Enzymes involved in denitrification

The reductive steps in denitrification involve several oxidoreductases, namely ni-trate, nitrite, NO and N2O reductases (Figure 8). Nitrate respiration is mediated by the dissimilatory nitrate reductases Nar and Nap [129]. The membrane-bound ni-trate reductase Nar is composed of 3 subunits and is encoded by narGHI [108, 129].

The cytoplasmic domain consists of theα- andβ-subunits, while theγ-subunit forms a membrane anchor [129]. The α-subunit contains a 4Fe-4S cluster and a

pyra-1 General introduction

nopterin cofactor, which is the site of nitrate reduction [129, 195]. The β-subunit is a globular-protein with 4 iron sulfur clusters (1 3Fe-4S, 3 4Fe-4S) which transfers electrons to the α-subunit [129, 195]. The γ-subunit consists of 5 transmembrane α-helices, a periplasmic N-terminus and a cytoplasmic C-terminus. It contains 2 b-type hemes that transfer electrons from the quinol pool to theβ-subunit [129]. A proton motive force is generated by consumption of protons in the cytoplasm and by the release of protons from the quinone pool as electrons are transfered to NarI [83, 129, 195]. The periplasmic nitrate reductase Nap is a heterodimer consisting of the 2 subunits NapA and NapB and is encoded by the napFDAGHBC operon [108, 129]. napA encodes the catalytic 90-kD subunit, which contains a molybde-num cofactor and a [4Fe-4S] center, whilenapB encodes a smaller 13-19-kD subunit with two-heme cytochromec which transfers electrons to the molybdopterin of the catalytic subunit [108].

Two types of respiratory nitrite reductases are used by denitrifiers to reduce ni-trite to NO: The copper-containing NirK and the cytochrome-cd1-containing NirS [195]. The enzymes are evolutionary unrelated and show no clear pattern of tax-onomic distribution, until now no organism is known that harbors both types of nitrite reductase [52, 195]. Even though both types seem to perform identical func-tions, there are indications that NirK- and NirS-type denitrifiers respond differently to changes in environmental conditions, thus allowing for niche differentiation of denitrifiers [60]. NirK is a homotrimeric periplasmatic enzyme that contains a type 1 and a type 2 copper center in each monomer. Nitrite binds to the type 2 copper center and is reduced to NO by electrons transfered from the type 1 copper center [83, 112, 195]. Several of NirK-type nitrite reductases are known, the NirK I is most intensively studied and often detected in denitrifying isolates. However, additional forms of NirK exist, and it is so far unresolved how widespread those forms are

1.3 Processes involved in greenhouse gas production in anoxic peatland soils

in denitrifiers [33, 61, 112]. NirS is a homodimeric periplasmatic enzyme with a prosthetic heme cand heme d1 in each cytochrome cd1 monomer [83, 195]. Nitrite binds to the d1-heme and is reduced to NO by electrons from cytochrome c₅₅₁ via the c-heme [83, 195]. NirK and NirS are found in Bacteria as well as in Archaea [83, 195].

Bacterial respiratory NO reductases are membrane-bound heme-nonheme Fe en-zymes and can be devided into two groups: a about 450 amino acid long variant scNor (also called cNor) receives electrons from cytochrome c, while the about 760 amino acid long variant lcNor (also called qNor) receives electrons from quinol [196].

Both scNor and lcNOR coexist with NirK or NirS [61, 196]. scNor is a heterodimer consisting of the catalytic subunit NorB and a c-type cytochrome NorC, which are encoded by norCB [196]. The long chain lcNOR is encoded by norZ. Compared to NorB it contains an N-terminal extension that acts as a quinol oxdiase [196].

NO reductases are homologues of heme-copper oxidases, however the NO reductase itself does not translocate protons, even though the electron flow to the reductase is linked to proton extrusion [196]. In Archaea, all known NO reductases belong to the scNor type [83, 196]. However, in many archaeal genomes, nonorZ homologues are detected, thus there might be alternative NO reductases in Archaea [83].

Bacterial N₂O reductases (NosZ) are multicopper homodimeric enzymes encoded by nosZDFY that are periplasmatic or membrane-bound in gram-negative and gram-positive Bacteria, respectively [83]. Each monomer of the NosZ homodimer contains two multi-copper centers CuA and CuZ [83, 132, 197]. CuZ is a [4Cu-2S]

copper-sulfur cluster that binds N2O while CuA is a mixed-valent binuclear copper-center that transfers electrons to the bound N2O molecule [132]. Some Bacteria cannot use exogenous N2O as the induction of N2O reductase genes requires NO to be present in the cell [197]. Some Archaea possess N₂O reductases that are

homo-1 General introduction

logues of the bacterial NosZ. In contrast to their bacterial conterparts these N2O reductases are membrane-bound and receive electrons from menaquinol [83, 197].

However, there are archaeal denitrifiers capable of N₂O reduction whose genomes do not harbornosZ homologues, thus there is likely a second archaeal type of N2O reductase [197]. Some denitrifiers lack N2O reductases, thus N2O is the endproduct of denitrification in those organisms [61, 197]. On the other hand, there are or-ganisms likeWolinella succinogenes that reduce N2O using a slightly modified N2O reductase without being capable of complete denitrification [197]. W. succinogenes reduces nitrate to nitrite and N2O to N2, but the intermediate steps are missing [197].

In fungal denitrification, the reductive enzymes are located in the mitochondria and are coupled to the mitochondrial electron transport chain to produce ATP [74]. The membrane-bound fungal nitrate reductase (Nar) reduces nitrate utilizing ubiquinol as electron donor [107, 165]. Properties of fungal Nar resemble those of the bacterial conterpart. However, no orthologues of the bacterial nar genes have so far been detected in fungal genomes, indicating that fungal Nar is evolutionary distinct from bacterial Nar [165]. In contrast, the fungal nitrite reductase genenirK detected in the genomes of many denitrifying fungi is an orthologue to the bacterial nirK [68]. Fungal NirK is a copper-containing nitrite reductase loctaed in the mi-tochondrion [75, 107]. Fungal NO reductases are clearly distinct from prokaryotic NO reductases. Fungal Nor is a member of the cytochrome P450 superfamily and is thus called P450nor [107, 165]. The reduction of NO is very different from the oxidation reactions of other P450 proteins, as those require further electron donat-ing proteins [165]. P450nor is a soluble enzyme that utilizes NADH or NADPH to reduce NO to N2O [165]. N2O is the major end product in fungal denitrification, even though some fungi are also capable of N₂ production [107, 151, 195]. So far, no

1.3 Processes involved in greenhouse gas production in anoxic peatland soils N2O reductase has been isolated from fungi or identified in fungal genomes [107].

1 General introduction