Mechanisms and Electron Transfer of Prokaryotic Nitrate

Reductases

Yiming Luo

Hwa Chong International School, Singapore, 269783, Singapore

Keywords: Prokaryotic Nitrate Reductases, Metalloenzyme, Special Cofactors, Electron Transfer.

Abstract: There are three types of prokaryotic nitrate reductases which are essential in the biological nitrogen cycle,

respectively membrane-bound respiratory nitrate reductase (Nar), periplasmic nitrate reductase (Nap) and

prokaryotic assimilatory nitrate reductase (Nas). This work summarizes the structures of these three types of

nitrate reductases, as well as their nitrate reduction mechanism and electron transfer pathways. They each

contain a molybdenum bis-molybdopterin guanine dinucleotide cofactor (Mo-bis-MGD) as the active site for

nitrate binding, and they use different numbers and types of iron–sulfur clusters to aid the electron transfer.

Nar and Nap use hemes and the quinol–quinone Q cycle as the electron source, whereas Nas relies on

flavodoxin or NADH. This work also presents some possible aspects for future research, such as the

uniqueness of molybdenum, the large potential barriers in the Nap electron transfer pathway, and unsolved

crystal structures of some enzyme subunits that have limited understanding.

1 INTRODUCTION

Nitrogen is an element necessary for all lifeforms

because it is a building block of many essential

biological molecules such as nucleic acids, amino

acids, and proteins. It occurs in various oxidation

states, such as dinitrogen (N

, 0) in the atmosphere,

ammonium (NH

, −3) in sediments and minerals,

nitrate (NO

−

, +5) as dissolved species in marine

environments (Bebout, Fogel, Cartigny, 2013), and

organic nitrogen compounds in organisms. Nitrogen

can be converted between these forms, referred to as

the nitrogen cycle. Nitrate reductases play an

essential role in the nitrogen cycle, by catalyzing the

reduction of nitrate to nitrite (NO

−

), which is ready

for further reduction:

−

+2H

+ 2e

−

→ NO

−

+ H

O, E

⦵

= +

420

(Equation 1)

The aim of this report is to provide a brief

introduction of the nitrogen cycle and nitrate

reduction pathways, summarize and compare the

structures of different prokaryotic nitrate reductases

and explore the mechanisms of the Mo-bis-MGD

active site, as well as the role of each special cofactor

in the entire electron transport chain.

2 COMMON METHODS TO

DETERMINE NITRATE

REDUCTASE STRUCTURE

Nitrate reductases are a category of metalloenzyme

with a molybdenum ion at each of their active sites.

Common methods to determine the structure of

nitrate reductases include X-ray crystallography,

specifically multiwavelength anomalous diffraction

(MAD), which provided high-resolution electron

density maps for the first crystal structure of

periplasmic nitrate reductase (Nap) (Dias, 1999).

This identified some unique cofactors present in

nitrate reductases: a molybdenum bis-molybdopterin

guanine dinucleotide cofactor (Mo-bis-MGD), and

an iron–sulfur cluster [4Fe–4S] (Figure 1). To

understand the coordination and redox activities of

the central Mo ion, electron paramagnetic resonance

(EPR) spectroscopy and extended X-ray absorption

fine structure (EXAFS) spectroscopy were used,

which facilitated the investigation of mechanisms of

catalysis (Butler, 1999).

Luo, Y.

Mechanisms and Electron Transfer of Prokaryotic Nitrate Reductases.

DOI: 10.5220/0011219400003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 505-513

ISBN: 978-989-758-595-1

505

3 THE BIOLOGICAL NITROGEN

CYCLE AND NITRATE

REDUCTASES

Nitrogen can be converted between different forms

through abiotic processes such as the production of

nitrogen monoxide (NO) by lightning:

+ O

→ 2NO (Equation 2)

Figure 1: First crystal structure of Nap (subunit NapA) of

bacterium Desulfovibrio desulfuricans (PDB: 2NAP)

obtained by MAD methods. The central Mo (IV) ion is

coordinated to two MGDs to form a Mo-bis-MGD cofactor,

as well as a cysteine residue Cys

140

. An iron–sulfur cluster

[4Fe–4S] is located nearby for electron transfer.

However, the focus of this report will be the

biological nitrogen cycle, where nitrogen is

circulated in the biosphere by organisms,

predominantly prokaryotes. Different nitrogen

compounds are simultaneously oxidized or reduced,

and the reductive process can be further divided into

two different pathways: nitrate assimilation and

denitrification (Cabello, 2009), or dissimilatory

nitrate reduction. In nitrate assimilation, NO

−

are

reduced to NH

, a nitrogen source for constructive

metabolism, via the only intermediate of NO

−

catalyzed by assimilatory nitrate reductase (Nas) and

nitrite reductase NirS:

−

Nas

⎯→⎯⎯

−

NirS

⎯→⎯⎯

(Equation 3)

In contrast to nitrate assimilation, denitrification

refers to the catabolic process where NO

−

acts as the

terminal electron acceptor in anaerobic respiration

instead of dioxygen (O

). The terminal products of

denitrification can be either nitrous oxide (N

O) or

, via the intermediate stages of NO

−

and NO. In

this case, NO

−

is reduced to NO

−

with the catalysis

of either Nap or membrane-bound respiratory nitrate

reductase (Nar), and the subsequent reduction are

catalyzed respectively by nitrite reductases NirS and

NirK, NO reductases cNor and qNor, and N

reductase (Nos):

−

Nar/ Nap

⎯→⎯⎯⎯

−

NirS/ NirK

⎯→⎯⎯⎯

cNor/qNor

⎯→⎯⎯⎯

Nos

⎯→⎯⎯

(Equation 4)

This report will only discuss prokaryotic nitrate

reductases, i.e. Nas, Nap and Nar, among these

enzymes in nitrate reduction processes. A key

difference between Nap and Nar is that the active site

Mo-bis-MGD of Nap is in the periplasmic space,

while Nar’s is in the cytoplasm (Moreno-Vivián,

1999). Nar consumes protons to reduce NO

−

in the

cytoplasm and protons are translocated into the

periplasm through the oxidation of quinols to

quinones (QH

→ Q + 2H

+ 2e

−

) in the lipid bilayer

by the quinol-oxidizing subunit NarI, thereby

enhancing the electrochemical proton gradient to

facilitate adenosine triphosphate (ATP) synthesis,

while Nap reduces NO

−

in the periplasm so there is

no proton translocation (Kuypers, 2018) (Figure 2).

Figure 2: Difference in location and contribution of ATP synthesis between Nar and Nap. The active sites where Mo-bis-

MGD is present are represented by circles, i.e. NarG and NapA.

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4 SPECIAL COFACTORS

PRESENT IN PROKARYOTIC

NITRATE REDUCTASES

4.1 Mo-Bis-MGD

Mo-containing enzymes can be categorized into 3

families with similar general structures, all of which

contain at least one MGD molecule each (Figure 3),

respectively the xanthine oxidase family, the

dimethyl sulfoxide (DMSO) reductase family, and

the sulfite oxidase family (Moura, 2004) (Figure 4a).

Though eukaryotic nitrate reductases (eukNR)

belong to the sulfite oxidase family, all prokaryotic

nitrate reductases are from the DMSO reductase

family, where the central Mo ion is coordinated to

two MGD molecules and one or two other ligands

(Sparacino-Watkins, 2014) (Figure 4b).

Figure 3: The structure of MGD.

The two sulfhydryl groups (colored yellow in

Figure 3) are responsible for Mo-coordination.

Although thiols generally have no net charge in

neutral pH, sulfhydryl groups are deprotonated and

coordinate as thiolates in Mo-bis-MGD (Moura,

2004). This is because highly charged Mo ions can

greatly stabilize the conjugate base of thiols, thus

lowering the pK

value of sulfhydryl groups in MGD

(Lippard, 1994).

Figure 4: (a) General structures of the three Mo-containing enzyme families. X and Y represent any ligand, either singly or

doubly bonded to the Mo ion. The −OH ligands may be replaced by −OH

or =O. (b) Some examples of Mo active sites.

From left to right: NarG (oxidized form) from Escherichia coli (PDB: 1Q16), NapA (oxidized form) from Escherichia coli

(PDB: 2NYA), eukNR from Pichia angusta (PDB: 2BII). The first two (Nap, Nar) belong to the DMSO reductase family

while the last one (eukNR) belongs to the sulfite oxidase family.

5a 5b

Figure 5: Two different proposed mechanisms of nitrate reduction at the Mo-bis-MGD active site. Though both mechanisms

have been proposed, Figure 5b shows the more widely accepted mechanism.

Mechanisms and Electron Transfer of Prokaryotic Nitrate Reductases

507

The central Mo ion has variable oxidation states

of IV, V and VI. Two different mechanisms of nitrate

reduction at the Mo-bis-MGD active site have been

proposed (Sparacino-Watkins, 2014) (Figure 5a and

5b). Nitrate reduction starts from the reduced form,

where the Mo ion is in the oxidation state of IV or V

and is only penta-coordinated with an empty binding

site for NO

−

. Mo(V) ion will then gain an electron,

because the binding of NO

−

raises its redox

potential, making it more easily reduced (Richardson,

2007) (Figure 5b). Since nitrogen is reduced from +5

to +3, the coordinated NO

−

gains two electrons from

Mo(IV), oxidizing it to be Mo(VI), with electron

configuration [Kr]. The Mo-bis-MGD cofactor is

now in its oxidized form, where an oxo ligand is left

coordinated to Mo(VI). It is then protonated and

leaves as water, reducing Mo(VI) back to either

Mo(IV) (Figure 5a) or Mo(V) (Figure 5b) for further

reduction of NO

−

. Regardless of the pathway, two

electrons in total are transferred in each cycle.

However, in the crystal structure of E. coli NarG

(PDB: 1Q16, leftmost in Figure 4b), a bidentate

Asp

222

was discovered, which might block the

binding of NO

−

. Besides, it was concluded that the

active site was in its oxidized form, i.e. Mo(VI), but

the oxo ligand that appeared in the proposed

mechanism was absent. In fact, there are different E.

coli NarG Mo-bis-MGD structures in the Protein

Data Bank (Figure 6).

Figure 6: A different structure of the Mo-bis-MGD active site of E. coli NarG (PDB: 1R27).

Figure 6 shows another oxidized form of the

active site, where the bidentate Asp

222

is now only

singly coordinated to the Mo ion, and the other

carboxylate O is hydrogen-bonded to the ε-nitrogen

of His

546

. Although the two structures are different,

their amino acid sequences are identical. The

difference may be caused by the protonation state of

His

546

, or structural flexibility of the active site.

However, further investigation is required to

conclude whether both forms are involved in the

catalysis of nitrate reduction (Jormakka, 2004).

The proposed mechanisms in Figure 5 also have

other limitations: it does not take into account of

some Nap structures where there are other ligands

coordinated to Mo in addition to a cysteine. At

Rhodobacter sphaeroides Nap (PDB: 3ML1) Mo-

bis-MGD site, a sulfido group (S

2−

) is doubly bonded

to Mo; for Cupriavidus necator Nap (PDB: 2JIR), the

protein crystal structure shows an additional cyanide

ligand coordinated to Mo. They seem to interfere

with the binding of NO

−

, and may also affect the

shifting of oxidation state of the Mo ion. A sulfur

shift mechanism has been proposed to address the

first circumstance (Cerqueira, 2013) (Figure 7). The

Mo-bis-MGD site with a sulfido group is described

as the inactive state, but when a NO

−

substrate comes

in, the sulfido ligand will undergo structural changes

to the active state, where a binding site is vacated and

the catalytic reaction can take place as usual.

Figure 7: The sulfur shift mechanism.

Tungsten (W) and Mo are from the same group in

the periodic table, so they share similar chemical

properties, and DMSO reductase (DMSOR) can

function with both Mo and W (Stewart, 2000).

However, nitrate reductases from the DMSO

reductase family, would become inefficient if the Mo

ion of Mo-bis-MGD is replaced by W (Sparacino-

Watkins, 2014). The reason is still unclear, but one

speculation is that W-containing enzymes typically

catalyze reactions with lower redox potentials (E

⦵

≤

−

420

mV) (Moura, 2004). Therefore, they may not

work for the reduction of NO

−

whose redox potential

is positive (+

420

mV).

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4.2 Iron–Sulfur Clusters

There are three most common iron–sulfur clusters,

respectively [2Fe–2S], [3Fe–4S], and [4Fe–4S]

(Broderick, 2004) (Figure 8a). Iron–sulfur clusters

are ancient structures developed during evolution.

While they are important protein cofactors primarily

responsible for electron transfer, they also have other

functions such as DNA damage repair (Brzóska,

2006). Iron–sulfur clusters transport electrons

through the alteration of Fe oxidation state between

+2 and +3, and since each cluster contains more than

one Fe ion, it may exhibit multiple overall oxidation

states (Beinert, 2000) (Figure 8b).

Figure 8: (a) Three common types of iron–sulfur clusters. L represents any ligand, often cysteine. Though [4Fe–4S] is cubic,

it is not a perfect cube. This is similar for [3Fe–4S]. (b) Electron transfer mechanisms in the three common iron–sulfur

clusters. The oxidation state of +2.5 is due to delocalized electrons, so they occur in pairs.

In prokaryotic nitrate reductases, all of the three

common types of iron–sulfur clusters are present or

possibly present. In NapA, there is a [4Fe–4S] near

the active site (Figures 1 & 9b), which is the same for

NarG. In NarH, the electron transfer subunit, there

are three more [4Fe–4S] and one [3Fe–4S], which is

at the side nearer to NarI, where two heme B are

located (Figure 9a). On the contrary, the

understanding of Nas is still limited and no crystal

structures of Nas have been determined, so it can only

be speculated based on genetic information that there

may be a [4Fe–4S] near Mo-bis-MGD and additional

[2Fe–2S] clusters (Richardson, 2007).

4.3 Hemes

Hemes are primarily composed of porphyrin rings,

with an iron ion chelated by four nitrogen atoms from

porphyrin’s pyrrole rings. Based on different side

chains, hemes are classified into different types, and

the most common ones include heme B and heme C.

Heme B is the precursor of other heme types and it is

not attached to the protein, while heme C is anchored

to the peptide chains through covalent bonding to

thiolate groups of cysteines (Kim, 2018) (Figures 10

& 9c). The iron center of heme is often axially

coordinated by histidine residues (Figure 9c),

Mechanisms and Electron Transfer of Prokaryotic Nitrate Reductases

509

because the protonation state of histidine can tune the

redox potential, spin state and reactivity of the Fe ion

(Bowman, 2008). Since the Fe ion has two oxidation

states, +2 and +3, hemes can also facilitate electron

transfer.

9a 9b 9c

Figure 9: (a) The overall structure of NarGHI of E. coli (PDB: 1Q16). All ligands binding to the labelled cofactors are hided.

(b) The overall structure of NapAB of Rhodobacter sphaeroides (PDB: 1OGY). Ligands binding to heme C are not hided.

Figure 10: Structures of heme B and heme C.

Hemes are only present in Nar and Nap, as two

different types. Two heme B are present in NarI and

two heme C are present in NapB. Though crystal

structures of NapC has not been determined, it also

contains heme C. Hemes in NapB only act as electron

carriers, but those in NapC and NarI have another

function, which is to oxidize quinols (including

ubiquinols and menaquinols) to their respective

quinone form, hence generating two electrons

necessary for NO

−

reduction (Gates, 2011). For Nar,

this process also releases two protons into the

periplasm, hence creating a proton gradient for ATP

synthesis (Figures 11 & 2). Although the possible

quinol binding sites have been suggested (Bertero,

2005), there are no studies on the mechanism of

quinol oxidation by NapC and NarI.

Figure 11: Chemical equation for oxidation of ubiquinols and menaquinols.

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5 ELECTRON TRANSFER IN

NITRATE REDUCTASES

5.1 Membrane-bound Respiratory

Nitrate Reductase (Nar)

To form a complete redox loop and convert quinones

back to quinols, another enzyme is required: formate

dehydrogenase N (Fdn). It is structurally highly

similar to Nar, with three subunits GHI, where FdnG

contains a Mo-bis-MGD and a [4Fe–4S] cluster as

NarG, FdnH contains four slightly different iron–

sulfur clusters compared to NarH, and FdnI anchors

the enzyme on plasma membrane as NarI, and

contains two heme B and Q sites where quinones are

reduced back to quinols (Figure 9a & 12a & 12b).

NarGHI often forms dimers while FdnGHI forms

trimers, and their orientations are different: the FdnG

active site is inside the periplasm (Bertero, 2011)

(Figure 12b).

12a 12b

Figure 12: (a) Crystal structure of E. coli FdnGHI (PDB: 1KQG). The structure shows a trimer, but the cofactors of only one

protein unit are shown. It also shows a quinone molecule at heme B, which is probably the substrate that is going to be

reduced. (b) Simplified representation of the positions of FdnGHI and NarGHI relative to the plasma membrane. Electron

transfers and the Q cycle are also shown.

Figure 13: Distances between each electron transfer cofactor and respective redox potential values for iron–sulfur clusters in

NarH. The values are for E. coli Nar, PDB: 1Q16.

The function of the FdnG active site is to oxidize

formates (HCOO

−

) to carbon dioxide (CO

) and a

proton, releasing two electrons which are then

transferred through the [4Fe–4S] clusters in the

electron transfer subunit FdnH to FdnI, where the Q

site is located. Quinones accept the two electrons and

are reduced to quinols, which are then used as the

electron donor for NO

−

reduction. In theory, this

process releases two protons into the periplasm, but

it seems that protons produced would be consumed

by quinone reduction at FdnI, and thus conserved in

the Q cycle. Despite this, there is still a net proton

movement from the cytoplasm towards periplasm,

because one proton is produced by the oxidation of

formates while two are consumed in the oxidation of

−

Mechanisms and Electron Transfer of Prokaryotic Nitrate Reductases

511

Some specific aspects of the route of electron

transfer in Nar are also intriguing. The electron

transfer cofactors are neatly aligned to form a

structure functioning like an electrical wire (Figure

9a), where the distance between adjacent cofactors

are all less than 14 Å, the distance limit for

physiological electron transfer (Bertero, 2011)

(Figure 13). However, the redox potential values

determined in several E. coli Nar iron–sulfur clusters

(Blasco, 2001) are not consistently arranged in a

thermodynamically favorable order, with several

large potential barriers (Figure 13). The reason is still

unclear, but it may involve electron tunnelling (Page,

1999).

5.2 Periplasmic Nitrate Reductase

(Nap)

Instead of iron–sulfur clusters, NapB uses two heme

C molecules to facilitate electron transfer (Figure 9b).

It also has a quinol-oxidizing subunit NapC with two

heme C, whose crystal structure is yet to be

determined. NapC is also anchored to the membrane

(Figure 2).

Unlike Nar, it is believed that amino acid residues

also play a part in the electron transfer pathway in

Nap. In NapA, the distance between the Mo ion and

the adjacent [4Fe–4S] cluster is about 12 Å, fairly

close to the 14 Å limit, so a lysine residue which is

hydrogen-bonded to the −NH

group of one of the

MGD provides an electron transfer pathway

(Sparacino-Watkins, 2014) (Figure 14). However,

the mechanism is not stated.

Figure 14: The lysine residue (Lys

) which facilitates electron transfer in Nap of E. coli (PDB: 2NAP).

5.3 Prokaryotic Assimilatory Nitrate

Reductase (Nas)

Since the major function of Nas is very different from

that of Nar and Nap, its structure also deviates a lot

from Nar and Nap. However, the current

understanding on Nas is rather limited, and no crystal

structures of any subunit of Nas have been

determined.

Nas are free proteins located in the cytoplasm.

They do not use the Q cycle as the electron source to

reduce NO

−

, so they do not contain hemes. Instead,

Nas rely on flavodoxin or NADH as the electron

source. Flavodoxin-dependent Nas gains electrons

through the electron carrier flavin mononucleotide

(reduced form, FMNH

) via the oxidation FMNH

→

FMN + 2H

+ 2e

−

and it is composed of a single

subunit, while NADH-dependent Nas has one FAD-

containing subunit receiving electrons from the

oxidation of NADH through NADH → NAD

+ H

+ 2e

−

, and another catalytic subunit containing Mo-

bis-MGD. Iron–sulfur clusters [4Fe–4S], [3Fe–4S]

and [2Fe–2S] are probably all present for electron

transfer, according to genetic information (Moreno-

Vivián, 1999).

6 CONCLUSIONS

In conclusion, prokaryotic nitrate reductases all use a

Mo-bis-MGD cofactor at the active site to catalyze

nitrate reduction and iron–sulfur clusters to transfer

electrons. Nar and Nap use the Q cycle as the electron

source, so they both contain hemes, while Nas gains

electron through flavodoxin or NADH, where heme

is not required.

There are still lots of problems to solve in the field

of nitrate reductases. The most completely studied

nitrate reductase is Nar, because the crystal structure

of NarGHI is clear, and data about redox potentials

and distances between cofactors have all been

determined. On the contrary, there is little structural

information about Nas, where most are only

speculations. There are also other intriguing aspects

for future studies, such as the potential barrier inside

NarH, the mechanism of quinol oxidation, and why

Mo is the unique element involved in nitrate

reduction.

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