Nitrogen cycles Nitrate/nitrite Reductase Ammonia monooxygenase Hydroxylamine oxidoreductase

N 2 + 3H 2 ---> 2NH 3 ∆G’º = kJ/mol However, N-N triple bond is a significant kinetic barrier Bond energy = 930 kJ/mol N H e ATP ---> 2 NH ADP + 16 P i + H 2 Nitrogen is fixed by anaerobic bacteria 0 -3

FeMo Cofactor

Proposed mechanism for dinitrogen reduction

Nitrate:ferredoxin reductase

Dissimilatory/Respiratory Nitrite Reductase Siroheme and ferredoxin dependent looks and behaves a lot like cytochrome c oxidase NO > NO ----> NH 2 OH ----> NH 4 + Allows anaerobic organisms to respire electrons onto nitrite instead of oxygen

Dissimilatory Nitrite Reductase NO > NO Produces NO and N 2 O - potent greenhouse gasses NO ---> N 2 O Nitrite reductase, multicopper enzyme Nitric oxide reductase, heme dependent N 2 O ---> N Nitrous oxide reductase, multicopper enzyme

Biological Nitrification NH 3 ---> NO > NO NH 3 + O e H + ---> NH 2 OH + H 2 O Ammonia monooxygenase (related to methane monooxygenase) NO > NO 3 - is non-enzymatic

NH 2 OH + H 2 O ---> NO H+ + 4 e - Hydroxylamine oxidoreductase (heme dependent) 2 of these electrons are transferred to cytochrome bc 1 Ultimately they land on O 2 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 + NH ATP ---> glutamine + ADP + P i + H + Glutamate synthase glutamine +  KG + NADPH + H + ---> 2glutamate + NADP + Net reaction  KG + NADPH + NH ATP ---> glutamate + NADP + + H 2 O + ADP + P i Need catalytic amounts of glutamate! There has to be another way!

Glutamine synthase

Glutamate synthase

What about the reverse reaction of glutamate dehydrogenase? glutamate + NADP + + H 2 O --->  KG + NH 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 Carbonic phosphoric anhydride Bicarbonate

Carbamoylphosphate synthase: step 2 Carbonic phosphoric anhydride Carbamoylphosphate Carbamate

Ornithine transcarbamoylase + P i Ornithine Citrulline These are  -amino acids

Argininosuccinate synthase: step 1 Citrulline Citrullyl-AMP

Argininosuccinate synthase: step 2 Citrullyl-AMP Argininosuccinate

Argininosuccinate lyase Argininosuccinate Arginine Fumarate

Arginase

The aspartate/malate shuttle

In respiration asp/mal shuttle Moves NADH into the mitochondrion with net movement of one H + in but also moves NH 3 out! For every asp out a mal goes in and NH 3 is moved out to cytoplasm

Sulfur Assimilation S > S ---> SO > SO

Sulfide oxidation by photosynthetic microbes e - from sulfide

S 2- + cyt c 553(ox) ---> S + cyt c 553(red) Sulfide dehydrogenase Sulfide:quinone oxidoreductase S 2- + UQ (ox) ---> S + UQH 2(red)

S > S ---> SO 4 2- 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 SOR S + O > HSO H + Non-enzymatic disproportionation 3S + 3H 2 O ----> HSO HS - + 3H + Sum 4S + O 2 + 4H 2 O ----> 2HSO HS - + 4H + Non-enzymatic reaction with more S S + HSO > S 2 O H +

Sulfite oxidase converts sulfite to sulfate

Thiosulfate:quinone oxidoreductase Tetrathionate S 4 O H 2 O ---> S 3 O SO H + Tetrathionate hydrolase S 3 O > S 2 O S Spontaneous disproportionation 2S 2 O 3 2- S 4 O 6 2-

How to assimilate sulfate? typo

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 ferredoxin related to cytochrome c oxidase Catalyzes the reduction of sulfite to sulfide

Sulfide is assimilated by incorporation into cysteine

Acetylserine lyase

Steegborn, C. et al. J. Biol. Chem. 1999;274: Overview of the transsulfuration reactions

Cystathionine-  -synthase

Cystathionine-  -lyase Homocysteine

Methionine synthase

In mammals cysteine is made from methionine

SAM-methyltransferase/S-adenosylhomocysteine hydrolase

Cystathionine-  -synthase

Cystathionine-  -lyase

Cysteine desulfurylase: looks like serine dehydratase