS

Mo

N+

Mo

N+

Mo

N+

Mo

N+

Mo

N+

H+

Mo

NH

FeS

H+

Mo

NH2

FeS

H+

Mo

NH3

FeS

Nitrate:ferredoxin reductase

S

Mo4+ O

O-

N+

O

O-

S

Mo6+ O

O-

N

O

O-

S

Mo6+ O

O2-

S

Mo6+ O

OH-

H+

H-

S

Mo4+O

H2O

Dissimilatory/Respiratory Nitrite Reductase

Siroheme and ferredoxin dependent looks and behaves a lot

like cytochrome c oxidase

NO2- ----> NO ----> NH2OH ----> NH4

+

Allows anaerobic organisms to respire electrons onto nitrite instead of oxygen

+3 +1 -1 -3

Dissimilatory Nitrite Reductase

NO2- ----> NO

Produces NO and N2O - potent greenhouse gasses

+3 +2

NO ---> N2O+2 +1

Nitrite reductase, multicopper enzyme

Nitric oxide reductase, heme dependent

N2O ---> N2

+1 0

Nitrous oxide reductase, multicopper enzyme

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

NH2OH + H2O ---> NO2- + 5 H+ + 4 e-

Hydroxylamine oxidoreductase (heme dependent)

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!

H

COO-

CH2

CH2

NH3+

O O-

ATP

H

COO-

CH2

CH2

NH3+

O OPO32-

H

NH2

BH

COO-

CH2

CH2

NH3+

O NH2

Glutamine synthase

Glutamate synthaseCOO-

C+H3N H

CH2

CH2

C

NH2O

E

S

HB

H A

COO-

C+H3N H

CH2

CH2

C

OSE

+NH3

H2O

COO-

C+H3N H

CH2

CH2

C

O-O

COO-

CO

CH2

CH2

C

O-O

COO-

C+H2N

CH2

CH2

C

O-O

H-HA

COO-

C+H3N H

CH2

CH2

C

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

N

NN

N

NH2

O

OHOH

HH

HH

OP-O

O

O

P-O O

O

P O

O-

-OC

O

O-HO

N

NN

N

NH2

O

OHOH

HH

HH

OP-O

O

O

P-O O

O-

C

O

OHO P

O

O-

O-

Carbamoylphosphate synthase: step 1

Carbonic phosphoric anhydride

Bicarbonate

Carbamoylphosphate synthase: step 2

N

NN

N

NH2

O

OHOH

HH

HH

OP-O

O

O

P-O O

O

P O

O-

-O

C

O

OHO P

O

O-

O-

HN

H

H

H2N

C

O

O

H

BN

NN

N

NH2

O

OHOH

HH

HH

OP-O

O

O

P-O O

O-H2N

C

O

O P

O

O-

O-

Carbonic phosphoric anhydride

Carbamoylphosphate

Carbamate

Ornithine transcarbamoylase

+ Pi

OrnithineCitrulline

These are -amino acids

N+ CH2 CH2 CH2 CH

NH3+

COO-

H2N C

O

O P

O

O-

O-

H

H

HB

HN CH2 CH2 CH2 C

H

NH3+

COO-C

O

H2N

Argininosuccinate synthase: step 1

N

NN

N

NH2

O

OHOH

HH

HH

OP-O

O

O

P-O O

O

P O

O-

-O

N

NN

N

NH2

O

OHOH

HH

HH

OPO

OH

O

H C

COO-

NH3+

CH2

CH2

CH2

NH

C

NH

H

B

O-

P-O O

O

P O

O-

-O

NH2+

C

NH

CH2

CH2

CH2

CH NH3+

COO-

O

Citrulline Citrullyl-AMP

Argininosuccinate synthase: step 2

Citrullyl-AMP

N C

COO-

H

CH2

COO-

H

H

H

B

B

N

NN

N

NH2

O

OHOH

HH

HH

OPO

OH

ONH2+

C

NH

CH2

CH2

CH2

CH NH3+

COO-

N

NN

N

NH2

O

OHOH

HH

HH

OP-O

OH

O

NH2+

C

NH

CH2

CH2

CH2

CH NH3+

COO-

HN C

COO-

CH2

COO-

H

Argininosuccinate

NH2+

C

NH

CH2

CH2

CH2

CH NH3+

COO-

HN C

COO-

C

COO-

H

HH

B

A

H

NH2+

C

NH

CH2

CH2

CH2

CH NH3+

COO-

NH2

C

C

-OOC H

H COO-

Argininosuccinate lyase

Argininosuccinate Arginine

Fumarate

NH2+

C

NH

CH2

CH2

CH2

CH NH3+

COO-

NH2H

OH

B NH2

C

NH

CH2

CH2

CH2

CH NH3+

COO-

NH2O

H

B

H

A

NH2

C

NH2

CH2

CH2

CH2

CH NH3+

COO-

NH2

O

Arginase

The aspartate/malate shuttle

In respiration asp/mal shuttle

Moves NADH into the mitochondrionwith net movement of one H+ in

but also moves NH3 out!

For every asp out a mal goes inand NH3 is moved out to cytoplasm

Sulfur Assimilation

S2- ---> S ---> SO32- ---> SO4

2-

-2 0 +4 +6

Sulfide oxidation by photosynthetic microbes

e- from sulfide

e- from sulfide

S2- + cyt c553(ox) ---> S + cyt c553(red)

Sulfide dehydrogenase

Sulfide:quinone oxidoreductase

S2- + UQ(ox) ---> S + UQH2(red)

S2- ---> S ---> SO42-

In 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

SORS + O2 ----> HSO3

- + H+

Non-enzymatic disproportionation3S + 3H2O ----> HSO3

- + 2HS- + 3H+

Sum4S + O2 + 4H2O ----> 2HSO3

- + 2HS- + 4H+

Non-enzymatic reaction with more SS + HSO3

- ----> S2O32- + H+

Sulfite oxidase converts sulfite to sulfate

S

Mo6+S

O2-

O

O

S O-

O-

S

Mo4+S

O-

O

O

S+ O-

O-

S

Mo4+S

-O

O

O

S O-

OO

H2O

Fe3+

S

Mo5+S O

-OH

cytochrome c

S

Mo5+S O

-OH

Fe3+S

Mo6+S O

O-H

HH

B

B

Thiosulfate:quinone oxidoreductase

Tetrathionate

S4O62- + H2O ---> S3O3

2- + SO42- + 2H+

Tetrathionate hydrolase

S3O32- ---> S2O3

2- + S

Spontaneous disproportionation

2S2O32- S4O6

2-

How to assimilate sulfate?

typo

E S

H

S

H

-O S

O

O

O P

O

O-

A

B

E S

S

H

S

O

O

O-

B

E S

S

S

O

O-

O-

H-

H+

E S

H

S

H

NADPH reduces the disulfide

PAPS Reductase

Sulfate/ite respiring organisms use dissimilatory sulfate/ite reductase

This is a respiratory enzyme that couples sulfate/ite reduction with NADPH

oxidation via ferredoxinrelated to cytochrome c oxidase

Catalyzes the reduction of sulfite to sulfide

Sulfide is assimilated by incorporation into cysteine

NH+

O-

2-O3PO

CH O

H C

COO-

NH3+

C

O

H

H

Schiff BaseFormation

NH+

O-

2-O3PO

CH

H C

COO-

NH+

C

O

H

HB

HA

NH+

O-

2-O3PO

CH

C

COO-

NH+

C H

H

+ acetate

Serine

O

CH3

O

CH3

HS-

NH+

O-

2-O3PO

CH

C

COO-

NH+

C H

H

SHH+

H

Acetylserine lyase

+H3NC

COO-

H

S

CH3

+H3NC

COO-

H

C O-

O

+H3NC

COO-

H

C H

O

+H3NC

COO-

H

CH2

HO

+H3NC

COO-

H

CH2

OC

O

H2C

CH2

COO-

Aspartate Aspartate-γ-semialdehyde

HomoserineO-succinylhomoserine

succinyl-CoA

+H3NC

COO-

H

CH2

SCH2

CCOO-+H3N

H

Cystathionine

cysteine

succinate

Cystathionineγ-synthase

+H3NC

COO-

H

CH2

HS

Homocysteine

Cystathionineβ-lyase

Pyruvate + NH3

S-adenosylmethionine

S-adenosylhomocysteine

serine

Cystathionineβ-synthase

cysteine+

-ketobutyrate

Cystathionineγ-lyase

Methionine

Steegborn, C. et al. J. Biol. Chem. 1999;274:12675-12684

Overview of the transsulfuration reactions

Cystathionine-γ-synthaseCOO-

H

NH+

H2C O C

H2C

H2C COO-

H

NH+

B

COO-

NH+

H2C O C

H2C

H2C COO-

H

NH

H2C

H2C O

O

H2C

H2C COO-

O

O

H

COO-

NH3+

H2C

H2C

H+

COO-

NH+

H2C O C

H2C

H2C COO-

HC

H

NH+

O

H2C

Cystathionine-γ-synthase

COO-

NH+

H2C O C

H2C

H2C COO-

HC

H

NH+

O

CH

H

B

COO-

NH+

CH2

-O CH2C

H2C COO-

HC

H

NH+

O

CH

H SH2C

COO-

H

NH3+B

COO-

NH

H2C

HC

H

NH+

CH

SH2C

COO-

H

NH3+

H+COO-

NH+

H2C

HC

H

NH+

H2C S

H2C

COO-

H

NH3+

Cystathionine-γ-synthase

COO-

NH+

H2C

HC

H

NH+

H2C S

H2C

COO-

H

NH3+

BH+

COO-

NH+

H2C

HC

NH+

H2C S

H2C

COO-

H

NH3+

H

Cystathionine-β-lyase

COO-

H

NH3+

H2C S

H2C

H2C

COO-

H

NH3+

COO-

H

NH+

H2C S

H2C

H2C

COO-

H

NH3+H

NH+

B

COO-

NH+

H2C S

H2C

H2C

COO-

H

NH3+H

NH

H+COO-

NH+

CH2 HSH2C

H2C

COO-

H

NH3+H

NH+

HN

CH2

COO-

H

B

H+

H3C

NH2+

COO-

Homocysteine

N

HN

HNCH3

Co+NN

N N

HA

Co3+NN

N N

CH3

Co3+NN

N N

CH3

+H3NC

COO-

H

S

H

B

Co+NN

N N

+H3NC

COO-

H

S

CH3

Methionine synthase

In mammals cysteine is made from methionine

+H3NC

COO-

H

S

CH3

+H3NC

COO-

H

C O-

O

+H3NC

COO-

H

C H

O

+H3NC

COO-

H

CH2

HO

+H3NC

COO-

H

CH2

OC

O

H2C

CH2

COO-

Aspartate Aspartate-γ-semialdehyde

HomoserineO-succinylhomoserine

succinyl-CoA

+H3NC

COO-

H

CH2

SCH2

CCOO-+H3N

H

Cystathionine

cysteine

succinate

Cystathionineγ-synthase

+H3NC

COO-

H

CH2

HS

Homocysteine

Cystathionineβ-lyase

Pyruvate + NH3

S-adenosylmethionine

S-adenosylhomocysteine

serine

Cystathionineβ-synthase

cysteine+

-ketobutyrate

Cystathionineγ-lyase

Methionine

H

COO-

NH3+

H2C S+

OA

OH OH

H2C

CH3

Nu-

H

COO-

NH3+

H2C S

OA

OH OH

H2C

Nu-CH3H

O H B

H+

H

COO-

NH3+

H2C SH

OA

OH OH

H2C HO

SAM-methyltransferase/S-adenosylhomocysteine hydrolase

HOH2C

COO-

H

NH3+

H2C

COO-

H

NH+H

NH+

B

HOH2C

COO-

NH+H

NH

H2C

COO-

NH+

NH+

HO

H+

COO-

NH3+

HH2C

H2C S

H

B

H+

COO-

H

NH3+

H2C S

H2C

H2C

COO-

H

NH+H

NH+

Cystathionine-β-synthase

COO-

H

NH3+

H2C S

H2C

H2C

COO-

H

NH3+

COO-

H

NH3+

H2C S

H2C

H2C

COO-

H

NH+H

NH+

B

COO-

H

NH3+

H2C S

H2C

H2C

COO-

NH+H

NH

H+

COO-

H

NH3+

H2C S

H2C

H2C

COO-

NH+

NH+

Cystathionine-γ-lyase

Cystathionine-γ-lyase

COO-

H

NH3+

H2C S

H2C C

H

COO-

NH+

NH+

H

B

COO-

H

NH3+

H2C S

H2C C

H

COO-

NH

NH+

H+

COO-

H

NH3+

H2C SH H2C C

H

COO-

N

NH+

HB

H3C CH

COO-

N

NH+

H+

COO-

NH2+

H

B

H+

COO-

NH2+

COO-

O

Cysteine desulfurylase: looks like serine dehydratase

H C

COO-

NH3+

CH2 SH

NH+

O-

2-O3PO

CH O Schiff Base

Formation

H C

COO-

NH+

CH2 SH

NH+

O-

2-O3PO

CH

B HA

H2S

C

COO-

NH+

NH+

O-

2-O3PO

CH

CH2

Schiff BaseHydrolysis

C

COO-

NH2

NH+

O-

2-O3PO

CH

CH2O


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